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The importance of 3D protein structure in proteolytic processing is well known. However, despite the plethora of existing methods for predicting proteolytic sites, only a few of them utilize the structural features of potential substrates as predictors. Moreover, to our knowledge, there is currently no method available for predicting the structural susceptibility of protein regions to proteolysis. We developed such a method using data from CutDB, a database that contains experimentally verified proteolytic events. For prediction, we utilized structural features that have been shown to influence proteolysis in earlier studies, such as solvent accessibility, secondary structure, and temperature factor. Additionally, we introduced new structural features, including length of protruded loops and flexibility of protein termini. To maximize the prediction quality of the method, we carefully curated the training set, selected an appropriate machine learning method, and sampled negative examples to determine the optimal positive-to-negative class size ratio. We demonstrated that combining our method with models of protease primary specificity can outperform existing bioinformatics methods for the prediction of proteolytic sites. We also discussed the possibility of utilizing this method for bioinformatics prediction of other post-translational modifications. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is a ubiquitous post-translational modification controlled by the opposing activities of protein kinases and phosphatases, which regulate diverse biological processes in all kingdoms of life. One of the key challenges to a complete understanding of phosphoregulatory networks is the unambiguous identification of kinase and phosphatase substrates. Liquid chromatography-coupled mass spectrometry (LC-MS/MS) and associated phosphoproteomic tools enable global surveys of phosphoproteome changes in response to signaling events or perturbation of phosphoregulatory network components. Despite the power of LC-MS/MS, it is still challenging to directly link kinases and phosphatases to specific substrate phosphorylation sites in many experiments. Here, we survey common LC-MS/MS-based phosphoproteomic workflows for identifying protein kinase and phosphatase substrates, noting key advantages and limitations of each. We conclude by discussing the value of inducible degradation technologies coupled with phosphoproteomics as a new approach that overcomes some limitations of current methods for substrate identification of kinases, phosphatases, and other regulatory enzymes. [ABSTRACT FROM AUTHOR]

Amino acid decarboxylases convert amino acids into different biogenic amines which regulate diverse biological processes. Therefore, identifying the substrates of amino acid decarboxylases is critical for investigating the function of the decarboxylases, especially for the new genes predicted to be amino acid decarboxylases. In the present work, we have established a simple and efficient method to identify the substrates and enzymatic activity of amino acid decarboxylases based on LC-MS methods. We chose GAD65 and AADC as models to validate our method. GAD65 and AADC were expressed in HEK 293T cells and purified through immunoprecipitation. The purified amino acid decarboxylases were subjected to enzymatic reaction with different substrate mixtures in vitro. LC-MS analysis of the reaction mixture identified depleted or accumulated metabolites, which corresponded to candidate enzyme substrates and products, respectively. Our method successfully identified the substrates and products of known amino acid decarboxylases. In summary, our method can efficiently identify the substrates and products of amino acid decarboxylases, which will facilitate future amino acid decarboxylase studies. [ABSTRACT FROM AUTHOR]

The ubiquitin (Ub) ligase E6AP, encoded by the UBE3A gene, has been causally associated with human diseases including cervical cancer and Angelman syndrome, a neurodevelopmental disorder. Yet, our knowledge about disease-relevant substrates of E6AP is still limited, presumably because at least some of these interactions are rather transient, a phenomenon observed for many enzyme-substrate interactions. Here, we introduce a novel approach to trap such potential transient interactions by combining a stable E6AP-Ub conjugate mimicking the active state of this enzyme with photo-crosslinking (PCL) followed by affinity enrichment coupled to mass spectrometry (AE-MS). To enable PCL, we equipped Ub with diazirine moieties at distinct positions. We validated our PCL assisted AE-MS approach by identification of known (e. g. PSMD4, UCHL5) and potential new (e. g. MSH2) substrates of E6AP. Our findings suggest that PCL assisted AE-MS is indeed suited to identify substrates of E6AP, thereby providing insights into E6AP-associated pathologies, and, potentially, of other enzymes of the Ub-conjugating system., (© 2025 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

