ChemBioChem

Cover image for Vol. 18 Issue 10

Editor: Meghan Campbell; Editorial Board Chairs: Thomas Carell, Donald Hilvert, Barbara Imperiali

Online ISSN: 1439-7633

Associated Title(s): ChemCatChem, ChemMedChem, ChemPhysChem, ChemSusChem

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May 04, 2017

Two recent VIP papers on mass spectrometry and mannosidase inhibitors

Very Important Paper: Mapping the Interactions of Selective Biochemical Probes of Antibody Conformation by Hydrogen--Deuterium Exchange Mass Spectrometry

Ulrike Leurs, Hermann Beck, Lea Bonnington, Ingo Lindner, Ewa Pol, Kasper Rand*

A major challenge during the development of therapeutic antibodies (IgG) is the assessment of changes to their higher-order structure. Now, K. Rand (University of Copenhagen, Denmark) and co-workers investigate the use of engineered Fab fragments, designed to bind to conserved regions of the IgG that are prone to undergo conformational changes. The selectivity of each probe to bind to distinct correctly folded parts of the IgG was identified by a combination of hydrogen--deuterium exchange mass spectrometry experiments (HDX-MS) and electron transfer dissociation (ETD). The use of these biochemical probes was also demonstrated in a surface plasmon resonance assay, which showed that their binding is sensitive to conformational changes occurring in IgG upon oxidation.

Very Important Paper: Selective Manipulation of Discrete Mannosidase Activities in the Endoplasmic Reticulum by Using Reciprocally Selective Inhibitors

Taiki Kuribara, Makoto Hirano, Gaetano Speciale, Spencer J. Williams, Yukishige Ito, Kiichiro Totani*

Around one third of N-linked glycoproteins produced in the cell fail to properly fold and must be detected and appropriately disposed of. Yet, the precise details of which glycan structures encode secretion and degradation signals are obscure. K. Totani (Seikei University, Japan) and co-workers report that known glycosidase inhibitors (deoxynojirimycin and deoxymannojirimycin) can selectively inhibit glycan-editing mannosidases provided their concentrations are carefully controlled. Using inhibitors to control the processing of a synthetic N-glycan in an ER-fraction, two discrete glycan processing pathways leading to secretion and degradation were discovered. This work provides fundamental insights into the signals that control the fate of glycoproteins.

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March 14, 2017

Very Important Paper: Identifying Unknown Enzyme-Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry

Kalli C. Catcott, Jing Yan, Wanlu Qu, Vicki H. Wysocki*, Zhaohui Sunny Zhou*

The enzyme-substrate complex is inherently transient, rendering its detection difficult. Therefore, V. Wysocki (The Ohio State University, U.S.A.), Z.S. Zhou (Northeastern University, USA) and co-workers developed a framework, IsoLAIT (Isotope-Labeled, Activity-based Identification and Tracking), for bisubstrate systems. Using this method a common substrate, such as S-adenosylmethionine for methyltransferases, is replaced by an analogue (e.g., S-adenosylvinthionine) that, as a probe, creates a tightly bound [enzyme•substrate-probe] complex upon catalysis by thiopurine-S-methyltransferase. Then, this persistent complex is identified by native mass spectrometry from the cellular milieu without separation. Furthermore, the probe’s isotope pattern flags even unknown substrates and enzymes. IsoLAIT can be broadly applicable for other enzyme systems, particularly those catalyzing group transfer and with multiple substrates.

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December 19, 2016

Very Important Paper: A Bifunctional Amino Acid Enables Both Covalent Chemical Capture and Isolation of in Vivo Protein--Protein Interactions

Cassandra M. Joiner+, Meghan E. Breen+, James Clayton, and Anna K. Mapp*

In vivo covalent chemical capture by using photo-activatable unnatural amino acids is a powerful tool for the identification of transient protein--protein interactions (PPIs) in their native environment, however, the isolation and characterization of the crosslinked complexes can be challenging. Now, A.K. Mapp (University of Michigan, U.S.A.) and co-workers report a strategy using the bifunctional unnatural amino acid, BPKyne, to capture and directly label transient PPIs in their native environment. Functionalization after crosslinking with a biotin--azide probe enabled the isolation of transcriptional protein complexes from yeast cells. BPKyne expands the toolbox for the discovery of novel PPIs in live cells and can be extended to other biological systems.

