Cystic Fibrosis (CF), the most common lethal autosomic recessive disorder among Caucasians, is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. CFTR, the protein encoded by this gene, is a polytopic integral membrane protein that functions as a cAMP-activated chloride (Cl^-) channel and regulator of other channels at the apical membrane of epithelial cells. CFTR is a somewhat atypical member of the ATP binding cassette (ABC) transporters superfamily – ABCC7.

This review outlines the application of Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) and hydrogen-deuterium exchange (HDX) to study rapid, activity-linked conformational transitions in proteins. The method is implemented on a microfluidic chip which incorporates all sample handling steps required for a ‘bottom-up’ HDX workflow: A capillary mixer for sub-second HDX labeling, a static mixer for HDX quenching, a microreactor for rapid protein digestion, and on-chip electrospray. By combining short HDX labeling pulses with rapid digestion, this approach can provide a detailed characterization of the structural transitions that occur during protein folding, ligand binding, post-translational modification, and catalytic turnover in enzymes. This broad spectrum of applications in areas largely inaccessible to conventional techniques makes microfluidics-enabled TRESI-MS/HDX a unique and powerful approach for investigating the dynamic basis of protein function.

Membranes define the identity of cells: they are crucial for a cell's perception of the environment, they regulate cellular homeostasis, and they function as hubs for extra- and intracellular signals. Countless lipids have been identified, but how they function in different cell organelles is still poorly defined. Dynamic processes such as membrane fusion, fission, aging, and elimination are being delineated, but are not fully understood. We know that localized lipid–protein and lipid–lipid interactions in membranes determine cell function and fate, but much remains to be learned about the exact nature of these processes.

The 2^nd EMBO conference on Catalytic Mechanisms by Biological Systems was held in Groningen, The Netherlands, from 7 to 10 October 2012. The conference was a continuation of the EMBO conference series on the theme Enzyme Mechanisms, of which the previous was held in 2010 in Hamburg, Germany. The goal of the conference was to provide an overview on recent developments and future perspectives in the area of molecular enzymology.

Summary

Mutations in human cationic trypsinogen cause hereditary pancreatitis by altering its proteolytic regulation of activation and degradation by chymotrypsin C (CTRC). CTRC stimulates trypsinogen autoactivation by processing the activation peptide to a shorter form, but also promotes degradation by cleaving the calcium-binding loop in trypsinogen. Mutations render trypsinogen resistant to CTRC-mediated degradation and/or increase processing of the activation peptide by CTRC. Here we demonstrate that the activation peptide mutations D19A, D22G, K23R and K23_I24insIDK robustly increased the rate of trypsinogen autoactivation, both in the presence and absence of CTRC. Degradation of the mutants by CTRC was unchanged, and processing of the activation peptide was increased fourfold in the D19A mutant only. Surprisingly, however, this increased processing had only a minimal effect on autoactivation. The tetra-aspartate motif in the trypsinogen activation peptide binds calcium (K_D of ~ 1.6 mm), which stimulates autoactivation. Unexpectedly, calcium binding was not compromised by any of the activation peptide mutations. Despite normal binding, autoactivation of mutants D22G and K23_I24insIDK was not stimulated by calcium. Finally, the activation peptide mutants exhibited reduced secretion from transfected cells, and secreted trypsinogen levels were inversely proportional with autoactivation rates. We conclude that D19A, D22G, K23R and K23_I24insIDK form a mechanistically distinct subset of hereditary pancreatitis-associated mutations that exert their effect primarily through direct stimulation of autoactivation, independently of CTRC. The potentially severe clinical impact of the markedly increased autoactivation is offset by diminished secretion, resulting in a clinical phenotype that is indistinguishable from typical hereditary pancreatitis.

Several small drugs and medicinal plant extracts, such as the Indian spice extract curcumin, have a wide range of useful pharmacological properties that cannot be ascribed to binding to a single protein target alone. The lipid bilayer membrane is thought to mediate the effects of many such molecules directly via perturbation of the plasma membrane structure and dynamics, or indirectly by modulating transmembrane protein conformational equilibria. Furthermore, for bioavailability, drugs must interact with and eventually permeate the lipid bilayer barrier on the surface of cells. Biophysical studies of the interactions of drugs and plant extracts are therefore of interest. Molecular dynamics simulations, which can access time and length scales that are not simultaneously accessible by other experimental methods, are often used to obtain quantitative molecular and thermodynamic descriptions of these interactions, often with complementary biophysical measurements. This review considers recent molecular dynamics simulations of small drug-like molecules with membranes, and provides a biophysical description of possible routes of membrane-mediated pharmacological effects of drugs. The review is not exhaustive, and we focus on molecules containing aromatic ring-like structures to develop our hypotheses. We also show that some drugs and anesthetics may have an effect on the lipid bilayer analogous to that of cholesterol.

Calmodulin (CaM) is a ubiquitous, highly conserved, eukaryotic protein that binds to and regulates a number of diverse target proteins involved in different functions such as metabolism, muscle contraction, apoptosis, memory, inflammation and the immune response. In this minireview, we analyze the large number of CaM-complex structures deposited in the Protein Data Bank (i.e. crystal and nuclear magnetic resonance structures) to gain insight into the structural diversity of CaM-binding sites and mechanisms, such as those for CaM-activated protein kinases and phosphatases, voltage-gated Ca²⁺-channels and the plasma membrane Ca²⁺-ATPase.

Three adhesion complexes span the sarcolemma and facilitate critical connections between the extracellular matrix and the actin cytoskeleton: the dystrophin– and utrophin–glycoprotein complexes and α7β1 integrin. Loss of individual protein components results in a loss of the entire protein complex and muscular dystrophy. Muscular dystrophy is a progressive, lethal wasting disease characterized by repetitive cycles of myofiber degeneration and regeneration. Protein-replacement therapy offers a promising approach for the treatment of muscular dystrophy. Recently, we demonstrated that sarcospan facilitates protein–protein interactions amongst the adhesion complexes and is an important potential therapeutic target. Here, we review current protein-replacement strategies, discuss the potential benefits of sarcospan expression, and identify important experiments that must be addressed for sarcospan to move to the clinic.

During the development, progression and dissemination of neoplastic lesions, cancer cells can hijack normal pathways and mechanisms. This includes the control of the function of cellular proteins through reversible post-translational modifications, such as ADP-ribosylation, phosphorylation, and acetylation. In the case of mono-ADP-ribosylation and poly-ADP-ribosylation, the addition of one or several units of ADP-ribose to target proteins occurs via two families of enzymes that can generate ADP-ribosylated proteins: the diphtheria toxin-like ADP-ribosyltransferase (ARTD) family, comprising 17 different proteins that are either poly-ADP-ribosyltransferases or mono-ADP-ribosyltransferases or inactive enzymes; and the clostridial toxin-like ADP-ribosyltransferase family, with four human members, two of which are active mono-ADP-ribosyltransferases, and two of which are enzymatically inactive. In line with a central role for poly-ADP-ribose polymerase 1 in response to DNA damage, specific inhibitors of this enzyme have been developed as anticancer therapeutics and evaluated in several clinical trials. Recently, in combination with the discovery of a large number of enzymes that can catalyse mono-ADP-ribosylation, the role of this modification has been linked to human diseases, such as inflammation, diabetes, neurodegeneration, and cancer, thus revealing the need for the development of specific ARTD inhibitors. This will provide a better understanding of the roles of these enzymes in human physiology and pathology, so that they can be targeted in the future to generate new and efficacious drugs. This review summarizes our present knowledge of the ARTD enzymes that are involved in mono-ADP-ribosylation reactions and that have roles in cancer biology. In particular, the well-documented role of macro-containing ARTD8 in lymphoma and the putative role of ARTD15 in cancer are discussed.

Enzymes in the glucose–methanol–choline (GMC) oxidoreductase superfamily catalyze the oxidation of an alcohol moiety to the corresponding aldehyde. In this review, the current understanding of the sugar oxidation mechanism in the reaction of pyranose 2-oxidase (P2O) is highlighted and compared with that of other enzymes in the GMC family for which structural and mechanistic information is available, including glucose oxidase, choline oxidase, cholesterol oxidase, cellobiose dehydrogenase, aryl-alcohol oxidase, and pyridoxine 4-oxidase. Other enzymes in the family that have been newly discovered or for which less information is available are also discussed. A large primary kinetic isotope effect was observed for the flavin reduction when 2-d-d-glucose was used as a substrate, but no solvent kinetic isotope effect was detected for the flavin reduction step. The reaction of P2O is consistent with a hydride transfer mechanism in which there is stepwise formation of d-glucose alkoxide prior to the hydride transfer. Site-directed mutagenesis of P2O and pH-dependence studies indicated that His548 is a catalytic base that facilitates the deprotonation of C2–OH in d-glucose. This finding agrees with the current mechanistic model for aryl-alcohol oxidase, glucose oxidase, cellobiose dehydrogenase, methanol oxidase, and pyridoxine 4-oxidase, but is different from that of cholesterol oxidase and choline oxidase. Although all of the GMC enzymes share similar structural folding and use the hydride transfer mechanism for flavin reduction, they appear to have subtle differences in the fine-tuned details of how they catalyze substrate oxidation.

Inferring knowledge about biological processes by a mathematical description is a major characteristic of Systems Biology. To understand and predict system's behavior the available experimental information is translated into a mathematical model. Since the availability of experimental data is often limited and measurements contain noise, it is essential to appropriately translate experimental uncertainty to model parameters as well as to model predictions. This is especially important in Systems Biology because typically large and complex models are applied and therefore the limited experimental knowledge might yield weakly specified model components. Likelihood profiles have been recently suggested and applied in the Systems Biology for assessing parameter and prediction uncertainty. In this article, the profile likelihood concept is reviewed and the potential of the approach is demonstrated for a model of the erythropoietin (EPO) receptor.

Nitrogen fixation is the vital biochemical process in which atmospheric molecular nitrogen is made available to the biosphere. The process is highly energetically costly and thus tightly regulated. The activity of the key enzyme, nitrogenase, is controlled by reversible mono-ADP-ribosylation of one of its components, the Fe protein. This protein provides the other component, the MoFe protein, with the electrons required for the reduction of molecular nitrogen. The Fe-protein is ADP-ribosylated and de-ADP-ribosylated by dinitrogenase reductase ADP-ribosyl transferase and dinitrogenase reductase activating glycohydrolase, respectively. Here we review the current biochemical and structural knowledge of this central regulatory reaction.

The families of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) function in a coordinated manner to regulate signal transduction events that are critical for cellular homeostasis. Aberrant tyrosine phosphorylation, resulting from disruption of either PTP or PTK function, has been shown to be the cause of major human diseases, including cancer and diabetes. Consequently, the characterization of small-molecule inhibitors of these kinases and phosphatases may not only provide molecular probes with which to define the significance of particular signaling events, but also may have therapeutic implications. BAY-11-7082 is an anti-inflammatory compound that has been reported to inhibit IκB kinase activity. The compound has an α,β-unsaturated electrophilic center, which confers the property of being a Michael acceptor; this suggests that it may react with nucleophilic cysteine-containing proteins, such as PTPs. In this study, we demonstrated that BAY-11-7082 was a potent, irreversible inhibitor of PTPs. Using mass spectrometry, we have shown that BAY-11-7082 inactivated PTPs by forming a covalent adduct with the active-site cysteine. Administration of the compound caused an increase in protein tyrosine phosphorylation in RAW 264 macrophages, similar to the effects of the generic PTP inhibitor sodium orthovanadate. These data illustrate that BAY-11-7082 is an effective pan-PTP inhibitor with cell permeability, revealing its potential as a new probe for chemical biology approaches to the study of PTP function. Furthermore, the data suggest that inhibition of PTP function may contribute to the many biological effects of BAY-11-7082 that have been reported to date.

The human hepcidin 25 (hep-25) and its isoform hepcidin 20 (hep-20) are histidine-containing, cystein rich, β-sheet structured peptides endowed with antimicrobial activity. We previously reported that, similar to other histidine-containing peptides, the microbicidal effects of hep-25 and hep-20 are highly enhanced at acidic pH. In the present study, we investigated whether pH influences the mode of action of hep-25 and hep-20 on Escherichia coli American Type Culture Collection 25922 and model membranes. A striking release of β-galactosidase by hepcidin-treated E. coli was observed at pH 5.0, whereas no inner membrane permeabilization capacity was seen at pH 7.4, even at bactericidal concentrations. Similar results were obtained by flow cytometry when assessing the internalization of propidium iodide by hepcidin-treated E. coli. Scanning electron microscope imaging revealed that both peptides induced the formation of numerous blebs on the surface of bacterial cells at acidic pH but not at neutral pH. Moreover, a phospholipid/polydiacetylene colourimetric vesicle assay revealed a more evident membrane damaging effect at pH 5.0 than at pH 7.4. The leakage of entrapped dextrans of increasing molecular size from liposomes was also assessed at pH 7.4. Consistent with the lack of β-galactosidase release from whole E. coli observed at such a pH value, evident leakage of only the smallest 4-kDa dextran (and not of dextrans of 20 or 70 kDa) was observed, indicating a poor ability of hepcidin peptides to permeabilize liposome vesicles at pH 7.4. Altogether, the data obtained in the present study using different approaches strongly suggest that the ability of hepcidins to perturb bacterial membranes is markedly pH-dependent.