MBL Associated Serine Protease-1 (MASP-1) is an abundant enzyme of the lectin complement pathway. MASP-1 cleaves numerous substrates like MASP-2, MASP-3, C2, C3i, fibrinogen, FXIII and prothrombin. It has thrombin-like specificity and can cleave thrombin substrates. Owing to its high concentration and relaxed substrate specificity, MASP-1 has substrates outside the complement system and can influence other proteolytic cascades and physiological processes. The unidentified substrates may assist us to ascertain the role(s) of MASP-1. In this study, we used a high-throughput N-terminomics method to identify substrates of MASP-1 from human plasma. We have identified 35 putative substrates of MASP-1. Among the identified proteins, alpha 2-antiplasmin, alpha-1-acid glycoprotein, antithrombin III, and siglec-6 were demonstrated to be cleaved by MASP-1. We have discussed the physiological relevance of cleavage of these substrates by MASP-1. The expression of Siglec-6 and MASP-1 has been reported in the B cells. Alpha-1-acid glycoprotein cleavage by MASP-1 may occur in the acute phase as it is known to be an inhibitor of platelet aggregation, whereas MASP-1 triggers platelet aggregation. The cleavage alpha2 antiplasmin by MASP-1 implies that MASP-1 may be promoting plasmin-mediated fibrinolysis. Our study supports that MASP-1 may be implicated in thrombosis as well as thrombolysis. • MBL Associated Serine Protease-1 (MASP-1)- vital enzyme of the lectin complement. • Atleast 35 putative substrates were identified using the N-terminomics strategy. • Predicting the influence of MASP-1 on proteolytic cascades, physiological processes. • MASP-1 may be implicated in thrombosis as well as thrombolysis. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is characterized by the invariably progressive deposition of fibrotic tissue in the lungs and overall poor prognosis. TG2 (transglutaminase 2) is an enzyme that crosslinks glutamine and lysine residues and is involved in IPF pathogenesis. Despite the accumulating evidence implicating TG2 as a critical enzyme, the causative function and direct target of TG2 relating to this pathogenesis remain unelucidated. Here, we clarified the distributions of TG2 protein/activity and conducted quantitative proteomics analyses of possible substrates crosslinked by TG2 on unfixed lung sections in a mouse pulmonary fibrosis model. We identified 126 possible substrates as markedly TG2-dependently increased in fibrotic lung. Gene ontology analysis revealed that these identified proteins were mostly enriched in the lipid metabolic process, immune system process, and protein transport. In addition, these proteins were enriched in 21 pathways, including phagosome, lipid metabolism, several immune responses, and protein processing in endoplasmic reticulum. Furthermore, the network analyses screened out the six clusters and top 20 hub proteins with higher scores, which are related to endoplasmic reticulum stress and peroxisome proliferator-activated receptor signals. Several enriched pathways and categories were identified, some of which were the same terms based on transcription analysis in IPF. Our results provide novel pathological molecular networks driven by protein crosslinking via TG2, which can lead to the development of new therapeutic targets for IPF. [ABSTRACT FROM AUTHOR]

The function and regulation of thrombin is a complex as well as an intriguing aspect of evolution and has captured the interest of many investigators over the years. The reported substrates of thrombin are coagulation factors V, VIII, XI, XIII, protein C and fibrinogen. However, these may not be all the substrate of thrombin and therefore its functional role(s), may not have been completely comprehended. The purpose of our study was to identify hitherto unreported substrates of thrombin from human plasma using a N-terminomics protease substrate identification method. We identified 54 putative substrates of thrombin of which 12 are already known and 42 are being reported for the first time. Amongst the proteins identified, recombinant siglec-6 and purified serum alpha-1-acid glycoprotein were validated by cleavage with thrombin. We have discussed the probable relevance of siglec-6 cleavage by thrombin in human placenta mostly because an upregulation in the expression of siglec-6 and thrombin has been reported in the placenta of preeclampsia patients. We also speculate the role of alpha-1-acid glycoprotein cleavage by thrombin in the acute phase as alpha-1-acid glycoprotein is known to be an inhibitor of platelet aggregation whereas thrombin is known to trigger platelet aggregation. [ABSTRACT FROM AUTHOR]

Poly-ADP-ribose polymerases (PARPs) are a family of enzymes responsible for transferring individual or chains of ADP-ribose subunits to substrate targets as a type of post-translational modification. PARPs regulate a wide variety of important cellular processes, ranging from DNA damage repair to antiviral response. However, most research to date has focused primarily on the polyPARPs, which catalyze the formation of ADP-ribose polymer chains, while the monoPARPs, which transfer individual ADP-ribose monomers, have not been studied as thoroughly. This is partially due to the lack of robust assays to measure mono-ADP-ribosylation in the cell. In this study, the recently developed MAR/PAR antibody has been shown to detect mono-ADP-ribosylation in cells, enabling the field to investigate the function and therapeutic potential of monoPARPs. In this study, the antibody was used in conjunction with engineered cell lines that overexpress various PARPs to establish a panel of assays to evaluate the potencies of literature-reported PARP inhibitors. These assays should be generally applicable to other PARP family members for future compound screening efforts. A convenient and generalizable workflow to identify and validate PARP substrates has been established. As an initial demonstration, aryl hydrocarbon receptor was verified as a direct PARP7 substrate and other novel substrates for this enzyme were also identified and validated. This workflow takes advantage of commercially available detection reagents and conventional mass spectrometry instrumentation and methods. Ultimately, these assays and methods will help drive research in the PARP field and benefit future therapeutics development. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphorylation and dephosphorylation are key regulatory mechanisms in eukaryotes. Protein tyrosine phosphorylation and dephosphorylation are catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), respectively. The combinatorial action of both PTKs and PTPs is essential for properly maintaining cellular functions. In this unit, we discuss different novel methods to identify PTP substrates. PTPs depend on specific invariant residues that enable binding to tyrosine-phosphorylated substrates and aid catalytic activity. Identifying PTP substrates has paved the way to understanding their role in distinct intracellular signaling pathways. Due to their high specific activity, the interaction between PTPs and their substrates is transient; therefore, identifying the physiological substrates of PTPs has been challenging. To identify the physiological substrates of PTPs, various PTP mutants have been generated. These PTP mutants, named "substrate-trapping mutants," lack catalytic activity but bind tightly to their tyrosine-phosphorylated substrates. Identifying the substrates for the PTPs will provide critical insight into the function of physiological and pathophysiological signal transduction. In this chapter, we describe interaction assays used to identify the PTP substrates., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Phosphorylation is a reversible post-translational modification that alters the functions of proteins to govern various cellular events, including cell signaling. Kinases catalyze the transfer of a phosphoryl group onto the hydroxyl residue of serine, threonine, and tyrosine, while phosphatases catalyze the removal. Unregulated kinase and phosphatase activity have been observed in various cancers and neurodegenerative diseases. Despite their importance in cell biology, the role of phosphatases in cellular events has yet to be fully characterized, partly due to the lack of tools to identify phosphatase-substrate pairs in a biological context. The method called kinase-catalyzed biotinylation to identify phosphatase substrates (K-BIPS) was developed to remedy the lack of information surrounding phosphatase biology, particularly focused on substrate identification. In the K-BIPS method, the γ-phosphoryl modified adenosine 5'-triphosphate (ATP) analog, ATP-biotin, is used by kinases to biotin-label phosphoproteins. Because phosphatases must initially remove a phosphoryl group for subsequent biotinylation by ATP-biotin, phosphatase substrates are identified in K-BIPS by comparing biotinylated proteins in the presence and absence of active phosphatases. K-BIPS has been used to discover novel substrates of both serine/threonine and tyrosine phosphatases. This chapter describes the K-BIPS method to enable the identification of substrates to any phosphatases of interest, which will augment studies of phosphatase biology., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

