The complexity of the human proteome is increasingly being deciphered by sophisticated, mainly mass spectrometry based techniques that have become an indispensable tool in life sciences. Within the human genome project ∼20.000 genes were characterized that are translated into ∼100.000 proteins. Furthermore, the protein function is fundamentally influenced by post-translational modifications (PTMs) that amplify the diversity up to an estimate of 1 million protein variants . However, the improved understanding of patho-physiological processes depends upon the in depth analysis of regulatory processes and PTMs represent a key examples of how genotype and phenotype are not uniquely directed by information that is present on the genome . Accordingly, the characterisation of PTMs like phosphorylation, acetylation, methylation, glycosylation and proteolytic processing has become a major focus of interest.
Specifically, the proteolytic processing of proteins and peptides is a common and irreversible post-translational modification. Proteases regulate multiple biological processes including cell differentiation, proteasomal degradation, inflammation, tissue remodelling, apoptosis, cell homeostasis, and coagulation. Consequently, the deregulation of proteolytic activity accounts for pathogenesis and progression of many diseases such as cardiovascular disease, inflammatory conditions, neurodegenerative disorders and cancer .
In this Special Issue, several groups of investigators present research and perspectives on proteomics and its applications to elucidate the link between proteases and disease and diagnostic as well as therapeutic options are discussed.
Patients’ blood samples are the most widely used specimens to make up diagnosis, prognosis or therapeutic monitoring for a multitude of different diseases. However, serum and plasma have high intrinsic proteolytic activity that is related to diverse exopeptidases as well as different endoproteases e.g. from the coagulation cascade and the complement system. Jambunathan et al. investigate this genuine proteolytic activity of serum and plasma specimens using a combinatorial library of internally quenched fluorogenic probes . Their results might give advice for the proper selection of respective anticoagulants to guarantee optimal praeanalytical conditions for various downstream applications. Furthermore, disease related protease activity in serum specimens can be revealed by monitoring the proteolytic processing of synthetic peptides ex vivo. The predominant cleavage of distinct reporter peptides in serum specimens of patients with colorectal tumors can be taken as diagnostic surrogate marker for tumor related proteolytic activity . Alternatively, Tempst and coworkers identified several aminopeptidase activities as biomarker candidates in urinary specimens of bladder cancer patients . The development of a targeted urinary aminopeptidase activity assays using fluorophoric substrates resulted in favourable classification accuracy of this promising approach. Another class of exoproteases are carboxypeptidases that play a crucial role not only in protein degradation but also in the synthesis of peptide hormones. Sapio et al. reviews the impact of dysregulated carboxyprotease activity with focus on neurological disorders ranging from obesity to epilepsy to neurodegeneration .
The in vivo trimming of peptide chains from their ends by amino- and carboxypeptidases is often associated with new biological activities. Huesgen et al. discuss that the investigation of the so called “terminome” is a promising new tool for biomarker discovery. Furthermore, the enrichment for N- and C-terminomes enables a much more sensitive analysis of proteolytical pathways that may outperform classical techniques like shotgun proteomics up to three orders of magnitude .
The in–depth characterization of protease substrate interaction is exemplarily shown for granzymes. These are well known serine proteases that are delivered from cytotoxic lymphocytes via perforin into the cytoplasm of target cells where they initiate cell death. Granzymes feature among the proteases that have been most widely studied by novel degradomic techniques . Joeckel et al. demonstrate how new approaches and techniques can identify new physiological granzyme substrates that indicate extracellular action and involvement of granzymes in non-cytotoxic processes .
As shown by Becker-Pauly et al. the role of orphan proteases can be assigned to precise biological functions by using sophisticated proteomics techniques. Exemplarily meprin α and meprin β were identified to be important for collagen assembly and deposition in skin, which makes them potential therapeutic targets in fibrotic conditions .
De Veer et al.  review our current state of knowledge on the involvement of proteases in skin diseases. They highlight that aberrant proteolytic activity is a common factor in a multitude of skin disorders that range in severity from relatively mild to life-threatening pathological conditions.
The tissue kallikrein family of serine proteases have functional roles in malignancies like prostate cancer and ovarian cancer. With mass spectrometry based techniques the protease substrate interaction can be deciphered and their potential as clinical biomarkers is reviewed by Fuhrman-Luck et al. .
Another class of proteases are cysteine cathepsins. These were generally considered to be involved mainly in the nonspecific bulk protein degradation that takes place within the lysosomes. However, new findings illustrate that they can also influence various other patho-physiological processes. Fonovic et al. elucidate the role of cathepsins for a broad variety of diseases such as atherosclerosis, osteoporosis, neurodegeneration, cancer and infectious diseases and respective therapeutic options are discussed accordingly . Aggrawal et al. focused on the role of cathepsin B and its pleiotropic role for the progression of many solid tumors and cathepsin B seems to be a promising druggable target .
Besides the characterisation of protease-substrate interaction the pathophysiological role of proteases can also be revealed by genetic association studies. The inherited decrease of factor seven activating protease (FSAP) activity does not only impact the vascular system and haemostasis but also has unfavourable effects on the progression of liver fibrosis, as outlined by Martinez-Palacian .
Furthermore, the impaired expression of endogenous protease inhibitors can also lead to deregulation of proteolytic activity. Cystatin C is directed against cathepsin proteases and is increased in lung tissue of patients with idiopathic pulmonary disease. Kasabova et al. show that this may reflect abnormal regulation of proteolytic activity of cathepsins, which leads to reduced degradation of extracellular matrix and in turn can promote the development of fibrosis .
Finally, new therapeutic options will hopefully arise from improved knowledge of dysregulated protease activity. Various therapeutically relevant protease inhibitors do already exist. For example, the angiotensin converting enzyme, thrombin or dipeptidylpeptidase 4 (DPP4) are drug target for arterial hypertension, coagulation disorders and type 2 diabetes respectively. The serinprotease fibroblast activation protein (FAP) is closely related to DPP4 and Hamson and coworkers review substrates, expression and targeting of FAP in malignant disease .
Taken together, the improved understanding of the disease-promoting function of deregulated protease activity will hopefully lead to improved diagnosis, prognosis and classification of patients that might enable new therapeutic options with targeted protease inhibitors.
We thank all authors for their contributions as well as the editorial team of Proteomics Clinical Applications for their continuous support of this Special Issue dedicated to “Proteases and Disease”.