Stress granules (SGs) are cytoplasmic foci that rapidly form when cells are exposed to stress. They transiently store mRNAs encoding house-keeping proteins and allow the selective translation of stress-response proteins (e.g. heat shock proteins). Besides mRNA, SGs contain RNA-binding proteins, such as T cell internal antigen-1 and poly(A)-binding protein 1, which can serve as characteristic SG marker proteins. Recently, some of these SG marker proteins were found to label pathological TAR DNA binding protein of 43 kDa (TDP-43)- or fused in sarcoma (FUS)-positive cytoplasmic inclusions in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. In addition, protein aggregates in other neurodegenerative diseases (e.g. tau inclusions in Alzheimer's disease) show a co-localization with T cell internal antigen-1 as well. Moreover, several RNA-binding proteins that are commonly found in SGs have been genetically linked to neurodegeneration. This suggests that SGs might play an important role in the pathogenesis of these proteinopathies, either by acting as a seed for pathological inclusions, by mediating translational repression or by trapping essential RNA-binding proteins, or by a combination of these mechanisms. This minireview gives an overview of the general biology of SGs and highlights the recently identified connection of SGs with TDP-43, FUS and other proteins involved in neurodegenerative diseases. We propose that pathological inclusions containing RNA-binding proteins, such as TDP-43 and FUS, might arise from SGs and discuss how SGs might contribute to neurodegeneration via toxic gain or loss-of-function mechanisms.

The proteasome is a protein complex responsible for the degradation of polyubiquitin-tagged proteins. Besides the removal of target proteins, the proteasome also participates in the regulation of gene transcription in both proteolytic and non-proteolytic fashion. In this study the effect of proteasome inhibition on the basal expression of monocyte chemotactic protein-1 induced protein 1 (MCPIP1) was examined. Treatment of HepG2 or HeLa cells with proteasome inhibitor MG-132 resulted in a significant increase of MCPIP1 expression, both at mRNA and protein level. Interestingly, MG-132 did not alter MCPIP1 stability. Instead, the observed protein increase was blocked by actinomycin D, suggesting the involvement of de novo mRNA synthesis in the increase of MCPIP1 protein following MG-132 treatment. Using several inhibitors we determined the participation of extracellular-signal-regulated kinase 1/2 and p38 kinases in MCPIP1 upregulation by MG-132. Our findings show for the first time the impact of proteasome inhibition on MCPIP1 protein expression by modulation of the activity of intracellular signaling pathways. Overexpression of MCPIP1-myc protein decreased the viability of HeLa cells but not HepG2 cells, which correlates with the increased susceptibility of HeLa cells to MG-132 toxicity. Notably, both MG-132 treatment and MCPIP1-myc overexpression led to the activation of apoptosis, as revealed by the induction of caspases 3/7 in both types of cell lines. This suggests the involvement of MCPIP1 upregulation in toxic properties of proteasome inhibition, which is an acknowledged approach to the treatment of several cancer types.

The mammalian diaphragm muscle is essential for respiration, and thus is one of the most critical skeletal muscles in the human body. Defects in diaphragm development leading to congenital diaphragmatic hernias (CDH) are common birth defects and result in severe morbidity or mortality. Given its functional importance and the frequency of congenital defects, an understanding of diaphragm development, both normally and during herniation, is important. We review current knowledge of the embryological origins of the diaphragm, diaphragm development and morphogenesis, as well as the genetic and developmental aetiology of diaphragm birth defects.

Telomeres are nucleoprotein structures found at the ends of linear chromosomes. Telomeric DNA shortens with each cell division, effectively restricting the proliferative capacity of human cells. Telomerase, a specialized reverse transcriptase, is responsible for de novo synthesis of telomeric DNA, and is the major physiological means by which mammalian cells extend telomere length. Telomerase activity in human soma is developmentally regulated according to cell type. Failure to tightly regulate telomerase has dire consequences: dysregulated telomerase activity is observed in more than 90% of human cancers, while haplo-insufficient expression of telomerase components underlies several inherited premature aging syndromes. Over the past decade, we have significantly improved our understanding of the structure–activity relationships between the two core telomerase components: telomerase reverse transcriptase and telomerase RNA. Genetic screening for telomerase deficiency syndromes has identified new partners in the biogenesis of telomerase and its catalytic functions. These data revealed a level of regulation complexity that is unexpected when compared with the other cellular polymerases. In this review, we summarize current knowledge on the structure–activity relationships of telomerase reverse transcriptase and telomerase RNA, and discuss how the biogenesis of telomerase provides additional regulation of its actions.

The human cytochrome P450 2D6 (CYP2D6) is one of the major human drug metabolizing enzymes and acts preferably on substrates containing a basic nitrogen atom. Testosterone − just as other steroids − is an atypical substrate and only poorly metabolized by CYP2D6. The present study intended to investigate the influence of the two active site residues 216 and 483 on the capability of CYP2D6 to hydroxylate steroids such as for example testosterone. All 400 possible combinatorial mutations at these two positions have been generated and expressed individually in Pichia pastoris. Employing whole-cell biotransformations coupled with HPLC-MS analysis the testosterone hydroxylase activity and regioselectivity of every single CYP2D6 variant was determined. Covering the whole sequence space, CYP2D6 variants with improved activity and so far unknown regio-preference in testosterone hydroxylation were identified. Most intriguingly and in contrast to previous literature reports about mutein F483I, the mutation F483G led to preferred hydroxylation at the 2β-position, while the slow formation of 6β-hydroxytestosterone, the main product of wild-type CYP2D6, was further reduced. Two point mutations have already been sufficient to convert CYP2D6 into a steroid hydroxylase with the highest ever reported testosterone hydroxylation rate for this enzyme, which is of the same order of magnitude as for the conversion of the standard substrate bufuralol by wild-type CYP2D6. Furthermore, this study is also an example for efficient human CYP engineering in P. pastoris for biocatalytic applications and to study so far unknown pharmacokinetic effects of individual and combined mutations in these key enzymes of the human drug metabolism.

Upon their discovery almost 40 years ago, isoforms of the lipid-activated protein kinase C (PKC) family were initially regarded only as downstream effectors of the second messengers calcium and diacylglycerol, undergoing activation upon phospholipid hydrolysis in response to acute stimuli. Subsequently, several isoforms were found to be associated with the inhibitory effects of lipid over-supply on glucose homeostasis, especially the negative cross-talk with insulin signal transduction, observed upon accumulation of diacylglycerol in insulin target tissues. The PKC family has therefore attracted much attention in diabetes and obesity research, because intracellular lipid accumulation is strongly correlated with defective insulin action and the development of type 2 diabetes. Causal roles for various isoforms in the generation of insulin resistance have more recently been confirmed using PKC-deficient mice. However, during characterization of these animals, it became increasingly evident that the enzymes play key roles in the modulation of lipid metabolism itself, and may control the supply of lipids between tissues such as adipose and liver. Molecular studies have also demonstrated roles for PKC isoforms in several aspects of lipid metabolism, such as adipocyte differentiation and hepatic lipogenesis. While the precise mechanisms involved, especially the identities of protein substrates, are still unclear, the emerging picture suggests that the currently held view of the contribution of PKC isoforms to metabolism is an over-simplification. Although PKCs may inhibit insulin signal transduction, these enzymes are not merely downstream effectors of lipid accumulation, but in fact control the fate of fatty acids, thus the tail wags the dog.

Myogenesis consists of a highly organized and regulated sequence of cellular processes aimed at forming or repairing muscle tissue. Several processes occur during myogenesis, including cell proliferation, migration, and differentiation. Cytokines, proteinases, cell adhesion molecules and growth factors are involved, either activating or inhibiting these events, and are modulated by a group of molecules called proteoglycans (PGs), which play critical roles in skeletal muscle physiology. Particularly interesting are some of the factors responsible for the fibrotic response associated with skeletal muscular dystrophies. Transforming growth factor-β and connective tissue growth factor have gained great attention as factors participating in the fibrotic response in skeletal muscle. This review is focused on the advances achieved in understanding the roles of proteoglycans as modulators of profibrotic growth factors in fibrosis associated with diseases such as skeletal muscle dystrophies.

This study explores the influence of long-range intra-protein electrostatic interactions on the conformation of calmodulin in solution. Ensemble Förster resonance energy transfer (FRET) is measured for calmodulin with a fluorophore pair incorporated specifically with a donor at residue 17 and an acceptor at position 117. This construct was generated by a combination of solid phase peptide synthesis, cloning, expression and native chemical ligation. This labelling method has not previously been used with calmodulin and represents a convenient method for ensuring the explicit positioning of the fluorophores. The ensemble FRET experiments reveal significant electrostatic repulsion between the globular domains in the calcium-free protein. At low salt, calmodulin has a relatively extended conformation and the distance between the domains is further increased by denaturation, by heat or by non-ionic denaturants. The repulsion between domains is screened by salt and is also diminished by calcium binding, which changes the protein net charge from −23 to −15. Compared with the calcium-free form at low salt, the FRET efficiency for the calcium-bound form has, on average, increased 10-fold. The conformation of the calcium form is insensitive to salt screening. These results imply that when the two globular domains of calmodulin interact with target, there is no significant free energy penalty due to electrostatic interactions.

The biotech industry is continuously seeking for new or improved biocatalysts. The success of these efforts is often hampered by the lack of an efficient screening assay. Thus, to be able to extend the number of enzymes available for industrial applications, high-throughput screening and selection methods are required. In the last few years an impressive range of screening and selection strategies has been developed. In this review, we will mainly focus on in vivo reporter systems in which the activity of a reporter is controlled by the activity of an enzyme of interest. Different mechanisms can be distinguished: (a) binding of the product of the enzymatic reaction to a transcriptional regulator and thereby turning on transcription of the reporter; (b) direct modification of a transcriptional regulator by the enzyme resulting in expression of the reporter; (c) binding of the product to a regulatory riboswitch or ribozyme, resulting in translation of the reporter; and (d) direct modification of the reporter by the enzyme, altering the reporter's activity. The choice for either a selection or a screening strategy depends on the type of reporter, e.g. providing antibiotic resistance (selection) or transmitting a fluorescent signal (screening). Although developing the specificity of each of these reporter-based selection or screening systems towards a certain enzymatic reaction is not yet straightforward, their adjustable modular design appears to be a promise for general applicability in the near future.

The fluorescent probes Nile Red (nonsteroidal dye) and 25-{N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino}-27-norcholesterol (25-NBD-cholesterol) (a cholesterol analog) were evaluated as novel substrates for steroid-converting oxidoreductases. Docking simulations with autodock showed that Nile Red fits well into the substrate-binding site of cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) (binding energy value of −8.3 kcal·mol⁻¹). Recombinant Saccharomyces cerevisiae and Yarrowia lipolytica, both expressing CYP17A1, were found to catalyze the conversion of Nile Red into two N-dealkylated derivatives. The conversion by the yeasts was shown to increase in the cases of coexpression of electron-donating partners of CYP17A1. The highest specific activity value (1.30 ± 0.02 min⁻¹) was achieved for the strain Y. lipolytica DC5, expressing CYP17A1 and the yeast's NADPH-cytochrome P450 reductase. The dye was also metabolized by pure CYP17A1 into the N-dealkylated derivatives, and gave a type I difference spectrum when titrated into low-spin CYP17A1. Analogously, docking simulations demonstrated that 25-NBD-cholesterol binds into the active site of the microbial cholesterol oxidase (CHOX) from Brevibacterium sterolicum (binding energy value of −5.6 kcal·mol⁻¹). The steroid was found to be converted into its 4-en-3-one derivative by CHOX (K_m and k_cat values were estimated to be 58.1 ± 5.9 μm and 0.66 ± 0.14 s⁻¹, respectively). The 4-en-3-one derivative was also detected as the product of 25-NBD-cholesterol oxidation with both pure microbial cholesterol dehydrogenase (CHDH) and a pathogenic bacterium, Pseudomonas aeruginosa, possessing CHOXs and CHDHs. These results provide novel opportunities for investigation of the structure–function relationships of the aforementioned oxidoreductases, which catalyze essential steps of steroid bioconversion in mammals (CYP17A1) and bacteria (CHOX and CHDH), with fluorescence-based techniques.

There is currently no cure for muscular dystrophies, although several promising strategies are in basic and clinical research. One such strategy is cell transplantation with satellite cells (or their myoblast progeny) to repair damaged muscle and provide dystrophin protein with the aim of preventing subsequent myofibre degeneration and repopulating the stem cell niche for future use. The present review aims to cover recent advances in satellite cell/myoblast therapy and to discuss the challenges that remain for it to become a realistic therapy.

Muscular dystrophy arises from ongoing muscle degeneration and insufficient regeneration. This imbalance leads to loss of muscle, with replacement by scar or fibrotic tissue, resulting in muscle weakness and, eventually, loss of muscle function. Human muscular dystrophy is characterized by a wide range of disease severity, even when the same genetic mutation is present. This variability implies that other factors, both genetic and environmental, modify the disease outcome. There has been an ongoing effort to define the genetic and molecular bases that influence muscular dystrophy onset and progression. Modifier genes for muscle disease have been identified through both candidate gene approaches and genome-wide surveys. Multiple lines of experimental evidence have now converged on the transforming growth factor-β (TGF-β) pathway as a modifier for muscular dystrophy. TGF-β signaling is upregulated in dystrophic muscle as a result of a destabilized plasma membrane and/or an altered extracellular matrix. Given the important biological role of the TGF-β pathway, and its role beyond muscle homeostasis, we review modifier genes that alter the TGF-β pathway and approaches to modulate TGF-β activity to ameliorate muscle disease.

The xylene ring of riboflavin (vitamin B₂) is assembled from two molecules of 3,4-dihydroxy-2-butanone 4-phosphate by a mechanistically complex process that is jointly catalyzed by lumazine synthase and riboflavin synthase. In Bacillaceae, these enzymes form a structurally unique complex comprising an icosahedral shell of 60 lumazine synthase subunits and a core of three riboflavin synthase subunits, whereas many other bacteria have empty lumazine synthase capsids, fungi, Archaea and some eubacteria have pentameric lumazine synthases, and the riboflavin synthases of Archaea are paralogs of lumazine synthase. The structures of the molecular ensembles have been studied in considerable detail by X-ray crystallography, X-ray small-angle scattering and electron microscopy. However, certain mechanistic aspects remain unknown. Surprisingly, the quaternary structure of the icosahedral β subunit capsids undergoes drastic changes, resulting in formation of large, quasi-spherical capsids; this process is modulated by sequence mutations. The occurrence of large shells consisting of 180 or more lumazine synthase subunits has recently generated interest for protein engineering topics, particularly the construction of encapsulation systems.