γ-Secretases mediate the regulated intramembrane proteolysis (RIP) of more than 150 integral membrane proteins. We developed an unbiased γ-secretase substrate identification (G-SECSI) method to study to what extent these proteins are processed in parallel. We demonstrate here parallel processing of at least 85 membrane proteins in human microglia in steady-state cell culture conditions. Pharmacological inhibition of γ-secretase caused substantial changes of human microglial transcriptomes, including the expression of genes related to the disease-associated microglia (DAM) response described in Alzheimer disease (AD). While the overall effects of γ-secretase deficiency on transcriptomic cell states remained limited in control conditions, exposure of mouse microglia to AD-inducing amyloid plaques strongly blocked their capacity to mount this putatively protective DAM cell state. We conclude that γ-secretase serves as a critical signaling hub integrating the effects of multiple extracellular stimuli into the overall transcriptome of the cell. [Display omitted] • G-SECSI: an unbiased approach for γ-secretase substrate identification • γ-Secretase substrates are central in intracellular microglial signaling • γ-Secretase maintains the basal states of in vitro and in vivo microglia models • γ-Secretase deficiency impedes disease-associated microglia (DAM) response The field has been lacking an unbiased approach for large-scale endogenous γ-secretase substrate identification. Hou et al. report a G-SECSI method and demonstrate that the γ-secretase substrate proteome is intimately connected to the intracellular signaling in microglia. γ-Secretase deficiency has profound effects on microglia in response to amyloid pathology. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to extracellular stimuli, among many others, and is deregulated in many diseases. Protein phosphorylation is coordinated by the opposing activities of protein kinases and protein phosphatases. In eukaryotic cells, most serine/threonine phosphorylation sites are dephosphorylated by members of the Phosphoprotein Phosphatase (PPP) family. However, we only know for a few phosphorylation sites which specific PPP dephosphorylates them. Although natural compounds such as calyculin A and okadaic acid inhibit PPPs at low nanomolar concentrations, no selective chemical PPP inhibitors exist. Here, we demonstrate the utility of endogenous tagging of genomic loci with an auxin-inducible degron (AID) as a strategy to investigate specific PPP signaling. Using Protein Phosphatase 6 (PP6) as an example, we demonstrate how rapidly inducible protein degradation can be employed to identify dephosphorylation sites and elucidate PP6 biology. Using genome editing, we introduce AID-tags into each allele of the PP6 catalytic subunit (PP6c) in DLD-1 cells expressing the auxin receptor Tir1. Upon rapid auxin-induced degradation of PP6c, we perform quantitative mass spectrometry-based proteomics and phosphoproteomics to identify PP6 substrates in mitosis. PP6 is an essential enzyme with conserved roles in mitosis and growth signaling. Consistently, we identify candidate PP6c-dependent dephosphorylation sites on proteins implicated in coordinating the mitotic cell cycle, cytoskeleton, gene expression, and mitogen-activated protein kinase (MAPK) and Hippo signaling. Finally, we demonstrate that PP6c opposes the activation of large tumor suppressor 1 (LATS1) by dephosphorylating Threonine 35 (T35) on Mps One Binder (MOB1), thereby blocking the interaction of MOB1 and LATS1. Our analyses highlight the utility of combining genome engineering, inducible degradation, and multiplexed phosphoproteomics to investigate signaling by individual PPPs on a global level, which is currently limited by the lack of tools for specific interrogation., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