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December 12, 2016

Very Important Paper: K-BILDS: A Kinase Substrate Discovery Tool

D. Maheeka Embogama and Mary Kay H. Pflum*

The discovery of kinase substrates has always been a challenge in biology. Now, D.M. Embogama and M.K.H. Pflum (Wayne State University, U.S.A.) have used kinase-catalyzed biotinylation with ATP-Biotin to develop a new substrate discovery tool entitled K-BILDS(kinase-catalyzed biotinylation with inactivated lysates for discovery of substrates). As a proof-of-concept, K-BILDS was applied to the identification of PKA substrates. From the analysis, more than 200 candidate PKA substrates were discovered, with roughly 20% of the hits identified as previously known substrates. To validate candidate PKA hits, in vitro kinase assays were carried out with three selected hits (BAG3, NASP, YWHAQ) to confirm PKA phosphorylation. These results established K-BILDS as a versatile tool to identify the kinase substrates of any kinase of interest.

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November 24, 2016

Very Important Paper: Exploring the Potent Inhibition of CTP Synthase by Gemcitabine-5'-Triphosphate

Gregory D. McCluskey, Samy Mohamady, Scott D. Taylor, Stephen L. Bearne*

The nucleoside analogue gemcitabine is used as a chemotherapy drug to treat lung, pancreatic, bladder, and breast cancers. One metabolite of this drug, gemcitabine-5'-triphosphate, is believed to deplete intracellular CTP levels by inhibiting CTP synthase, but the direct inhibition of this enzyme had not been demonstrated. Now, S.L. Bearne (Dalhousie University, Canada) and co-workers show that gemcitabine-5'-triphosphate is indeed a potent competitive inhibitor of Escherichia coli CTP synthase. Remarkably, the enzyme also acts on an abundant catabolite of the drug, 2',2'-difluoro-2'-deoxyuridine-5'-triphosphate, to convert it back to gemcitabine-5'-triphosphate, suggesting that CTP synthase may play a role in replenishing the active drug in vivo.

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November 16, 2016

Very Important Paper: Exploring the Potent Inhibition of CTP Synthase by Gemcitabine-5'-Triphosphate

Gregory D. McCluskey, Samy Mohamady, Scott D. Taylor, Stephen L. Bearne*

The nucleoside analogue gemcitabine is used as a chemotherapy drug to treat lung, pancreatic, bladder, and breast cancers. One metabolite of this drug, gemcitabine-5'-triphosphate, is believed to deplete intracellular CTP levels by inhibiting CTP synthase, but the direct inhibition of this enzyme had not been demonstrated. Now, S.L. Bearne (Dalhousie University, Canada) and co-workers show that gemcitabine-5'-triphosphate is indeed a potent competitive inhibitor of Escherichia coli CTP synthase. Remarkably, the enzyme also acts on an abundant catabolite of the drug, 2',2'-difluoro-2'-deoxyuridine-5'-triphosphate, to convert it back to gemcitabine-5'-triphosphate, suggesting that CTP synthase may play a role in replenishing the active drug in vivo.

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May 04, 2017
Two recent VIP papers on mass spectrometry and mannosidase inhibitors

March 14, 2017
Very Important Paper: Identifying Unknown Enzyme-Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry

December 19, 2016
Very Important Paper: A Bifunctional Amino Acid Enables Both Covalent Chemical Capture and Isolation of in Vivo Protein--Protein Interactions

December 12, 2016
Very Important Paper: K-BILDS: A Kinase Substrate Discovery Tool

November 24, 2016
Very Important Paper: Exploring the Potent Inhibition of CTP Synthase by Gemcitabine-5'-Triphosphate

November 16, 2016
Very Important Paper: Exploring the Potent Inhibition of CTP Synthase by Gemcitabine-5'-Triphosphate

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