Despite the sequencing of the human genome, the rate of innovative and successful drug discovery in the pharmaceutical industry has continued to decrease. Leaving aside regulatory matters, the fundamental and interlinked intellectual issues proposed to be largely responsible for this are: (a) the move from ‘function-first’ to ‘target-first’ methods of screening and drug discovery; (b) the belief that successful drugs should and do interact solely with single, individual targets, despite natural evolution's selection for biochemical networks that are robust to individual parameter changes; (c) an over-reliance on the rule-of-5 to constrain biophysical and chemical properties of drug libraries; (d) the general abandoning of natural products that do not obey the rule-of-5; (e) an incorrect belief that drugs diffuse passively into (and presumably out of) cells across the bilayers portions of membranes, according to their lipophilicity; (f) a widespread failure to recognize the overwhelmingly important role of proteinaceous transporters, as well as their expression profiles, in determining drug distribution in and between different tissues and individual patients; and (g) the general failure to use engineering principles to model biology in parallel with performing ‘wet’ experiments, such that ‘what if?’ experiments can be performed in silico to assess the likely success of any strategy. These facts/ideas are illustrated with a reasonably extensive literature review. Success in turning round drug discovery consequently requires: (a) decent systems biology models of human biochemical networks; (b) the use of these (iteratively with experiments) to model how drugs need to interact with multiple targets to have substantive effects on the phenotype; (c) the adoption of polypharmacology and/or cocktails of drugs as a desirable goal in itself; (d) the incorporation of drug transporters into systems biology models, en route to full and multiscale systems biology models that incorporate drug absorption, distribution, metabolism and excretion; (e) a return to ‘function-first’ or phenotypic screening; and (f) novel methods for inferring modes of action by measuring the properties on system variables at all levels of the ‘omes. Such a strategy offers the opportunity of achieving a state where we can hope to predict biological processes and the effect of pharmaceutical agents upon them. Consequently, this should both lower attrition rates and raise the rates of discovery of effective drugs substantially.

Research into fundamental principles and the testing of therapeutic hypotheses for treatment of human disease is commonly performed on mouse models of human diseases. Although this is often the only practicable approach, it carries a number of caveats arising from differences between the two species. This review focuses on the example of skeletal muscle disease, in particular muscular dystrophy, to identify some of the principal classes of obstacles to translation of data from mouse to humans. Of these, the difference in scale is one of the most commonly ignored, and is of particular interest because it has quite major repercussions for evaluation of some classes of intervention and of outcome criteria, while having comparatively little bearing on others. Likewise, inter-species differences and similarities in cell and molecular biological mechanisms underlying development, growth and response to pathological processes should be considered on an individual basis. An awareness of such distinctions is crucial if we are to avoid misjudging the likely applicability to humans of results obtained on mouse models.

Accumulation of amyloidogenic proteins such as Tau is a hallmark of neurodegenerative diseases including Alzheimer disease and fronto-temporal dementias. To link Tau pathology to cognitive impairments and defects in synaptic plasticity, we created four inducible Tau transgenic mouse models with expression of pro- and anti-aggregant variants of either full-length human Tau (hTau40/ΔK280 and hTau40/ΔK280/PP) or the truncated Tau repeat domain (Tau_RD/ΔK280 and Tau_RD/ΔK280/PP). Here we review the histopathological features caused by pro-aggregant Tau, and correlate them with behavioral deficits and impairments in synaptic transmission. Both pro-aggregant Tau variants cause Alzheimer-like features, including synapse loss, mis-localization of Tau into the somatodendritic compartment, conformational changes and hyperphosphorylation. However, there is a clear difference in the extent of Tau aggregation and neurotoxicity. While pro-aggregant full-length hTau40/ΔK280 leads to a ‘pre-tangle’ pathology, the repeat domain Tau_RD/ΔK280 causes massive formation of neurofibrillary tangles and neuronal loss in the hippocampus. However, both Tau variants cause co-aggregation of human and mouse Tau and similar functional impairments. Thus, earlier Tau pathological stages and not necessarily neurofibrillary tangles are critical for the development of cognitive malfunctions. Most importantly, memory and synapses recover after switching off expression of pro-aggregant Tau. The rescue of functional impairments correlates with the rescue of most Tau pathological changes and most strikingly the recovery of synapses. This implies that tauopathies as such are reversible, provided that amyloidogenic Tau is removed. Therefore, our Tau transgenic mice may serve as model systems for in vivo validation of therapeutic strategies and drug candidates with regard to cognition and synaptic function.

Cells encounter many physical, chemical and biological stresses that perturb plasma membrane integrity, warranting an immediate membrane repair response to regain cell homeostasis. Failure to respond properly to such perturbation leads to individual cell death, which may also produce systemic influence by triggering sterile immunological responses. In this review, we discuss recent progress on understanding the mechanisms underlying muscle cell membrane repair and the potential mediators of innate immune activation when the membrane repair system is defective, specifically focusing on pathology associated with dysferlin deficiency.

Mesenchymal stem cells have the potential to undergo commitment and differentiation into a variety of cell types, including osteoblasts, chondrocytes, myocytes and adipocytes. Growth differentiation factor 6 (Gdf6) is a member of the transforming growth factor β superfamily. We have examined the potential role of Gdf6 in adipogenesis of mesenchymal stem cells, and found that over-expression of Gdf6 induced commitment of pluripotent mesenchymal C3H10T1/2 cells to the adipocyte lineage. The type I receptor Bmpr1a and the type II receptors Bmpr2 and Acvr2a mediate the Gdf6 signaling pathway. RNAi silencing of Smad4 and p38 MAPK suggested that both Smad and p38 MAPK pathways are involved in this process. The expression of Runx1t1 was down-regulated in committed pre-adipocytes, and forced expression of Runx1t1 blocked the adipocytic commitment. The results demonstrate a role for Gdf6 in adipocytic commitment and differentiation.

S-palmitoylation is post-translational modification, which consists in the addition of a C16 acyl chain to cytosolic cysteines and which is unique amongst lipid modifications in that it is reversible. It can thus, like phosphorylation or ubiquitination, act as a switch. While palmitoylation of soluble proteins allows them to interact with membranes, the consequences of palmitoylation for transmembrane proteins are more enigmatic. We briefly review the current knowledge regarding the enzymes responsible for palmitate addition and removal. We then describe various observed consequences of membrane protein palmitoylation. We propose that the direct effects of palmitoylation on transmembrane proteins, however, might be limited to four non-mutually exclusive mechanistic consequences: alterations in the conformation of transmembrane domains, association with specific membrane domains, controlled interactions with other proteins and controlled interplay with other post-translational modifications.

The interactions of selected antibiotics with the trans-acting antigenomic delta ribozyme were mapped. Ribozyme with two oligonucleotide substrates was used, one uncleavable with deoxycytidine at the cleavage site, mimicking the initial state of ribozyme, and the other with an all-RNA substrate mimicking, after cleavage, the product state. Mapping was performed with a set of RNA structural probing methods: Pb²⁺ -induced cleavage, nuclease digestion, and the selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) approach. The experimental results combined with molecular modeling revealed different binding sites for neomycin B, amikacin and actinomycin D inside the ribozyme structure. Neomycin B, an aminoglycoside antibiotic, which strongly inhibited the catalytic properties of delta ribozyme, was bound to the pocket formed by the P1 stem, the P1.1 pseudoknot, and the J4/2 junction. Amikacin showed less effective binding to the ribozyme catalytic core, resulting in weak inhibition. Complexes of these aminoglycosides with Cu²⁺ ions were bound to the same ribozyme regions, but more effectively, showing lower K_d values. On the other hand, the Cu²⁺ complex of the cyclopeptide antibiotic actinonomycin D was preferentially intercalated into the P2 and the P4 double-stranded region, and was three times more potent in ribozyme inhibition than the free antibiotic. In addition, some differences in SHAPE reactivities between the ribozyme forms containing all-RNA and deoxycytidine-modified substrates in the J4/2 region were detected, pointing to different ribozyme conformations before and after the cleavage event.

Telomerase plays a pivotal role in the pathology of cancer by maintaining genome integrity, controlling cell proliferation, and regulating tissue homeostasis. Experimental data from genetically modified mice and human premature aging diseases clearly indicate that intact telomere function is crucial for cell proliferation and survival, whereas dysfunctional telomeres can lead to either cancer or aging pathologies, depending on the integrity of the cellular stress response pathways. The canonical function of telomerase reverse transcriptase is the synthesis of telomeric DNA repeats and the maintenance of telomere length. However, accumulating evidence indicates that telomerase reverse transcriptase may also exert some fundamental biological functions independently of its enzymatic activity in telomere maintenance. More recent studies have demonstrated that telomerase reverse transcriptase can act as a transcriptional modulator in the nucleus and exhibits RNA-dependent RNA polymerase activity in the mitochondria. Telomerase activation may have both telomere-dependent and telomere-independent implications for tumor progression. Many excellent reviews have described critical roles of telomere and telomerase in human cancer; this minireview will focus on the role of telomerase in cancer progression, with a special emphasis on the nontelomeric function of telomerase.

All sulfation reactions rely on active sulfate in the form of 3′-phospho-adenosine-5′-phosphosulfate (PAPS). In fungi, bacteria, and plants, the enzymes responsible for PAPS synthesis, ATP sulfurylase and adenosine-5′-phosphosulfate (APS) kinase, reside on separate polypeptide chains. In metazoans, however, bifunctional PAPS synthases catalyze the consecutive steps of sulfate activation by converting sulfate to PAPS via the intermediate APS. This intricate molecule and the related nucleotides PAPS and 3′-phospho-adenosine-5′-phosphate modulate the function of various enzymes from sulfation pathways, and these effects are summarized in this review. On the ATP sulfurylase domain that initially produces APS from sulfate and ATP, APS acts as a potent product inhibitor, being competitive with both ATP and sulfate. For the APS kinase domain that phosphorylates APS to PAPS, APS is an uncompetitive substrate inhibitor that can bind both at the ATP/ADP-binding site and the PAPS/APS-binding site. For human PAPS synthase 1, the steady-state concentration of APS has been modelled to be 1.6 μm, but this may increase up to 60 μm under conditions of sulfate excess. It is noteworthy that the APS concentration for maximal APS kinase activity is 15 μm. Finally, we recognized APS as a highly specific stabilizer of bifunctional PAPS synthases. APS most likely stabilizes the APS kinase part of these proteins by forming a dead-end enzyme–ADP–APS complex at APS concentrations between 0.5 and 5 μm; at higher concentrations, APS may bind to the catalytic centers of ATP sulfurylase. Based on the assumption that cellular concentrations of APS fluctuate within this range, APS can therefore be regarded as a key modulator of PAPS synthase functions.

A large number of industrially relevant enzymes depend upon nicotinamide cofactors, which are too expensive to be added in stoichiometric amounts. Existing NAD(P)H-recycling systems suffer from low activity, or the generation of side products. H₂-driven cofactor regeneration has the advantage of 100% atom efficiency and the use of H₂ as a cheap reducing agent, in a world where sustainable energy carriers are increasingly attractive. The state of development of H₂-driven cofactor-recycling systems and examples of their integration with enzyme reactions are summarized in this article. The O₂-tolerant NAD⁺-reducing hydrogenase from Ralstonia eutropha is a particularly attractive candidate for this approach, and we therefore discuss its catalytic properties that are relevant for technical applications.

Skeletal muscle mass increases during postnatal development through a process of hypertrophy, i.e. enlargement of individual muscle fibers, and a similar process may be induced in adult skeletal muscle in response to contractile activity, such as strength exercise, and specific hormones, such as androgens and β-adrenergic agonists. Muscle hypertrophy occurs when the overall rates of protein synthesis exceed the rates of protein degradation. Two major signaling pathways control protein synthesis, the IGF1–Akt–mTOR pathway, acting as a positive regulator, and the myostatin–Smad2/3 pathway, acting as a negative regulator, and additional pathways have recently been identified. Proliferation and fusion of satellite cells, leading to an increase in the number of myonuclei, may also contribute to muscle growth during early but not late stages of postnatal development and in some forms of muscle hypertrophy in the adult. Muscle atrophy occurs when protein degradation rates exceed protein synthesis, and may be induced in adult skeletal muscle in a variety of conditions, including starvation, denervation, cancer cachexia, heart failure and aging. Two major protein degradation pathways, the proteasomal and the autophagic–lysosomal pathways, are activated during muscle atrophy and variably contribute to the loss of muscle mass. These pathways involve a variety of atrophy-related genes or atrogenes, which are controlled by specific transcription factors, such as FoxO3, which is negatively regulated by Akt, and NF-κB, which is activated by inflammatory cytokines.

The oxidation of lipids has been shown to impact virtually all cellular processes. The paradigm has been that this involvement is due to interference with the functions of membrane-associated proteins. It is only recently that methodological advances in molecular-level detection and identification have begun to provide insights into oxidative lipid modification and its involvement in cell signaling as well as in major diseases and inflammation. Extensive evidence suggests a correlation between lipid peroxidation and degenerative neurological diseases such as Parkinson's and Alzheimer's, as well as type 2 diabetes and cancer. Despite the obvious relevance of understanding the molecular basis of the above ailments, the exact modes of action of oxidized lipids have remained elusive. In this minireview, we summarize recent findings on the biophysical characteristics of biomembranes following oxidative derivatization of their lipids, and how these altered properties are involved in both physiological processes and major pathological conditions. Lipid-bearing, oxidatively truncated and functionalized acyl chains are known to modify membrane bulk physical properties, such as thermal phase behavior, bilayer thickness, hydration and polarity profiles, as manifest in the altered structural dynamics of lipid bilayers, leading to augmented membrane permeability, fast lipid transbilayer diffusion (flip-flop), loss of lipid asymmetry (scrambling) and phase segregation (the formation of ‘rafts’). These changes, together with the generated reactive lipid derivatives, can be further expected to interfere with lipid–protein interactions, influencing metabolic pathways, causing inflammation, the execution phase in apoptosis and initiating pathological processes.

Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca²⁺ on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has an unambiguous preference for basic amino acids at the P₁ position of peptide substrates and that this is most probably a result of hydrogen bonding from the P₁ residue to Asp95 and Asp211 in TbMCA2. In addition, TbMCA2 also has a preference for charged residues at the P₂ and P₃ positions and for small residues at the prime side of a peptide substrate. Studies into the effects of Ca²⁺ on the enzyme revealed the presence of two Ca²⁺ binding sites and a reversible structural modification of the enzyme upon Ca²⁺ binding. In addition, the concentration of Ca²⁺ used for activation of TbMCA2 was found to produce a differential effect on the activity of TbMCA2, but only when a series of peptides that differed in P₂ were examined, suggesting that Ca²⁺ activation of TbMCA2 has a structural effect on the enzyme in the vicinity of the S₂ binding pocket. Collectively, these data give new insights into the substrate specificity and Ca²⁺ activation of TbMCA2. This provides important functional details and leads to a better understanding of metacaspases, which are known to play an important role in trypanosomes and make attractive drug targets due to their absence in humans.

An important mechanistic aspect of enzyme catalyzed amide bond hydrolysis is the specific orientation of the lone pair of the nitrogen of the scissile amide bond during catalysis. As discussed in the literature during the last decades, stereoelectronic effects cause the single lone pair in the formed tetrahedral intermediate to be situated in a non-productive conformation in the enzyme active site and hence nitrogen inversion or rotation is necessary. By discussing recent mechanistic findings in the literature relevant for the conformation of the lone pair of the reacting amide nitrogen atom, it will be demonstrated that nature has evolved at least two catalytic strategies to cope with the stereoelectronic constraints inherent to amide bond hydrolysis regardless of the fold or catalytic mechanism. One solution to the inversion problem is to stabilize the transition state of inversion by hydrogen bond formation; another is to introduce a concerted proton shuttle mechanism that avoids inversion and delivers a hydrogen to the lone pair. By using molecular modeling it is demonstrated that the H-bond strategy is general and can be expanded to include many amidases/proteases with important metabolic functions, including the proteasome. Some examples of the proton shuttle mechanism will also be mentioned. To complete the picture of efficient enzyme catalyzed amide bond hydrolysis, general interactions in the active site of these catalysts will be discussed. An expanded knowledge of the prerequisites of efficient amide bond hydrolysis beyond the oxyanion hole and the catalytic dyad/triad will be of importance for enzyme and drug design.

Calcium is a universal messenger regulating many physiological functions, including the ability of the cell to undergo orderly self-destruction upon completion of its function, called apoptosis. In physiopathological conditions such as cancer, apoptotic processes become deregulated, leading to apoptosis-resistant phenotypes. Recently, perturbations of cellular calcium homeostasis have been described in apoptosis-resistant cell phenotypes. Thereby, new molecular actors have been identified, offering more accurate research possibilities in the field of apoptosis resistance and providing the bases for more rational approaches to cancer treatments. In this review, we focus on the calcium-transporting protein-dependent pathways involved in apoptosis, which are deregulated by cancer. We present the calcium-transporting proteins involved in the deregulation of apoptosis, and those chemotherapies that target actors in calcium-induced apoptosis.

Satellite cells, the myogenic progenitors located at the myofibre surface, are essential for the repair of adult skeletal muscle. There is ample evidence for an age-linked decline in the number of satellite cells and performance in limb muscles. Hence, an effective means of activating and expanding the satellite cell pool may enhance muscle maintenance and reduce the impact of age-associated muscle deterioration (sarcopaenia). Accordingly, in the present study, we explored the beneficial effects of endurance exercise on satellite cells in young and old mice. Animals were subjected to an 8-week moderate-intensity treadmill-running approach that does not inflict apparent muscle damage (0° inclination, 11.5 m·min⁻¹ for 30 min·day⁻¹, 6 days·week⁻¹). Myofibres of extensor digitorum longus muscles were then isolated from exercised and sedentary mice and used for monitoring the number of satellite cells, as well as for harvesting individual satellite cells for clonal growth assays. We specifically focused on satellite cell pools of single myofibres, with the view that daily wear of muscles probably affects individual myofibres rather than causing overall muscle damage. We found an expansion of the satellite cell pool in the exercised groups compared to the sedentary groups, with the same increase (~ 1.6-fold) in both ages. The results of the present study are in agreement with our findings obtained using rat gastrocnemius, indicating the consistent effect of exercise on satellite cell expansion in limb muscles. The experimental paradigm established in the present study is useful for investigating satellite cell dynamics at the myofibre niche, as well as for broader investigations of the impact of physiologically and pathologically relevant factors on adult myogenesis.

Caffeic acid phenyl ester (CAPE) has been identified as an active component of propolis, a substance that confers diverse activities in cells of various origins. However, the molecular basis of CAPE-mediated cellular activity remains to be clarified. Here, we show that CAPE preferentially induced S- and G₂/M-phase cell-cycle arrests and initiated apoptosis in human cervical cancer lines. The effect was found to be associated with increased expression of E2F-1, as there is no CAPE-mediated induction of E2F-1 in the pre-cancerous cervical Z172 cells. CAPE also up-regulated the E2F-1 target genes cyclin A, cyclin E and apoptotic protease activating of factor 1 (Apaf-1) but down-regulated cyclin B and induced myeloid leukemia cell differentiation protein (Mcl-1). These results suggest the involvement of E2F-1 in CAPE-mediated growth inhibition and cell-cycle arrest. Transient transfection studies with luciferase reporters revealed that CAPE altered the transcriptional activity of the apaf-1 and mcl-1 promoters. Further studies using chromatin immunoprecipitation assays demonstrated that E2F-1 binding to the apaf-1 and cyclin B promoters was increased and decreased, respectively, in CAPE-treated cells. Furthermore, E2F-1 silencing abolished CAPE-mediated effects on cell-cycle arrest, apoptosis and related gene expression. Taken together, these results indicate a crucial role for E2F-1 in CAPE-mediated cellular activities in cervical cancer cells.

Haloalkane dehalogenases catalyze the hydrolysis of carbon–halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilizing residues that are important in substrate binding and stabilization of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenases, these residues are either a pair of tryptophans or a tryptophan–asparagine pair. The newly-isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 (EC 3.8.1.5) possesses a unique halide-stabilizing tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the structure and catalytic properties of the enzyme were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen-bonding patterns within the active sites of the wild-type and the mutant, as well as of the stabilization of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding by 3.7-fold, presumably as a result of the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the substrate specificity of the enzyme and reduced its K_0.5 for selected halogenated substrates by a factor of 2–4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilizes its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.

Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 μg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family.

Calpain-7 is a mammalian ortholog of a fungal non-classical calpain named PalB, which is an intracellular cysteine protease and functions in fungal alkaline adaptation in association with the endosomal sorting complex required for transport (ESCRT) system. Despite our previous finding [Osako Y et al. (2010) FEBS J 277, 4412–4426] of autolytic activity, neither physiological nor non-physiological substrates of calpain-7 have yet been identified, and experimentally useful substrates that show robust evidence of intermolecular proteolytic activity of calpain-7 are required. In this study, we found limited proteolysis of C-terminally truncated ALG-2-interacting protein X (ALIX; (ALIXΔC), but not full-length ALIX, when the mutant was co-over-expressed with calpain-7 in HEK293T cells and analyzed by western blotting. The extent of ALIXΔC cleavage by calpain-7 was enhanced by co-expression with several ESCRT proteins. We investigated whether fusion of casein, a commonly used substrate for a variety of proteases including calpains, to the Bro1 domain confers the ability to serve as a substrate of calpain-7, but no specific cleavage was observed. However, when domain 1 of calpastatin, an endogenous inhibitory protein of ubiquitous classical calpains, was fused with the Bro1 domain, the fusion protein was cleaved at the C-terminal border of subdomain B (an inhibitory center for calpains) of calpastatin. These results demonstrate for the first time that calpain-7 has limited proteolytic activity and substrate preference. Moreover, the proteolytic assay system developed enabled us to perform mutational analysis of calpain-7, which revealed the importance of not only the N-terminal microtubule-interacting and trafficking (MIT) domains but also the C-terminal C2 domain-like domains for proteolytic activity.

The most severe form of human malaria is caused by the parasite Plasmodium falciparum. Despite the current need, there is no effective vaccine and parasites are becoming resistant to most of the antimalarials available. Therefore, there is an urgent need to discover new drugs from targets that have not yet suffered from drug pressure with the aim of overcoming the problem of new emerging resistance. Membrane transporters, such as P. falciparum Ca²⁺-ATPase 6 (PfATP6), the P. falciparum sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA), have been proposed as potentially good antimalarial targets. The present investigation focuses on: (a) the large-scale purification of PfATP6 for maintenance of its enzymatic activity; (b) screening for PfATP6 inhibitors from a compound library; and (c) the selection of the best inhibitors for further tests on P. falciparum growth in vitro. We managed to heterologously express in yeast and purify an active form of PfATP6 as previously described, although in larger amounts. In addition to some classical SERCA inhibitors, a chemical library of 1680 molecules was screened. From these, we selected a pool of the 20 most potent inhibitors of PfATP6, presenting half maximal inhibitory concentration values in the range 1–9 μm. From these, eight were chosen for evaluation of their effect on P. falciparum growth in vitro, and the best compound presented a half maximal inhibitory concentration of ~ 2 μm. We verified the absence of an inhibitory effect of most of the compounds on mammalian SERCA1a, representing a potential advantage in terms of human toxicity. The present study describes a multidisciplinary approach allowing the selection of promising PfATP6-specific inhibitors with good antimalarial activity.

Candida albicans is the most common opportunistic fungal pathogen and its apoptosis is inducible by environmental stress. Based on our previous finding that transcription factor Cap1p was involved in baicalein-induced apoptosis, the present study aimed to further clarify the role of Cap1p in apoptosis by observing the impact of CAP1 deletion on cell fate. It was found that apoptotic stimulation with amphotericin B, acetic acid and hydrogen peroxide increased the number of apoptotic and necrotic cells, caspase activity and the accumulation of reactive oxygen species, whereas it decreased the mitochondrial membrane potential and intracellular ATP level in the cap1Δ/Δ mutant. The cell fate was, at least partly, caused by glutathione depletion and attenuation of the expression of the glutathione reductase gene in the cap1Δ/Δ mutant. Collectively, our data suggest that Cap1p participated in the apoptosis of C. albicans by regulating the expression of the glutathione reductase gene and glutathione content.

The classical picture of enzyme catalysis relies on controlling the entropic and enthalpic contributions by manipulating reaction barriers and co-locating reactants and cofactors to facilitate the reaction chemistry. Catalysis is linked inextricably to the geometry of the enzyme–substrate complex and the chemical/physical properties of the active site, and probably to dynamical contributions that guide reactants along the desired reaction coordinate. Coenzyme B₁₂-dependent enzymes have remarkable catalytic power and unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. Here we discuss recent developments that are beginning to provide atomistic insight into how coenzyme B₁₂-dependent enzymes steer reactants along the reaction coordinate. Such insight will ultimately generate ‘movies’ of the catalytic process across all relevant time scales. In the longer term, this will enable more predictive engineering of this class of enzyme to achieve new and desirable chemical outcomes.

Aging of an organism is associated with the functional decline of tissues and organs, as well as a sharp decline in the regenerative capacity of stem cells. A prevailing view holds that the aging rate of an individual depends on the ratio of tissue attrition to tissue regeneration. Therefore, manipulations that favor the balance towards regeneration may prevent or delay aging. Skeletal muscle is a specialized tissue composed of postmitotic myofibers that contract to generate force. Satellite cells are the adult stem cells responsible for skeletal muscle regeneration. Recent studies on the biology of skeletal muscle and satellite cells in aging have uncovered the critical impact of systemic and niche factors on stem cell functionality and demonstrated the capacity of aged satellite cells to rejuvenate and increase their regenerative potential when exposed to a youthful environment. Here we review the current literature on the coordinated relationship between cell extrinsic and intrinsic factors that regulate the function of satellite cells, and ultimately determine tissue homeostasis and repair during aging, and which encourage the search for new anti-aging strategies.

The surface of mammalian cells is neither smooth nor flat and cells have several times more plasma membrane than the minimum area required to accommodate their shape. We discuss the biological function of this apparent excess membrane that allows the cells to migrate and undergo shape changes and probably plays a role in signal transduction. Methods for studying membrane folding and topography – atomic force microscopy, scanning ion conductance microscopy, fluorescence polarization microscopy and linear dichroism – are described and evaluated. Membrane folding and topography is frequently ignored when interpreting microscopy data. This has resulted in several misconceptions regarding for instance colocalization, membrane organization and molecular clustering. We suggest simple ways to avoid these pitfalls and invoke Occam's razor – that simple explanations are preferable to complex ones. Topography, i.e. deviations from a smooth surface, should always be ruled out as the cause of anomalous data before other explanations are presented.

Ryanodine receptors (RyRs) are the largest known ion channels. They are Ca²⁺ release channels found primarily on the sarcoplasmic reticulum of myocytes. Several hundred mutations in RyRs are associated with skeletal or cardiomyocyte disease in humans. Many of these mutations can now be mapped onto the high resolution structures of individual RyR domains and on full-length tetrameric cryo-electron microscopy structures. A closely related Ca²⁺ release channel, the inositol 1,4,5-trisphospate receptor (IP₃R), shows a conserved structural architecture at the N-terminus, suggesting that both channels evolved from an ancestral unicellular RyR/IP₃R. The functional insights provided by recent structural studies for both channels will aid in the development of rationale treatments for a myriad of Ca²⁺-signaled malignancies.