ABC transporters represent a large family of ATP-driven transmembrane transporters involved in uni- or bidirectional transfer of a variety of substrates. Divided in 7 families, they represent 48 transporter proteins, several of which are associated with human disease. Among the latter is ABCC6, a unidirectional exporter protein primarily expressed in liver and kidney. ABCC6 deficiency causes pseudoxanthoma elasticum (PXE), characterised by calcification and fragmentation of elastic fibres, resulting in oculocutaneous and cardiovascular symptoms. Unique among connective tissue disorders, the relation between the ABCC6 transporter and ectopic mineralization in PXE remains enigmatic, not in the least because of lack of knowledge on the substrate(s) of ABCC6 and its unusual expression pattern. Because many features, including structure and transport mechanism, are shared by ABC transporters, it is worthwhile to evaluate if and to what extent the knowledge on the (patho)physiology of other transporters may provide useful clues towards understanding the (patho)physiological role of ABCC6. In this paper, we summarize relevant knowledge and methods for analysis on ABC transporters which may be useful for the further study of ABCC6.

Lysine fatty acylation is a protein posttranslational modification (PTM) that has been linked to various important biological processes. HDAC11, the sole member of class IV of histone deacetylases (HDACs), has been shown to have high lysine defatty-acylase activity. In order to better understand the functions of lysine fatty acylation and its regulation by HDAC11, it is important to identify the physiological substrates of HDAC11. This can be achieved through profiling the interactome of HDAC11 using a stable isotope labeling with amino acids in cell culture (SILAC) proteomics strategy. Here we describe a detailed method on using SILAC to identify the interactome of HDAC11. This method can be similarly used to identify the interactome, and thus potential substrates, of other PTM enzymes., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Abstract: Phosphorylation of proteins is a prevalent post-translational modification, which affects intracellular signaling in many ways. About 2% of all eukaryotic genes code for protein kinases catalyzing phosphorylation events. Despite technological advances that have made it possible to identify thousands of phosphorylation sites simultaneously, identification of the substrates of a given kinase remains an exceptionally challenging task. Here, we summarize approaches for substrate identification that make use of genetically engineered ‘analog-sensitive’ kinases. [Copyright &y& Elsevier]

Abstract: A microarray-based mix-and-measure, nonradioactive multiplex method with real-time detection was used for substrate identification, assay development, assay optimisation, and kinetic characterization of protein kinase A (PKA). The peptide arrays included either up to 140 serine/threonine-containing peptides or a concentration series of a smaller number of peptides. In comparison with existing singleplex assays, data quality was high, variation in assay conditions and reagent consumption were reduced considerably, and assay development could be accelerated because phosphorylation kinetics were monitored simultaneously on 4, 12, or 96 arrays. PKA was shown to phosphorylate many peptides containing known PKA phosphorylation sites as well as some new substrates. The kinetic behavior of the enzyme and the mechanism of inhibition by AMP–PNP, staurosporin, and PKA inhibitor peptide on the peptide microarray correlated well with data from homogeneous assays. Using this multiplex setup, we showed that the kinetic parameters of PKA and the potency of PKA inhibitors can be affected by the sequence of the peptide substrate. The technology enables kinetic monitoring of kinase activity in a multiplex setting such as a cell or tissue lysate. Finally, this high-throughput method allows fast identification of peptide substrates for serine/threonine kinases that are still uncharacterized. [Copyright &y& Elsevier]

Abstract: Phosphatase of Regenerating Liver-3 (PRL-3) is a small protein tyrosine phosphatase considered an appealing therapeutic cancer target due to its involvement in metastatic progression. However, despite its importance, the direct molecular targets of PRL-3 action are not yet known. Here we report the identification of Ezrin as a specific and direct cellular substrate of PRL-3. In HCT116 colon cancer cell line, Ezrin was identified among the cellular proteins whose phosphorylation level decreased upon ectopic over-expression of wtPRL-3 but not of catalytically inactive PRL-3 mutants. Although PRL-3 over-expression in HCT116 cells appeared to affect Ezrin phosphorylation status at both tyrosine residues and Thr567, suppression of the endogenous protein by RNA interference pointed to Ezrin-Thr567 as the residue primarily affected by PRL-3 action. In vitro dephosphorylation assays suggested Ezrin-Thr567 as a direct substrate of PRL-3 also proving this enzyme as belonging to the dual specificity phosphatase family. Furthermore, the same effect on levels of pThr567, but not on pTyr residues, was observed in endothelial cells pointing to Ezrin-pThr567 dephosphorylation as a mean through which PRL-3 exerts its function in promoting tumor progression as well as in the establishment of the new vasculature needed for tumor survival and expansion. [Copyright &y& Elsevier]