Lipids have highly diverse functions that go beyond cellular membrane structure and energy storage. One of the great challenges in lipid research will be to understand how the enormous complexity of lipid homeostasis is maintained. Genetic approaches combined with mass spectrometry-based lipidomics will help to elucidate how cells create and maintain their nonrandom lipid distribution within tissues, cells, organelles and lipid bilayers. Lipid homeostasis is crucial for many cellular processes and we are currently only beginning to understand the specific functions of lipids and the local environment that they create.

The cellular prion protein (PrP^C) plays important roles in neurodegenerative diseases. First, it is the well-established substrate for the conformational conversion into its pathogenic isoform (PrP^Sc) giving rise to progressive and fatal prion diseases. Moreover, several recent reports highlight important roles of PrP^C in other neurodegenerative conditions such as Alzheimer's disease. Since PrP^C is subject to proteolytic processing, here we discuss the two main cleavage events under physiological conditions, α-cleavage and shedding. We focus on how these cleavages and the resulting fragments may impact prion diseases as well as other neurodegenerative proteinopathies. Finally, we discuss the recently identified sheddase of PrP^C, namely the metalloprotease ADAM10, with regard to therapeutic potential against neurodegenerative diseases.

The Ca²⁺ ATPases of the plasma membrane (PMCA pumps) export Ca²⁺ from all eukaryotic cells. In mammals they are the products of four separate genes. PMCA types 1 and 4 are distributed ubiquitously; PMCA types 2 and 3 are restricted to some tissues, the most important being the nervous system. Alternative splicing at two sites greatly increases the number of pump isoforms. The two ubiquitous isoforms are no longer considered as only housekeeping pumps as they also perform tissue-specific functions. The PMCAs are classical P-type pumps, their reaction cycle repeating that of all other pumps of the family. Their 3D structure has not been solved, but molecular modeling on SERCA pump templates shows the essential structural pattern of the latter. PMCAs are regulated by calmodulin, which interacts with high affinity with their cytosolic C-terminal tail. A second calmodulin-binding domain with lower affinity is present in some splicing variants of the pump. The PMCAs are essential to the regulation of cellular Ca²⁺, but the all-important Ca²⁺ signal is ambivalent: defects in its control generate various pathologies, the most thoroughly studied being those of genetic origin. Genetic defects of PMCA function produce disease phenotypes: the best characterized is a form of deafness in mice and in humans linked to PMCA2 mutations. A cerebellar X-linked human ataxia has recently been found to be caused by a mutation in the calmodulin-binding domain of PMCA3.

Adult skeletal muscle contains a resident population of stem cells, termed satellite cells, that exist in a quiescent state. In response to an activating signal (such as physical trauma), satellite cells enter the cell cycle and undergo multiple rounds of proliferation, followed by differentiation, fusion, and maturation. Over the last 10–15 years, our understanding of the transcriptional regulation of this stem cell population has greatly expanded, but there remains a dearth of knowledge with regard to the initiating signal leading to these changes in transcription. The recent renewed interest in the metabolic regulation of both cancer and stem cells, combined with previous findings indicating that satellite cells preferentially colocalize with blood vessels, suggests that satellite cell function may be regulated by changes in cellular metabolism. This review aims to describe what is currently known about satellite cell metabolism during changes in cell fate, as well as to describe some of the exciting findings in other cell types and how these might relate to satellite cells.

The muscle-specific basic helix–loop–helix proteins MyoD, Myf5, myogenin (Myog) and MRF4 constitute the myogenic regulatory factor (MRF) family of transcription factors that drive muscle gene expression during myogenesis. Having evolved from a single ancestral gene, the spatial and temporal specificity of expression for each family member has been used to define a hierarchical relationship between the four MRFs. Molecular characterization of two of the MRFs (MyoD and Myog) suggests an important distinction between these factors, whereby MyoD establishes an open chromatin structure at muscle-specific genes, whereas Myog drives high levels of transcription of genes within this open chromatin state. Furthermore, recent data have provided an additional distinction between MRF function with respect to cell cycle regulation. Indeed, MyoD has been shown to directly activate genes involved in cell cycle progression, leading to myoblast proliferation. In contrast, Myog has antiproliferative activity through the activation of genes that shut down the cell proliferation machinery, leading to cell cycle exit and myoblast differentiation. Although the transcriptional activities of MyoD and Myog synergize to drive muscle differentiation, it is the expression of Myog that sets in motion a gene expression program that constitutes a ‘point of no return’, leading to cell cycle exit. In this review, we compare and contrast the current literature with respect to MRF function, with a particular emphasis on the differential role of MRFs in modulating the cell cycle.

The chitinolytic machinery of Serratia marcescens is one of the best known enzyme systems for the conversion of insoluble polysaccharides. This machinery includes four chitin-active enzymes: ChiC, an endo-acting non-processive chitinase; ChiA and ChiB, two processive chitinases moving along chitin chains in opposite directions; and CBP21, a surface-active CBM33-type lytic polysaccharide monooxygenase that introduces chain breaks by oxidative cleavage. Furthermore, an N-acetylhexosaminidase or chitobiase converts the oligomeric products from the other enzymes to monomeric N-acetylglucosamine. Here we discuss the catalytic mechanisms of these enzymes as well as the structural basis of each enzyme's specific role in the chitin degradation process. We also discuss how knowledge of this enzyme system may be extrapolated to other enzyme systems for conversion of insoluble polysaccharides, in particular conversion of cellulose by cellulases and GH61-type lytic polysaccharide monooxygenases.

Muscular dystrophies are genetic disorders characterized by skeletal muscle wasting and weakness. Although there is no effective therapy, a number of experimental strategies have been developed over recent years and some of them are undergoing clinical investigation. In this review, we highlight recent developments and key challenges for strategies based upon gene replacement and gene/expression repair, including exon-skipping, vector-mediated gene therapy and cell therapy. Therapeutic strategies for different forms of muscular dystrophy are discussed, with an emphasis on Duchenne muscular dystrophy, given the severity and the relatively advanced status of clinical studies for this disease.

The phosphoinositide 3-kinase (PI3K) signaling pathway is crucial for cell growth, proliferation, metabolism, and survival, and is frequently deregulated in human cancer, including ~ 70% of breast tumors. PIK3CA, the gene encoding the catalytic subunit p110α of PI3K, is mutated in ~ 30% of breast cancers. However, the exact mechanism of PIK3CA-evoked breast tumorigenesis has not yet been defined. Genetically engineered mouse models are valuable for examining the initiation, development and progression of cancer. Transgenic mice harboring hotspot mutations in p110α have helped to elucidate breast cancer pathogenesis and increase our knowledge about molecular and cellular alterations in vivo. They are also useful for the development of therapeutic strategies. Here, we describe current mouse models of mutant PIK3CA in the mammary gland, and discuss differences in tumor latency and pathogenesis.

Mammalian skeletal muscle is notable for both its highly ordered biophysical structure and its regenerative capacity following trauma. Critical to both of these features is the specialized muscle extracellular matrix, comprising both the multiple concentric sheaths of connective tissue surrounding structural units from single myofibers to whole muscles and the dense interstitial matrix that occupies the space between them. Extracellular matrix-dependent interactions affect all activities of the resident muscle stem cell population (the satellite cells), from maintenance of quiescence and stem cell potential to the regulation of proliferation and differentiation. This review focuses on the role of the extracellular matrix in muscle regeneration, with a particular emphasis on regulation of satellite-cell activity.

Adult skeletal muscle has the remarkable property of regenerating after damage, owing to satellite cells and myogenic precursor cells becoming committed to adult myogenesis to rebuild the muscle. This process is accompanied by the continuing presence of macrophages, from the phagocytosis of damaged myofibres to the full re-formation of new myofibres. In recent years, there has been huge progress in our understanding of the roles of macrophages during skeletal muscle regeneration, notably concerning their effects on myogenic precursor cells. Here, we review the most recent knowledge acquired on monocyte entry into damaged muscle, the various macrophage subpopulations, and their respective roles during the sequential phases of muscle repair. We also discuss the role of macrophages after exercise-induced muscle damage, notably in humans.

Ketosteroid isomerase (Δ⁵–3–keto steroid isomerase or steroid Δ–isomerase) is a highly efficient enzyme at the centre of current debates on enzyme catalysis. We have modelled the reaction mechanism of the isomerization of 3–oxo-Δ⁵–steroids into their Δ⁴-conjugated isomers using high-level combined quantum mechanics/molecular mechanics (QM/MM) methods, and semi-empirical QM/MM molecular dynamics simulations. Energy profiles were obtained at various levels of QM theory (AM1, B3LYP and SCS–MP2). The high-level QM/MM profile is consistent with experimental data. QM/MM dynamics simulations indicate that active site closure and desolvation of the catalytic Asp38 occur before or during formation of dienolate intermediates. These changes have a significant effect on the reaction barrier. A low barrier to reaction is found only when the active site is closed, poising it for catalysis. This conformational change is thus integral to the whole process. The effects on the barrier are apparently largely due to changes in solvation. The combination of high-level QM/MM energy profiles and QM/MM dynamics simulation shows that the reaction involves active site closure, desolvation of the catalytic base, efficient isomerization and re-opening of the active site. These changes highlight the transition between the ligand binding/releasing form and the catalytic form of the enzyme. The results demonstrate that electrostatic interactions (as a consequence of pre-organization of the active site) are crucial for stabilization during the chemical reaction step, but closure of the active site is essential for efficient catalysis to occur.

Endocytosis describes the processes by which proteins, peptides and solutes, and also pathogens, enter the cell. Endocytosed material progresses to endosomes. Genetic studies in yeast, worms, flies and mammals have identified a set of universally conserved proteins that are essential for early-to-late endosome transition and lysosome biogenesis, and for endolysosomal trafficking pathways, including autophagy. The two Vps-C complexes CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) perform diverse biochemical functions in endocytosis: they tether membranes, interact with Rab GTPases, activate and proof-read SNARE assembly to drive membrane fusion, and possibly attach endosomes to the cytoskeleton. In addition, several of the CORVET and HOPS subunits have diversified in metazoans, and probably form additional specialized complexes to accomodate the higher complexity of trafficking pathways in these cells. Recent studies offer new insights into the complex relationships between CORVET and HOPS complexes and other factors of the endolysosomal pathway. Interactions with V-ATPase, the ESCRT machinery, phosphoinositides, the cytoskeleton and the Rab switch suggest an intricate cooperative network for endosome maturation. Accumulating evidence supports the view that endosomal tethering complexes implement a regulatory logic that governs endomembrane identity and dynamics.

The muscular dystrophies comprise more than 30 clinical disorders that are characterized by progressive skeletal muscle wasting and degeneration. Although the genetic basis for many of these disorders has been identified, the exact mechanism for pathogenesis generally remains unknown. It is considered that disturbed levels of reactive oxygen species (ROS) contribute to the pathology of many muscular dystrophies. Reactive oxygen species and oxidative stress may cause cellular damage by directly and irreversibly damaging macromolecules such as proteins, membrane lipids and DNA; another major cellular consequence of reactive oxygen species is the reversible modification of protein thiol side chains that may affect many aspects of molecular function. Irreversible oxidative damage of protein and lipids has been widely studied in Duchenne muscular dystrophy, and we have recently identified increased protein thiol oxidation in dystrophic muscles of the mdx mouse model for Duchenne muscular dystrophy. This review evaluates the role of elevated oxidative stress in Duchenne muscular dystrophy and other forms of muscular dystrophies, and presents new data that show significantly increased protein thiol oxidation and high levels of lipofuscin (a measure of cumulative oxidative damage) in dysferlin-deficient muscles of A/J mice at various ages. The significance of this elevated oxidative stress and high levels of reversible thiol oxidation, but minimal myofibre necrosis, is discussed in the context of the disease mechanism for dysferlinopathies, and compared with the situation for dystrophin-deficient mdx mice.

Two libraries of simultaneous double mutations in the active site region of an esterase from Bacillus stearothermophilus were constructed to improve the enantioselectivity in the hydrolysis of tetrahydrofuran-3-yl acetate. As screening of large mutant libraries is hampered by the necessity for GC/MS analysis, mutant libraries were designed according to a ‘small but smart’ concept. The design of focused libraries was based on data derived from a structural alignment of 3317 amino acid sequences of α/β-hydrolase fold enzymes with the bioinformatic tool 3dm. In this way, the number of mutants to be screened was substantially reduced as compared with a standard site-saturation mutagenesis approach. Whereas the wild-type esterase showed only poor enantioselectivity (E = 4.3) in the hydrolysis of (S)-tetrahydrofuran-3-yl acetate, the best variants obtained with this approach showed increased E-values of up to 10.4. Furthermore, some variants with inverted enantiopreference were found.

The dysregulation of PI3K/AKT/mTORC1 signalling and/or hyperactivation of MYC are observed in a high proportion of human cancers, and together they form a ‘super signalling’ network mediating malignancy. A fundamental downstream action of this signalling network is up-regulation of ribosome biogenesis and subsequent alterations in the patterns of translation and increased protein synthesis, which are thought to be critical for AKT/MYC-driven oncogenesis. We have demonstrated that AKT and MYC cooperate to drive ribosomal DNA (rDNA) transcription and ribosome biogenesis, with AKT being essential for rDNA transcription and in vitro survival of lymphoma cells isolated from a MYC-driven model of B-cell lymphoma (Eμ-Myc) [Chan JC et al., (2011) Science Signalling 4, ra56]. Here we show that the allosteric AKT inhibitor MK-2206 rapidly and potently antagonizes rDNA transcription in Eμ-Myc B-cell lymphomas in vivo, and this is associated with a rapid reduction in indicators of disease burden, including spleen weight and the abundance of tumour cells in both the circulation and lymph nodes. Extended treatment of tumour-bearing mice with MK-2206 resulted in a significant delay in disease progression, associated with increased B-cell lymphoma apoptosis. Our findings suggest that malignant diseases characterized by unrestrained ribosome biogenesis may be vulnerable to therapeutic strategies that target the PI3K/AKT/mTORC1/MYC growth control network.