Abstract: Protein ligand docking has recently been investigated as a tool for protein function identification, with some success in identifying both known and unknown substrates of proteins. However, identifying a protein''s substrate when cross-docking a large number of enzymes and their cognate ligands remains a challenge. To explore a more limited yet practically important and timely problem in more detail, we have used docking for identifying the substrates of a single protein family with remarkable substrate diversity, the short-chain dehydrogenases/reductases. We examine different protocols for identifying candidate substrates for 27 short-chain dehydrogenase/reductase proteins of known catalytic function. We present the results of docking >900 metabolites from the human metabolome to each of these proteins together with their known cognate substrates and products, and we investigate the ability of docking to (a) reproduce a viable binding mode for the substrate and (b) to rank the substrate highly amongst the dataset of other metabolites. In addition, we examine whether our docking results provide information about the nature of the substrate, based on the best-scoring metabolites in the dataset. We compare two different docking methods and two alternative scoring functions for one of the docking methods, and we attempt to rationalise both successes and failures. Finally, we introduce a new protocol, whereby we dock only a set of representative structures (medoids) to each of the proteins, in the hope of characterising each binding site in terms of its ligand preferences, with a reduced computational cost. We compare the results from this protocol with our original docking experiments, and we find that although the rank of the representatives correlates well with the mean rank of the clusters to which they belong, a simple structure-based clustering is too naïve for the purpose of substrate identification. Many clusters comprise ligands with widely varying affinities for the same protein; hence important candidates can be missed if a single representative is used. [Copyright &y& Elsevier]

The use of RNA interference (RNAi) has provided the study of cell signalling with a specific and relatively easily applicable method of silencing individual components of signalling pathways. RNAi mediated gene muting was combined with two-dimensional electrophoresis (2DE) and immuno-blotting analysis (IB) to study phospho-signalling downstream of the protein kinase, Akt. NIH3T3 mouse fibroblasts were transiently transfected with short interfering RNAs (siRNAs) to Akt with resulting suppression of Akt expression. Down-regulation of Akt reduced the cellular content of phosphorylated FKHR, enhanced GSK3 β phosphorylation, and increased the sensitivity of the cells to apoptotic agents. Stable transfection of short hairpin RNAs inserted into a stable expression vector, pRETRO-SUPER (pRS), also proved to be a successful method of downregulating Akt1, and resulted in an altered phosphorylation pattern and a reduced rate of cell proliferation. Akt-regulated phosphorylation was identified by comparison of extracts from Akt RNAi transfected cells with extracts from cells transfected with pRS vector alone. While Akt muting suppressed some phospho-modifications, others were enhanced. The␣PDGF-induced tyrosine phosphorylation cascades were, surprisingly, found to be influenced by RNAi-mediated Akt muting. Tyrosine phosphorylation of some proteins were reduced in cells with down-regulated Akt1 activity, suggesting the existence of either a downstream tyrosine kinase positively regulated by Akt1, or a negatively regulated tyrosine phosphatase, positioned centrally in the cross-talk between the Akt1 regulated signalling pathways and the growth factor induced phospho-tyrosine pathways. Our results illustrate the analytical potential of combining RNAi-mediated regulator muting with 2DE-based analysis of phospho-signalling, and suggest that such a combination could become a highly efficient tool for the identification and characterisation of new kinase substrates. [ABSTRACT FROM AUTHOR]

Loss-of-function mutations in the secreted enzyme ADAMTS7 (a disintegrin and metalloproteinase with thrombospondin motifs 7) are associated with protection for coronary artery disease. ADAMTS7 catalytic inhibition has been proposed as a therapeutic strategy for treating coronary artery disease; however, the lack of an endogenous substrate has hindered the development of activity-based biomarkers. To identify ADAMTS7 extracellular substrates and their cleavage sites relevant to vascular disease, we used TAILS (terminal amine isotopic labeling of substrates), a method for identifying protease-generated neo-N termini. We compared the secreted proteome of vascular smooth muscle and endothelial cells expressing either full-length mouse ADAMTS7 WT, catalytic mutant ADAMTS7 E373Q, or a control luciferase adenovirus. Significantly enriched N-terminal cleavage sites in ADAMTS7 WT samples were compared to the negative control conditions and filtered for stringency, resulting in catalogs of high confidence candidate ADAMTS7 cleavage sites from our three independent TAILS experiments. Within the overlap of these discovery sets, we identified 24 unique cleavage sites from 16 protein substrates, including cleavage sites in EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1/Fibulin-3). The ADAMTS7 TAILS preference for EFEMP1 cleavage at the amino acids 123.124 over the adjacent 124.125 site was validated using both endogenous EFEMP1 and purified EFEMP1 in a binary in vitro cleavage assay. Collectively, our TAILS discovery experiments have uncovered hundreds of potential substrates and cleavage sites to explore disease-related biological substrates and facilitate activity-based ADAMTS7 biomarker development., Competing Interests: Conflicts of interest B. T. M., N. H. E., and Y. X. are named inventors on patent applications relating to ADAMTS7 assays and compounds. B. T. M., A. A., and N. H. E are named inventors on a patent application relating to ADAMTS7 biomarkers. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is characterized by the invariably progressive deposition of fibrotic tissue in the lungs and overall poor prognosis. TG2 (transglutaminase 2) is an enzyme that crosslinks glutamine and lysine residues and is involved in IPF pathogenesis. Despite the accumulating evidence implicating TG2 as a critical enzyme, the causative function and direct target of TG2 relating to this pathogenesis remain unelucidated. Here, we clarified the distributions of TG2 protein/activity and conducted quantitative proteomics analyses of possible substrates crosslinked by TG2 on unfixed lung sections in a mouse pulmonary fibrosis model. We identified 126 possible substrates as markedly TG2-dependently increased in fibrotic lung. Gene ontology analysis revealed that these identified proteins were mostly enriched in the lipid metabolic process, immune system process, and protein transport. In addition, these proteins were enriched in 21 pathways, including phagosome, lipid metabolism, several immune responses, and protein processing in endoplasmic reticulum. Furthermore, the network analyses screened out the six clusters and top 20 hub proteins with higher scores, which are related to endoplasmic reticulum stress and peroxisome proliferator-activated receptor signals. Several enriched pathways and categories were identified, some of which were the same terms based on transcription analysis in IPF. Our results provide novel pathological molecular networks driven by protein crosslinking via TG2, which can lead to the development of new therapeutic targets for IPF.

Protein phosphorylation is the most widespread type of post-translational modification and is properly controlled by protein kinases and phosphatases. Regarding the phosphorylation of serine (Ser) and threonine (Thr) residues, relatively few protein Ser/Thr phosphatases control the specific dephosphorylation of numerous substrates, in contrast with Ser/Thr kinases. Recently, protein Ser/Thr phosphatases were reported to have rigid substrate recognition and exert various biological functions. Therefore, identification of targeted proteins by individual protein Ser/Thr phosphatases is crucial to clarify their own biological functions. However, to date, information on the development of methods for identification of the substrates of protein Ser/Thr phosphatases remains scarce. In turn, substrate-trapping mutants are powerful tools to search the individual substrates of protein tyrosine (Tyr) phosphatases. This review focuses on the development of novel methods for the identification of Ser/Thr phosphatases, especially small C-terminal domain phosphatase 1 (Scp1), using peptide-displayed phage library with AlF4−/BeF3−, and discusses the identification of putative inhibitors. [ABSTRACT FROM AUTHOR]

The transglutaminase (TGase) family consists of eight isozymes that catalyze Ca2+-dependent crosslink formation between glutamine and lysine residues of proteins. In the pathogenesis of various chronic diseases, among the TGase isozymes, TG2 in particular is upregulated and contributes to a critical role in fibrosis development and progression via the stabilization of extracellular matrix proteins and activation of TGF-β. Although TG2 has been considered a key enzyme in fibrosis, the causative role of TG2 and involvement of other isozymes remain unclear. We have recently developed a comprehensive analysis method targeting the isozyme-specific substrates of TGase in liver and kidney fibrosis. In this review article, we introduce a previously developed method for determining the activity and tissue distribution of TGase and for the detecting and identification of TGase substrates in an isozyme-specific manner. Using our comprehensive analysis method, we newly characterized the overlapping profile data regarding potential substrates of TG1 and TG2 that have been identified in liver and kidney fibrosis to date. Our results obtained by comparing the specificity and similarity of potential TGase substrates between different tissue fibrosis models provide a deeper understanding regarding the specific and common pathways in disease pathogenesis and progression. • Transglutaminases catalyze the crosslink formation between glutamine and lysine residues. • TG2, among transglutaminase family, contribute to a critical role in tissue fibrosis. • We introduce the method for measuring isozyme-specific transglutaminase activity. • We recently developed methods for detection and identification of transglutaminase substrates. • We evaluated the specificity and similarity of possible transglutaminase substrates identified. [ABSTRACT FROM AUTHOR]

The family of radical SAM RNA-methylating enzymes comprises a large group of proteins that contains only a few functionally characterized members. Several enzymes in this family have been implicated in the regulation of translation and antibiotic susceptibility, emphasizing their significance in bacterial physiology and their relevance to human health. While few characterized enzymes have been shown to modify diverse RNA substrates, highlighting potentially broad substrate scope within the family, many enzymes in this class have no known substrates. The precise knowledge of RNA substrates and modification sites for uncharacterized family members is important for unraveling their biological function. Here, we describe a strategy for substrate identification that takes advantage of mechanism-based cross-linking between the enzyme and its RNA substrates, which we named individual-nucleotide-resolution cross-linking and immunoprecipitation combined with mutational profiling with sequencing (miCLIP-MaPseq). Identification of the position of the modification site is achieved using thermostable group II intron reverse transcriptase (TGIRT), which introduces a mismatch at the site of the cross-link.

Specific E3 ligases target tumor suppressors for degradation. Inhibition of such E3 ligases may be an important approach to cancer treatment. RNF146 is a RING domain and PARylation-dependent E3 ligase that functions as an activator of the β-catenin/Wnt and YAP/Hippo pathways by targeting the degradation of several tumor suppressors. Tankyrases 1 and 2 (TNKS1/2) are the only known poly-ADP-ribosyltransferases that require RNF146 to degrade their substrates. However, systematic identification of RNF146 substrates have not yet been performed. To uncover substrates of RNF146 that are targeted for degradation, we generated RNF146 knockout cells and TNKS1/2-double knockout cells and performed proteome profiling with label-free quantification as well as transcriptome analysis. We identified 160 potential substrates of RNF146, which included many known substrates of RNF146 and TNKS1/2 and 122 potential TNKS-independent substrates of RNF146. In addition, we validated OTU domain-containing protein 5 and Protein mono-ADP-ribosyltransferase PARP10 as TNKS1/2-independent substrates of RNF146 and SARDH as a novel substrate of TNKS1/2 and RNF146. Our study is the first proteome-wide analysis of potential RNF146 substrates. Together, these findings not only demonstrate that proteome profiling can be a useful general approach for the systemic identification of substrates of E3 ligases but also reveal new substrates of RNF146, which provides a resource for further functional studies., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Nie et al.)