Recombinant Ca²⁺–ATPase was expressed in Saccharomyces cerevisiae with a biotin-acceptor domain linked to its C–terminus by a thrombin cleavage site. We obtained 200 μg of ~ 70% pure recombinant sarcoendoplasmic reticulum Ca²⁺–ATPase isoform 1a (SERCA1a) from a 6–L yeast culture. The catalytic cycle of SERCA1a was followed in real time using rapid scan FTIR spectroscopy. Different intermediate states (Ca₂E1P and Ca₂E2P) of the recombinant protein were accumulated using different buffer compositions. The difference spectra of their formation from Ca₂E1 had the same spectral features as those from the native rabbit SERCA1a. The enzyme-specific activity for the active enzyme fraction in both samples was also similar. The results show that the recombinant protein obtained from the yeast-based expression system has similar structural and dynamic properties as native rabbit SERCA1a. It is now possible to apply this expression system together with IR spectroscopy to the investigation of the role of individual amino acids.

Endocytosis and subsequent membrane traffic through endosomes are cellular processes that are integral to eukaryotic evolution, and numerous human diseases are associated with their dysfunction. Consequently, it is important to untangle the molecular machineries that regulate membrane dynamics and protein flow in the endocytic pathway. Central in this context is class III phosphatidylinositol 3–kinase, an evolutionarily conserved enzyme complex that phosphorylates phosphatidylinositol into phosphatidylinositol 3–phosphate. Phosphatidylinositol 3–phosphate recruits specific effector proteins, most of which contain FYVE or PX domains, to promote endocytosis, endosome fusion, endosome motility and endosome maturation, as well as cargo sorting to lysosomes, the biosynthetic pathway or the plasma membrane. Here we review the functions of key phosphatidylinositol 3–phosphate effectors in regulation of endocytic membrane dynamics and protein sorting.

Muscular dystrophies are heritable and heterogeneous neuromuscular disorders characterized by the primary wasting of skeletal muscle, usually caused by mutations in the proteins forming the link between the cytoskeleton and the basal lamina. As a result of mutations in the dystrophin gene, Duchenne muscular dystrophy patients suffer from progressive muscle atrophy and an exhaustion of muscular regenerative capacity. No efficient therapies are available. The evidence that adult stem cells were capable of participating in the regeneration of more than their resident organ led to the development of potential stem cell treatments for degenerative disorder. In the present review, we describe the different types of myogenic stem cells and their possible use for the progression of cell therapy in Duchenne muscular dystrophy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with motor neuron degeneration, muscle atrophy and paralysis. Although numerous pathological mechanisms have been elucidated, ALS remains an invariably fatal disease in the absence of any effective therapy. The heterogeneity of the disease and the failure to develop satisfactory therapeutic protocols reinforce the view that ALS is a multi-factorial and multi-systemic disease. Thus, a better understanding of the pathogenic mechanisms and study of the potential pathological relationship between the various cellular processes is required to ensure efficacious therapy. The pathogenic mechanisms associated with ALS are reviewed, and the strengths and limitations of some new therapeutic approaches are discussed.

Cellular calcium signaling plays important roles in several signal transduction pathways that control proliferation, differentiation and apoptosis. In epithelial cells calcium signaling is initiated mainly by calcium release from endoplasmic-reticulum-associated intracellular calcium pools. Because calcium is accumulated in the endoplasmic reticulum by sarco/endoplasmic reticulum calcium ATPases (SERCA), these enzymes play a critical role in the control of calcium-dependent cell activation, growth and survival. We investigated the modulation of SERCA expression and function in human lung adenocarcinoma cells. In addition to the ubiquitous SERCA2 enzyme, the SERCA3 isoform was also expressed at variable levels. SERCA3 expression was selectively enhanced during cell differentiation in lung cancer cells, and marked SERCA3 expression was found in fully differentiated normal bronchial epithelium. As studied by using a recombinant fluorescent calcium probe, induction of the expression of SERCA3, a lower calcium affinity pump, was associated with decreased intracellular calcium storage, whereas the amplitude of capacitative calcium influx remained unchanged. Our observations indicate that the calcium homeostasis of the endoplasmic reticulum in lung adenocarcinoma cells presents a functional defect due to decreased SERCA3 expression that is corrected during pharmacologically induced differentiation. The data presented in this work show, for the first time, that endoplasmic reticulum calcium storage is anomalous in lung cancer cells, and suggest that SERCA3 may serve as a useful new phenotypic marker for the study of lung epithelial differentiation.

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Small leucine-rich proteoglycans (SLRPs) are involved in a variety of biological and pathological processes. This review focuses on their regulatory roles in matrix assembly. SLRPs have protein cores and hypervariable glycosylation with multivalent binding abilities. During development, differential interactions of SLRPs with other molecules result in tissue-specific spatial and temporal distributions. The changing expression patterns play a critical role in the regulation of tissue-specific matrix assembly and therefore tissue function. SLRPs play significant structural roles within extracellular matrices. In addition, they play regulatory roles in collagen fibril growth, fibril organization and extracellular matrix assembly. Moreover, they are involved in mediating cell–matrix interactions. Abnormal SLRP expression and/or structures result in dysfunctional extracellular matrices and pathophysiology. Altered expression of SLRPs has been found in many disease models, and structural deficiency also causes altered matrix assembly. SLRPs regulate assembly of the extracellular matrix, which defines the microenvironment, modulating both the extracellular matrix and cellular functions, with an impact on tissue function.

The stromal-specific proteoglycan decorin has emerged in recent years as a critical regulator of tumor initiation and progression. Decorin regulates the biology of various types of cancer by modulating the activity of several receptor tyrosine kinases coordinating growth, survival, migration, and angiogenesis. Decorin binds to surface receptors for epidermal growth factor and hepatocyte growth factor with high affinity, and negatively regulates their activity and signaling via robust internalization and eventual degradation. The insulin-like growth factor (IGF)-I system plays a critical role in the regulation of cell growth both in vivo and in vitro. The IGF-I receptor (IGF-IR) is also essential for cellular transformation, owing to its ability to enhance cell proliferation and protect cancer cells from apoptosis. Recent data have pointed to a role of decorin in regulating the IGF-I system in both nontransformed and transformed cells. Significantly, there is a surprising dichotomy in the mechanism of decorin action on IGF-IR signaling, which differs considerably between physiological and pathological cellular models. In this review, we summarize the current knowledge on decorin regulation of the IGF-I system in normal and transformed cells, and discuss possible decorin-based therapeutic approaches to target IGF-IR-driven tumors.

Decorin, a secreted small leucine-rich proteoglycan, acts as a tumor repressor in a variety of cancers, mainly by blocking the action of several receptor tyrosine kinases such as the receptors for hepatocyte, epidermal and insulin-like growth factors. In the present study we investigated the effects of decorin in an experimental model of thioacetamide-induced hepatocarcinogenesis and its potential role in modulating the signaling of platelet-derived growth factor receptor-α (PDGFRα). Genetic ablation of decorin in mice led to enhanced tumor prevalence and a higher tumor count compared with wild-type mice. These findings correlated with decreased levels of the cyclin-dependent kinase inhibitor p21^Waf1/Cip1 and concurrent activation (phosphorylation) of PDGFRα in the hepatocellular carcinomas generated in the decorin-null vis-à-vis wild-type mice. Notably, in normal liver PDGFRα localized primarily to the membrane of nonparenchymal cells, whereas in the malignant counterpart PDGFRα was expressed by the malignant cells at their cell surfaces. This process was facilitated by a genetic background lacking endogenous decorin. Double immunostaining of the proteoglycan and the receptor revealed only minor colocalization, leading to the hypothesis that decorin would bind to the natural ligand PDGF rather than to the receptor itself. Indeed, we found, using purified proteins and immune-blot assays, that decorin binds to PDGF. Collectively, our findings support the idea that decorin acts as a secreted tumor repressor during hepatocarcinogenesis by hindering the action of another receptor tyrosine kinase, such as the PDGFRα, and could be a novel therapeutic agent in the battle against liver cancer.

An emerging body of evidence indicates that secreted proteoglycans act as signaling molecules, in addition to their canonical function in maintaining and regulating the architecture of various extracellular matrices. Proteoglycans interact with a number of receptors that regulate growth, motility and immune response. In part, as a consequence of their complex structure, proteoglycans can induce crosstalk among various families of receptors and can also interact with natural receptor ligands, often blocking and sequestering their bioactivity. In their soluble form, originating from either partial proteolytic processing or through de novo synthesis by activated cells, some proteoglycans can become potent danger signals, denoting tissue stress and injury. Recently, it has been shown that proteoglycans, especially those belonging to the small leucine-rich and hyaluronan-binding gene families as well as the glycosaminoglycan hyaluronan, act as endogenous ligands of the toll-like receptors, a group of central receptors regulating innate immunity. Furthermore, proteoglycans can activate intracellular inflammasomes and trigger sterile inflammation. In this review, we critically assess the signaling events induced by the proteoglycans biglycan, decorin, lumican and versican as well as hyaluronan during inflammation. We discuss the intriguing emerging notion that, in spite of structural diversity of biglycan, decorin, versican and hyaluronan, all of them signal through the same toll-like receptors, albeit triggering differential responses and biological outcomes. Finally, we review the modes of action of these endogenous ligands of toll-like receptors and their ability to specifically modify the final signaling events and the inflammatory response.

Heparan sulfate proteoglycans (HSPGs) have essential functions during embryonic development and throughout postnatal life. To exert these functions, they undergo a series of processing reactions by heparan-sulfate-modifying enzymes (HSMEs), which endows them with highly modified heparan sulfate (HS) domains that provide specific docking sites for a large number of bioactive molecules. The development and antigen-dependent differentiation of normal B lymphocytes, as well as the growth and progression of B-lineage malignancies, are orchestrated by an array of growth factors, cytokines and chemokines many of which display HS binding. As discussed in this review, tightly regulated HSPG expression is a requirement for normal B cell maturation, differentiation and function. In addition, the HSPG syndecan-1 functions as a versatile co-receptor for signals from the bone marrow microenvironment, essential for the survival of long-lived plasma cells and multiple myeloma (MM) plasma cells. Targeting of HSMEs or HS chains on MM cells increases their sensitivity to drugs currently used in MM treatment, including bortezomib, lenalidomide or dexamethasone. Taken together, these findings render the HS biosynthetic machinery a promising target for MM treatment.

Vascular endothelial growth factor (VEGF)-stimulated angiogenesis depends on a cross-talk mechanism involving VEGF receptor 2 (VEGFR2), vascular endothelial (VE)-cadherin and the αVβ3 integrin. Because we have shown that αVβ3 integrin activation is dependent on its incorporation, along with the insulin-like growth factor-1 receptor (IGF1R) kinase, into a ternary receptor complex organized by the matrix receptor syndecan-1 (Sdc1), we questioned the role of this core complex in VEGF-stimulated angiogenesis. We find that the Sdc1-coupled ternary receptor complex is required for VEGF signalling and for stimulation of vascular endothelial cell migration by vascular endothelial cadherin (VE-cadherin) engagement. VE-cadherin binding to Fc/VE-cadherin extracellular domain chimera activates Sdc1-coupled IGF1R and αvβ3 integrin; this depends on VEGFR2 and c-Src activated by the cadherin. Blocking homotypic VE-cadherin engagement disrupts VEGF-stimulated cell migration, which is restored by clustering the cadherin in the absence of cell–cell adhesion. This cadherin-dependent stimulation requires VEGFR2 and IGF1R and is blocked by synstatin (SSTN)_92–119, a peptide that competitively disrupts the Sdc1-coupled ternary complex and prevents the αVβ3 integrin activation required for VEGFR2 activation. VEGFR2-stimulated angiogenesis in the mouse aortic ring explant assay is disrupted by SSTN, although only early in the process, suggesting that IGF1R coupling to Sdc1 and αVβ3 integrin comprises a core activation mechanism activated by VE-cadherin that is necessary for VEGFR2 and integrin activation in the initial stages of endothelial cell dissemination during angiogenesis.

The syndecans are a family of heparan sulfate-decorated cell-surface proteoglycans: matrix receptors with roles in cell adhesion and growth factor signaling. Their heparan sulfate chains recognize ‘heparin-binding’ motifs that are ubiquitously present in the extracellular matrix, providing the means for syndecans to constitutively bind and cluster to sites of cell–matrix adhesion. Emerging evidence suggests that specialized docking sites in the syndecan extracellular domains may serve to localize other receptors to these sites as well, including integrins and growth factor receptor tyrosine kinases. A prototype of this mechanism is capture of the αvβ3 integrin and insulin-like growth factor 1 receptor (IGF1R) by syndecan-1 (Sdc1), forming a ternary receptor complex in which signaling downstream of IGF1R activates the integrin. This Sdc1-coupled ternary receptor complex is especially prevalent on tumor cells and activated endothelial cells undergoing angiogenesis, reflecting the up-regulated expression of αvβ3 integrin in such cells. As such, much effort has focused on developing therapeutic agents that target this integrin in various cancers. Along these lines, the site in the Sdc1 ectodomain that is responsible for capture and activation of the αvβ3 or αvβ5 integrins by IGF1R can be mimicked by a short peptide called ‘synstatin’, which competitively displaces the integrin and IGF1R kinase from the syndecan and inactivates the complex. This review summarizes our current knowledge of the Sdc1-coupled ternary receptor complex and the efficacy of synstatin as an emerging therapeutic agent to target this signaling mechanism.