The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and Clpp -/- mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondria N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP., Competing Interests: Conflict of interest—Authors declare no competing interests., (© 2020 Hofsetz et al.)

Mycobacterial Ser/Thr protein kinases (STPKs) play a critical role in signal transduction pathways that ultimately determine mycobacterial growth and metabolic adaptation. Identification of key physiological substrates of these protein kinases is, therefore, crucial to better understand how Ser/Thr phosphorylation contributes to mycobacterial environmental adaptation, including response to stress, cell division, and host-pathogen interactions. Various substrate detection methods have been employed with limited success, with direct targets of STPKs remaining elusive. Recently developed mass spectrometry (MS)-based phosphoproteomic approaches have expanded the list of potential STPK substrate identifications, yet further investigation is required to define the most functionally significant phosphosites and their physiological importance. Prior to the application of MS workflows, for instance, GarA was the only known and validated physiological substrate for protein kinase G (PknG) from pathogenic mycobacteria. A subsequent list of at least 28 candidate PknG substrates has since been reported with the use of MS-based analyses. Herein, we integrate and critically review MS-generated datasets available on novel STPK substrates and report new functional and subcellular localization enrichment analyses on novel candidate protein kinase A (PknA), protein kinase B (PknB) and PknG substrates to deduce the possible physiological roles of these kinases. In addition, we assess substrate specificity patterns across different mycobacterial STPKs by analyzing reported sets of phosphopeptides, in order to determine whether novel motifs or consensus regions exist for mycobacterial Ser/Thr phosphorylation sites. This review focuses on MS-based techniques employed for STPK substrate identification in mycobacteria, while highlighting the advantages and challenges of the various applications., (© 2020 Baros et al.)

Here we describe the use of a decellularized ECM produced in vitro by BALB/c 3T3 fibroblasts for the identification of ADAMTS substrates. Seeding of ADAMTS protease-producing HEK cells on top of the cell-free ECM followed by analysis of the conditioned medium by liquid chromatography tandem mass spectrometry (LC-MS/MS), allows for screening of ADAMTS substrates without prior purification of full-length protease.

Cell surface proteolysis controls numerous biological processes including cell-cell attachment and the communication between cells. The membrane-tethered families of matrix metalloproteinases (MT-MMPs) and disintegrin metalloproteinases (ADAMs) are major enzymes involved in the cleavage of molecules at the cell surface, and their activity is finely regulated by their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). The biological function of a metalloproteinase closely depends on the subset of substrates that it cleaves. Similarly, molecular processes that are regulated by a specific TIMP strictly depend on its unique inhibitory profile.Herein, we describe a mass spectrometry-based method for the quantitative analysis of protein abundance in conditioned media of cultured cells that is particularly suited for substrate identification of membrane-tethered metalloproteinases and for the identification of membrane proteins whose cleavage is regulated by TIMPs. This unbiased proteomic method represents a valuable tool to investigate biological functions of metalloproteinases and TIMPs at the "omic" level.

Protein N termini unambiguously identify truncated, alternatively translated or modified proteoforms with distinct functions and reveal perturbations in disease. Selective enrichment of N-terminal peptides is necessary to achieve proteome-wide coverage for unbiased identification of site-specific regulatory proteolytic processing and protease substrates. However, many proteolytic processes are strictly confined in time and space and therefore can only be analyzed in minute samples that provide insufficient starting material for current enrichment protocols. Here we present High-efficiency Undecanal-based N Termini EnRichment (HUNTER), a robust, sensitive and scalable method for the analysis of previously inaccessible microscale samples. HUNTER achieved identification of >1000 N termini from as little as 2 μg raw HeLa cell lysate. Broad applicability is demonstrated by the first N-terminome analysis of sorted human primary immune cells and enriched mitochondrial fractions from pediatric cancer patients, as well as protease substrate identification from individual Arabidopsis thaliana wild type and Vacuolar Processing Enzyme-deficient mutant seedlings. We further implemented the workflow on a liquid handling system and demonstrate the feasibility of clinical degradomics by automated processing of liquid biopsies from pediatric cancer patients., (© 2019 Weng et al.)