Syndecan-1 is a cell surface heparan sulfate proteoglycan with various biological functions relevant to tumor progression and inflammation, including cell–cell adhesion, cell–matrix interaction, and cytokine signaling driving cell proliferation and motility. Syndecan-1 is a prognostic factor in breast cancer, and has a predicitive value for neodadjuvant chemotherapy. It is still poorly understood how syndecan-1 integrates matrix-dependent and cytokine-dependent signaling processes in the tumor microenvironment. Here, we evaluated the potential role of syndecan-1 in modulating matrix-dependent breast cancer cell migration in the presence of interleukin-6, and its potential involvement in resistance to irradiation in vitro. MDA-MB-231 breast cancer cells were transiently transfected with syndecan-1 small interfering RNA or control reagents, and this was followed by stimulation with interleukin-6 or irradiation. Cellular responses were monitored by adhesion, migration and colony formation assays, as well as analysis of cell signaling. Syndecan-1 depletion increased cell adhesion to fibronectin. Increased migration on fibronectin was significantly suppressed by interleukin-6, and GRGDSP peptides inhibited both adhesion and migration. Interleukin-6-induced activation of focal adhesion kinase and reduction of constitutive nuclear factor kappaB signaling were decreased in syndecan-1-deficient cells. Focal adhesion kinase hyperactivation in syndecan-1-depleted cells was associated with dramatically reduced radiation sensitivity. We conclude that loss of syndecan-1 leads to enhanced activation of β₁-integrins and focal adhesion kinase, thus increasing breast cancer cell adhesion, migration, and resistance to irradiation. Syndecan-1 deficiency also attenuates the modulatory effect of the inflammatory microenvironment constituent interleukin-6 on cancer cell migration.

Sustained pressure overload induces heart failure, the main cause of mortality in the Western world. Increased understanding of the underlying molecular mechanisms is essential to improve heart failure treatment. Despite important functions in other tissues, cardiac proteoglycans have received little attention. Syndecan-4, a transmembrane heparan sulfate proteoglycan, is essential for pathological remodeling, and we here investigated its expression and shedding during heart failure. Pressure overload induced by aortic banding for 24 h and 1 week in mice increased syndecan-4 mRNA, which correlated with mRNA of inflammatory cytokines. In cardiac myocytes and fibroblasts, tumor necrosis factor-α, interleukin-1β and lipopolysaccharide through the toll-like receptor-4, induced syndecan-4 mRNA. Bioinformatical and mutational analyses in HEK293 cells identified a functional site for the proinflammatory nuclear factor-κB transcription factor in the syndecan-4 promoter, and nuclear factor-κB regulated syndecan-4 mRNA in cardiac cells. Interestingly, tumor necrosis factor-α, interleukin-1β and lipopolysaccharide induced nuclear factor-κB-dependent shedding of the syndecan-4 ectodomain from cardiac cells. Overexpression of syndecan-4 with mutated enzyme-interacting domains suggested enzyme-dependent heparan sulfate chains to regulate shedding. In cardiac fibroblasts, lipopolysaccharide reduced focal adhesion assembly, shown by immunohistochemistry, suggesting that inflammation-induced shedding affects function. After aortic banding, a time-dependent cardiac recruitment of T lymphocytes was observed by measuring CD3, CD4 and CD8 mRNA, which was reduced in syndecan-4 knockout hearts. Finally, syndecan-4 mRNA and shedding were upregulated in failing human hearts. Conclusively, our data suggest that syndecan-4 plays an important role in the immune response of the heart to increased pressure, influencing cardiac remodeling and failure progression.

Estradiol (E2)–estrogen receptor (ER) actions are implicated in initiation, growth and progression of hormone-dependent breast cancer. Crosstalk between ERs, epidermal growth factor receptor (EGFR) and/or insulin-like growth factor receptor (IGFR) is critical for the observed resistance to endocrine therapies. Cell surface heparan sulfate proteoglycans (HSPGs) are principal mediators of cancer cell properties and the E2–ER pathway as well as those activated by EGFR and IGFR have significant roles in regulating the expression of certain cell surface HSPGs, such as syndecan-2 (SDC-2), syndecan-4 (SDC-4) and glypican-1. In this study, we therefore evaluated the role of EGFR-IGFR signaling on the constitutive expression and E2-mediated expression of ERs and HSPGs as well as the effect of E2–ERs and IGFR/EGFR-mediated cell migration in ERα+ (MCF-7) and ERβ+ (MDA-MB-231) breast cancer cells using specific intracellular inhibitors of EGFR and IGFR. We report that the expression of ERα is mainly enhanced by IGFR, whereas ERβ expression is mainly coordinated by EGFR. Moreover, constitutive SDC-2 expression in ERα+ and ERβ+ cells is mainly mediated through the IGFR, whereas in ERα+ E2-treated cells EGFR is the active one. In contrast, SDC-4 expression is regulated by IGFR in the presence and absence of E2. E2 also seems to diminish the inhibitory effect of EGFR and IGFR inhibitors in breast cancer cell migration. These data suggest that the coordinated action of ERs with EGFR and/or IGFR is of crucial importance, providing potential targets for designing and developing novel multi-potent agents for endocrine therapies.

The evolution of the fibroblast growth factor (FGF)–FGF receptor (FGFR) signalling system has closely followed that of multicellular organisms. The abilities of nine FGFs (FGF-1 to FGF-9; examples of FGF subfamilies 1, 4, 7, 8, and 9) and seven FGFRs or isoforms (FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, FGFR3c, and FGFR4) to support signalling in the presence of heparin, a proxy for the cellular heparan sulfate coreceptor, were assembled into a network. A connection between two FGFRs was defined as their mutual ability to signal with a particular FGF. The network contained a core of four receptors (FGFR1c, FGFR2c, FGFR3c, and FGFR4) with complete connectivity and high redundancy. Analysis of the wider network indicated that neither FGF-3 nor FGF-7 was well connected to this core of four receptors, and that divergence of a precursor of FGF subgroups 1, 4 and 9 from FGF subgroup 8 may have allowed expansion from a three-member FGFR core signalling system to the four-member core network. This increases by four-fold the number of possible signalling combinations. Synchrotron radiation CD spectra of the FGFs with heparin revealed no overall common structural change, suggesting the existence of distinct heparin-binding sites throughout the FGFs. The approach provides a potential method of identifying agents capable of influencing particular FGF–FGFR combinations, or areas of the signalling network, for experimental or therapeutic purposes.

Keratan sulfate (KS) is an important glycosaminoglycan that is found in cartilage, reproductive tissues, and neural tissues. Corneal KS glycosaminoglycan is found N-linked to lumican, keratocan and mimecan proteoglycans, and has been widely studied by investigators interested in corneal development and diseases. Recently, the availability of corneal KS has become severely limited, owing to restrictions on the shipment of bovine central nervous system byproducts across international borders in an effort to prevent additional cases of mad cow disease. We report a simple method for the purification of multi-milligram quantities of bovine corneal KS, and characterize its structural properties. We also examined its protein-binding properties, and discovered that corneal KS bound with high affinity to fibroblast growth factor-2 and sonic hedgehog, a growth factor and a morphogen involved in corneal development and healing.

Heparanase is an endoglucuronidase that cleaves heparan sulfate chains of proteoglycans. In many malignancies, high heparanase expression and activity correlate with an aggressive tumour phenotype. A major consequence of heparanase action in cancer is a robust up-regulation of growth factor expression and increased shedding of syndecan-1 (a transmembrane heparan sulfate proteoglycan). Substantial evidence indicates that heparanase and syndecan-1 work together to drive growth factor signalling and regulate cell behaviours that enhance tumour growth, dissemination, angiogenesis and osteolysis. Preclinical and clinical studies have demonstrated that therapies targeting the heparanase/syndecan-1 axis hold promise for blocking the aggressive behaviour of cancer.

Recent years have seen a growing body of evidence that enzymatic remodeling of heparan sulfate proteoglycans profoundly affects a variety of physiological and pathological processes, including inflammation, neovascularization, and tumor development. Heparanase is the sole mammalian endoglycosidase that cleaves heparan sulfate. Extensively studied in cancer progression and aggressiveness, heparanase was recently implicated in several inflammatory disorders as well. Although the precise mode of heparanase action in inflammatory reactions is still not completely understood, the fact that heparanase activity is mechanistically important both in malignancy and in inflammation argues that this enzyme is a candidate molecule linking inflammation and tumorigenesis in inflammation-associated cancers. Elucidation of the specific effects of heparanase in cancer development, particularly when inflammation is a causal factor, will accelerate the development of novel therapeutic/chemopreventive interventions and help to better define target patient populations in which heparanase-targeting therapies could be particularly beneficial.

Syndecans are transmembrane heparan sulfate proteoglycans with roles in cell proliferation, differentiation, adhesion, and migration. They have been associated with multiple functions in tumour progression, through their ability to interact with a wide range of ligands as well as other receptors, which makes them key effectors in the pericellular microenvironment. Extracellular shedding of syndecans by tumour-associated matrix metalloproteinases (MMPs) may have an important role in tumour progression. Such ectodomain shedding generates soluble ectodomains that may function as paracrine or autocrine effectors, or as competitive inhibitors of the intact proteoglycan. Tumour-associated MMPs are shown here to cleave the ectodomains of human syndecan-1 and syndecan-4. Two membrane proximal regions of both syndecan-1 and syndecan-4 are favoured MMP cleavage sites, six and 15 residues from the transmembrane domain. Other sites are 35–40 residues C-terminal from the heparan sulfate chain substitution sites in both syndecans. The MT1-MMP cleavage sites in syndecan-1 and syndecan-4 were confirmed by site-directed mutagenesis. These findings provide insights into the characteristics of syndecan shedding.

Similar to most proteinases, matrix metalloproteinases (MMP) do not recognize a consensus cleavage site. Thus, it is not surprising that, in a defined in vitro reaction, most MMPs can act on a wide range of proteins, including many extracellular matrix proteins. However, the findings obtained from in vivo studies with genetic models have demonstrated that individual MMPs act on just a few extracellular protein substrates, typically not matrix proteins. The limited, precise functions of an MMP imply that mechanisms have evolved to control the specificity of proteinase:substrate interactions. We discuss the possibility that interactions with the glycosaminoglycan chains of proteoglycans may function as allosteric regulators or accessory factors directing MMP catalysis to specific substrates. We propose that understanding how the activity of specific MMPs is confined to discreet compartments and targeted to defined substrates via interactions with other macromolecules may provide a means of blocking potentially deleterious MMP-mediated processes at the same time as sparing any beneficial functions.

Serglycin (SG) is mainly expressed by hematopoetic cells as an intracellular proteoglycan. Multiple myeloma cells constitutively secrete SG, which is also localized on the cell surface in some cell lines. In this study, SG isolated from myeloma cells was found to interact with collagen type I (Col I), which is a major bone matrix component. Notably, myeloma cells positive for cell-surface SG (csSG) adhered significantly to Col I, compared to cells lacking csSG. Removal of csSG by treatment of the cells with chondroitinase ABC or blocking of csSG by an SG-specific polyclonal antibody significantly reduced the adhesion of myeloma cells to Col I. Significant up-regulation of expression of the matrix metalloproteinases MMP–2 and MMP–9 at both the mRNA and protein levels was observed when culturing csSG-positive myeloma cells on Col I-coated dishes or in the presence of soluble Col I. MMP–9 and MMP–2 were also expressed in increased amounts by myeloma cells in the bone marrow of patients with multiple myeloma. Our data indicate that csSG of myeloma cells affects key functional properties, such as adhesion to Col I and the expression of MMPs, and imply that csSG may serve as a potential prognostic factor and/or target for pharmacological interventions in multiple myeloma.

Pathological neovascularization relies on an imbalance between potent proangiogenic agents and equally effective antiangiogenic cues. Collectively, these factors contribute to an angiogenic niche within the tumor microenvironment. Oncogenic events and hypoxia contribute to augmented levels of angiokines, and thereby activate the so-called angiogenic switch to promote aggressive tumorigenic and metastatic growth. Soluble decorin functions as a paracrine pan-inhibitor of receptor tyrosine kinases, such as Met and epidermal growth factor receptor, and thus is capable of suppressing angiogenesis under normoxia. This leads to noncanonical repression of hypoxia-inducible factor 1-alpha and vascular endothelial growth factor A (VEGFA), and concurrent induction of thrombospondin-1. The substantial induction of endogenous tumor cell-derived thrombospondin-1, a potent antiangiogenic effector, led us to the discovery of an unexpected secretory phenotype occurring very rapidly (within 5 min) after decorin treatment of the triple-negative basal breast carcinoma cell line MDA-MB-231. Surprisingly, the effect was not mediated by Met receptor antagonism, as initially hypothesized, but required epidermal growth factor receptor signaling to achieve swift and robust thrombospondin-1 release. Furthermore, this effect was ultimately dependent on the prompt degradation of Ras homolog gene family member A, via the 26S proteasome, leading to direct inactivation of Rho-associated coiled-coil containing protein kinase 1. The latter led to derepression of thrombospondin-1 secretion. Collectively, these data provide a novel mechanistic role for Rho-associated coiled-coil containing protein kinase 1, in addition to providing the first conclusive evidence of decorin exclusively targeting a receptor tyrosine kinase to achieve a specific effect. The overall effects of soluble decorin on the tumor microenvironment would cause an immediately-early as well as a sustained antiangiogenic response in vivo.

Lumican is a member of the small leucine-rich proteoglycan family. It is present in numerous extracellular matrices of different tissues, such as muscle, cartilage, and cornea. In skin, lumican is present as a glycoprotein. It plays a critical role in collagen fibrillogenesis, as shown by knocking out of its gene in mice. A direct link between lumican expression and melanoma progression and metastasis has been demonstrated. Lumican was shown to impede tumour cell migration and invasion by directly interacting with the α₂β₁ integrin. In addition, an active sequence of the lumican core protein, called lumcorin, was identified as being responsible for inhibition of melanoma cell migration. Lumican was also shown to exert angiostatic properties by downregulating the proteolytic activity associated with endothelial cell membranes, particularly matrix metalloproteinase (MMP)-14 and MMP-9. Globally, lumican appears to be a potent agent for inhibiting tumour progression rather than tumorigenesis. However, progressive changes in proteoglycans occur in the tumour environment. The complexity and diversity of proteoglycan structure might be responsible for a variety of functions that regulate cell behaviour. Through their core protein and their glycosaminoglycan chains, proteoglycans can interact with growth factors and chemokines. These interactions affect cell signalling, motility, adhesion, growth, and apoptosis. This review summarizes recent data concerning lumican control of tumour progression in different cancers, with a particular focus on its interactions with MMPs and integrins. Its potential therapeutic implications are discussed.

During progression to heart failure (HF), myocardial extracellular matrix (ECM) alterations and tissue inflammation are central. Lumican is an ECM-localized proteoglycan associated with inflammatory conditions and known to bind collagens. We hypothesized that lumican plays a role in the dynamic alterations in cardiac ECM during development of HF. Thus, we examined left ventricular cardiac lumican in a mouse model of pressure overload and in HF patients, and investigated expression, regulation and effects of increased lumican in cardiac fibroblasts. After 4 weeks of aortic banding, mice were divided into groups of hypertrophy (AB) and HF (ABHF) based on lung weight and left atrial diameter. Sham-operated mice were used as controls. Accordingly, cardiac lumican mRNA and protein levels were increased in mice with ABHF. Similarly, cardiac biopsies from patients with end-stage HF revealed increased lumican mRNA and protein levels compared with control hearts. In vitro, mechanical stretch and the proinflammatory cytokine interleukin-1β increased lumican mRNA as well as secreted lumican protein from cardiac fibroblasts. Stimulation with recombinant glycosylated lumican increased collagen type I alpha 2, lysyl oxidase and transforming growth factor-β1 mRNA, which was attenuated by costimulation with an inhibitor of the proinflammatory transcription factor NFκB. Furthermore, lumican increased the levels of the dimeric form of collagen type I, decreased the activity of the collagen-degrading enzyme matrix metalloproteinase-9 and increased the phosphorylation of fibrosis-inducing SMAD3. In conclusion, cardiac lumican is increased in experimental and clinical HF. Inflammation and mechanical stimuli induce lumican production by cardiac fibroblasts and increased lumican altered molecules important for cardiac remodeling and fibrosis in cardiac fibroblasts, indicating a role in HF development.

Glioblastoma, a malignant brain cancer, is characterized by abnormal activation of receptor tyrosine kinase signalling pathways and a poor prognosis. Extracellular proteoglycans, including heparan sulfate and chondroitin sulfate, play critical roles in the regulation of cell signalling and migration via interactions with extracellular ligands, growth factor receptors and extracellular matrix components, as well as intracellular enzymes and structural proteins. In cancer, proteoglycans help drive multiple oncogenic pathways in tumour cells and promote critical tumour–microenvironment interactions. In the present review, we summarize the evidence for proteoglycan function in gliomagenesis and examine the expression of proteoglycans and their modifying enzymes in human glioblastoma using data obtained from The Cancer Genome Atlas (http://cancergenome.nih.gov/). Furthermore, we demonstrate an association between specific proteoglycan alterations and changes in receptor tyrosine kinases. Based on these data, we propose a model in which proteoglycans and their modifying enzymes promote receptor tyrosine kinase signalling and progression in glioblastoma, and we suggest that cancer-associated proteoglycans are promising biomarkers for disease and therapeutic targets.

Down syndrome (DS) is a common birth defect characterized by the trisomy of chromosome 21. DS-affected umbilical cords (UCs) of fetuses show altered architecture of the extracellular matrix. Overexpression of the chromosome 21 genes encoding the collagen type VI (COLVI) chains α1(VI) and α2(VI), COL6A1 and COL6A2, respectively, has also reported to occur in the nuchal skin of DS fetuses. The aim of this study was therefore to evaluate the COLVI content in euploid and DS-affected UCs and human skin fibroblasts, and to investigate the relationships between COLVI and hyaluronan (HA) and HA synthase-2 (HAS2). We found that the UCs of DS fetuses showed denser staining of COLVI and increased COL6A2 expression at both early and term gestational ages. In vitro expression studies in DS-derived fibroblasts showed similarly increased amounts of α1(VI) and α2(VI) chains at the protein and transcriptional level, supporting the hypothesis of the gene dosage effect. Furthermore, increased levels of HA and HAS2 were also found in DS-derived skin fibroblast cultures. Notably, silencing of COL6A2 in DS-derived cells resulted in downregulation of HAS2, with a simultaneous decrease in secreted HA. Exogenous addition of COLVI to normal fibroblasts did not have any effect on HAS2 expression. In conclusion, UCs and skin fibroblasts in DS show significant increases in COLVI and HA; the overexpression of COL6A2 in DS tissue and cells is closely related to the increased expression of HAS2. These data may explain the DS phenotypes and their effects in organ tissue maturation.

The presence of iduronic acid in chondroitin/dermatan sulfate changes the properties of the polysaccharides because it generates a more flexible chain with increased binding potentials. Iduronic acid in chondroitin/dermatan sulfate influences multiple cellular properties, such as migration, proliferation, differentiation, angiogenesis and the regulation of cytokine/growth factor activities. Under pathological conditions such as wound healing, inflammation and cancer, iduronic acid has diverse regulatory functions. Iduronic acid is formed by two epimerases (i.e. dermatan sulfate epimerase 1 and 2) that have different tissue distribution and properties. The role of iduronic acid in chondroitin/dermatan sulfate is highlighted by the vast changes in connective tissue features in patients with a new type of Ehler–Danlos syndrome: adducted thumb-clubfoot syndrome. Future research aims to understand the roles of the two epimerases and their interplay with the sulfotransferases involved in chondroitin sulfate/dermatan sulfate biosynthesis. Furthermore, a better definition of chondroitin/dermatan sulfate functions using different knockout models is needed. In this review, we focus on the two enzymes responsible for iduronic acid formation, as well as the role of iduronic acid in health and disease.

Glycosaminoglycans (GAGs) vary widely in disaccharide and oligosaccharide content in a tissue-specific manner. Nonetheless, there are common structural features, such as the presence of highly sulfated non-reducing end domains on heparan sulfate (HS) chains. Less clear are the patterns of expression of GAGs on specific cell types. Leukocytes are known to express GAGs primarily of the chondroitin sulfate (CS) type. However, little is known regarding the properties and structures of the GAG chains, their variability among normal subjects, and changes in structure associated with disease conditions. We isolated peripheral blood leukocyte populations from four human donors and extracted GAGs. We determined the relative and absolute disaccharide abundances for HS and CS GAGs classes using size exclusion chromatography-mass spectrometry (SEC-MS). We found that all leukocytes express HS chains with a level of sulfation that is more similar to heparin than to organ-derived HS. The levels of HS expression follows the trend T cells/B cells > monocytes/natural killer cells > polymorphonuclear leukocytes (PMNs). In addition, CS abundances were considerably higher than total HS but varied considerably in a leukocyte cell type-specific manner. Levels of CS were higher for myeloid lineage cells (PMNs and monocytes) than for lymphoid cells (B, T and natural killer (NK) cells). This information establishes the range of GAG structures expressed on normal leukocytes and is necessary for subsequent inquiry into disease conditions.

Glycosaminoglycans, including chondroitin sulfate (CS), dermatan sulfate, and heparan sulfate, attached to proteoglycans at the surface of tumor cells play key roles in malignant transformation and metastasis. A Lewis lung carcinoma (LLC)-derived tumor cell line with high metastatic potential shows a higher proportion of E disaccharide units, d-glucuronic acid–GalNAc(4,6-O-disulfate), in CS chains than LLC cells with low metastatic potential, suggesting that E units in the CS chains contribute to the metastatic potential. In fact, the metastasis of LLC to mouse lungs is drastically inhibited by preadministration of CS-E or a phage display antibody specific for CS-E. However, the molecular mechanism underlying the pulmonary metastasis involving CS chains remained to be elucidated. Recently, receptor for advanced glycation end-products (RAGE), which is predominantly expressed in the lung, was identified as a functional receptor for CS chains containing E units. RAGE strongly interacted with not only CS-E but also heparan sulfate in vitro. The interaction with CS-E required a decasaccharide length and a cluster of basic amino acids. Intriguingly, antibody against RAGE robustly inhibited the pulmonary metastasis of not only LLC but also B16 melanoma cells, which also colonize mouse lungs after injection into a tail vein. Thus, CS chains containing E units are involved in the metastatic process, and RAGE is a critical receptor for glycosaminoglycan chains expressed at the tumor cell surface. Hence, RAGE and glycosaminoglycans are potential targets of drugs for pulmonary metastasis and a number of other pathological conditions involving RAGE in the pathogenetic mechanism.

Glypican-3 (GPC3) is a member of the glypican family. Glypicans are proteoglycans that are attached to the cell surface by a glycosyl-phosphatidylinositol anchor. They regulate the signaling activity of several growth factors, including Wnts. This regulation is based on the ability of glypicans to stimulate or inhibit the interaction of these growth factors with their respective signaling receptors. It has been clearly established that whereas GPC3 is expressed by most hepatocellular carcinomas (HCCs), this glypican is not detected in normal and cirrhotic liver, or in benign hepatic lesions. Consequently, immunostaining of liver biopsies for GPC3 is currently being used by clinical pathologists to confirm HCC diagnosis when the malignant nature of the lesion is difficult to establish. In addition to being a marker of HCC, GPC3 plays a role in the progression of the disease. GPC3 promotes the growth of HCC by stimulating canonical Wnt signaling. It has been proposed that this stimulation is based on the ability of GPC3 to increase the binding of Wnt to its signaling receptor, Frizzled. Two therapeutic approaches for HCC that target GPC3 are currently being tested in phase II clinical trials. One of them is based on the use of a humanized GPC3 monoclonal antibody that inhibits the in vivo growth of HCC xenografts by inducing antibody-dependent cellular cytotoxicity. The second approach employs a vaccine that consists of two GPC3-derived peptides that induce cytotoxic T lymphocytes against these peptides. Targeting of GPC3 might offer a new tool for the treatment of HCC.

Cell surface heparan sulfate proteoglycans (HSPGs), syndecans and glypicans, play crucial roles in the functional properties of cancer cells, such as proliferation, adhesion, migration and invasion. Platelet-derived growth factor (PDGF)/PDGF receptor (PDGF-R) mediated signaling, on the other hand, is highly associated with cancer progression. Specifically, PDGF-Rα and PDGF-Rβ expressions documented in breast cancer tissue specimens as well as breast cancer cell lines are correlated with tumor aggressiveness and metastasis. Imatinib (Glivec^®) is a tyrosine kinase inhibitor specific for PDGF-Rs, c-ΚΙΤ and BCR-ABL. In this study we evaluated the effects of imatinib on the properties of breast cancer cells as well as on the expression of HSPGs in the presence and absence of PDGF-BB. These studies have been conducted in a panel of three breast cancer cell lines of low and high metastatic potential. Our results indicate that imatinib exerts a significant inhibitory effect on breast cancer cell proliferation, invasion and migration as well as on the cell surface expression of HSPGs even after exposure of PDGF. These effects depend on the aggressiveness of breast cancer cells and the type of HSPG. It is suggested that imatinib may be of potential therapeutic usefulness in breast cancer regimes.

Proteoglycans are ubiquitous dynamic molecules that are made up of a protein core to which specific linear glycosylation structures, known as glycosaminoglycans, have been covalently coupled. They have roles in many biological and pathological processes, which have been shown to be dependent on events involving the protein component and/or the glycosaminoglycan chains. This review focuses on the literature describing the recombinant expression and production of proteoglycans known to be present in the extracellular, cell surface and intracellular environments with an emphasis on how the structure of the molecule relates to its biological function and how this relationship has been explored using recombinant DNA technology for clinical applications.

Proteoglycans, comprised of a core protein to which glycosaminoglycan chains are covalently linked, are an important structural and functional family of macromolecules found in the extracellular matrix. Advances in our understanding of biological interactions have lead to a greater appreciation for the need to design tissue engineering scaffolds that incorporate mimetics of key extracellular matrix components. A variety of synthetic and semisynthetic molecules and polymers have been examined by tissue engineers that serve as structural, chemical and biological replacements for proteoglycans. These proteoglycan mimetics have been referred to as neoproteoglycans and serve as functional and therapeutic replacements for natural proteoglycans that are often unavailable for tissue engineering studies. Although neoproteoglycans have important limitations, such as limited signaling ability and biocompatibility, they have shown promise in replacing the natural activity of proteoglycans through cell and protein binding interactions. This review focuses on the recent in vivo and in vitro tissue engineering applications of three basic types of neoproteoglycan structures, protein–glycosaminoglycan conjugates, nano-glycosaminoglycan composites and polymer–glycosaminoglycan complexes.

Heparin has been the most commonly used anticoagulant drug for nearly a century. The drug heparin is generally categorized into three forms according to its molecular weight: unfractionated (UF, average molecular weight 13 000), low molecular weight (average molecular weight 5000) and ultra-low-molecular-weight heparin (ULMWH, average molecular weight 2000). An overdose of heparin may lead to very dangerous bleeding in patients. Protamine sulfate may be administered as an antidote to reverse heparin's anticoagulant effect. However, there is no effective antidote for ULMWH. In the current study, we examine the use of human N-acetylglucosamine 6-sulfatase (NG6S), expressed in Chinese hamster ovary cells, as a reversal agent for ULMWH. NG6S removes a single 6-O-sulfo group at the non-reducing end of the ULMWH Arixtra^® (fondaparinux), effectively removing its ability to bind to antithrombin and preventing its inhibition of coagulation factor Xa. These results pave the way to developing human NG6S as an antidote for neutralizing the anticoagulant activity of ULMWHs.