Vascular endothelial protein tyrosine phosphatase (VE-PTP, PTPRB) is a receptor type phosphatase that is crucial for the regulation of endothelial junctions and blood vessel development. We and others have shown recently that VE-PTP regulates vascular integrity by dephosphorylating substrates that are key players in endothelial junction stability, such as the angiopoietin receptor TIE2, the endothelial adherens junction protein VE-cadherin and the vascular endothelial growth factor receptor VEGFR2. Here, we have systematically searched for novel substrates of VE-PTP in endothelial cells by utilizing two approaches. First, we studied changes in the endothelial phosphoproteome on exposing cells to a highly VE-PTP-specific phosphatase inhibitor followed by affinity isolation and mass-spectrometric analysis of phosphorylated proteins by phosphotyrosine-specific antibodies. Second, we used a substrate trapping mutant of VE-PTP to pull down phosphorylated substrates in combination with SILAC-based quantitative mass spectrometry measurements. We identified a set of substrate candidates of VE-PTP, of which a remarkably large fraction (29%) is related to cell junctions. Several of those were found in both screens and displayed very high connectivity in predicted functional interaction networks. The receptor protein tyrosine kinase EPHB4 was the most prominently phosphorylated protein on VE-PTP inhibition among those VE-PTP targets that were identified by both proteomic approaches. Further analysis revealed that EPHB4 forms a ternary complex with VE-PTP and TIE2 in endothelial cells. VE-PTP controls the phosphorylation of each of these two tyrosine kinase receptors. Despite their simultaneous presence in a ternary complex, stimulating each of the receptors with their own specific ligand did not cross-activate the respective partner receptor. Our systematic approach has led to the identification of novel substrates of VE-PTP, of which many are relevant for the control of cellular junctions further promoting the importance of VE-PTP as a key player of junctional signaling., (© 2019 Drexler et al.)

Kallikrein-related peptidase 7 (KLK7) is a serine peptidase that is over expressed in ovarian cancer. In vitro functional analyses have suggested KLK7 to play a cancer progressive role, although monitoring of KLK7 expression has suggested a contradictory protective role for KLK7 in ovarian cancer patients. In order to help delineate its mechanism of action and thereby the functional roles, information on its substrate repertoire is crucial. Therefore, in this study a quantitative proteomics approach-PROtein TOpography and Migration Analysis Platform (PROTOMAP)-coupled with SILAC was used for in-depth analysis of putative KLK7 substrates from a representative ovarian cancer cell line, SKOV-3, secreted proteins. The Terminal Amine Isotopic Labeling of Substrates (TAILS) approach was used to determine the exact cleavage sites and to validate qPROTOMAP-identified putative substrates. By employing these two technically divergent approaches, exact cleavage sites on 16 novel putative substrates and two established substrates, matrix metalloprotease (MMP) 2 and insulin growth factor binding protein 3 (IGFBP3), were identified in the SKOV-3 secretome. Eight of these substrates were also identified on TAILS analysis of another ovarian cancer cell (OVMZ-6) secretome, with a further seven OVMZ-6 substrates common to the SKOV-3 qPROTOMAP profile. Identified substrates were significantly associated with the common processes of cell adhesion, extracellular matrix remodeling and cell migration according to the gene ontology (GO) biological process analysis. Biochemical validation supports a role for KLK7 in directly activating pro-MMP10, hydrolysis of IGFBP6 and cleavage of thrombospondin 1 with generation of a potentially bioactive N-terminal fragment. Overall, this study constitutes the most comprehensive analysis of the putative KLK7 degradome in any cancer to date, thereby opening new avenues for KLK7 research., (© 2019 Silva et al.)

Acute myeloid leukemia (AML) is an aggressive disease that is characterized by abnormal increase of immature myeloblasts in blood and bone marrow. The FLT3 receptor tyrosine kinase plays an integral role in hematopoiesis, and one third of AML diagnoses exhibit gain-of-function mutations in FLT3, with the juxtamembrane domain internal tandem duplication (ITD) and the kinase domain D835Y variants observed most frequently. Few FLT3 substrates or phosphorylation sites are known, which limits insight into FLT3's substrate preferences and makes assay design particularly challenging. We applied in vitro phosphorylation of a cell lysate digest (adaptation of the Kinase Assay Linked with Phosphoproteomics (KALIP) technique and similar methods) for high-throughput identification of substrates for three FLT3 variants (wild-type, ITD mutant, and D835Y mutant). Incorporation of identified substrate sequences as input into the KINATEST-ID substrate preference analysis and assay development pipeline facilitated the design of several peptide substrates that are phosphorylated efficiently by all three FLT3 kinase variants. These substrates could be used in assays to identify new FLT3 inhibitors that overcome resistant mutations to improve FLT3-positive AML treatment., Competing Interests: Conflict of interest: Dr. Laurie Parker owns equity in and serves on the Scientific Advisory Board for KinaSense, LLC. The University of Minnesota and Purdue University reviews and manages this relationship in accordance with their conflict of interest policies., (© 2019 Perez et al.)

The endoplasmic reticulum (ER), as a multifunctional organelle, plays crucial roles in lipid biosynthesis and calcium homeostasis as well as the synthesis and folding of secretory and membrane proteins. Therefore, it is of high importance to maintain ER homeostasis and to adapt ER function and morphology to cellular needs. Here, we show that signal peptide peptidase (SPP) modulates the ER shape through degradation of morphogenic proteins. Elevating SPP activity induces rapid rearrangement of the ER and formation of dynamic ER clusters. Inhibition of SPP activity rescues the phenotype without the need for new protein synthesis, and this rescue depends on a pre-existing pool of proteins in the Golgi. With the help of organelle proteomics, we identified certain membrane proteins to be diminished upon SPP expression and further show that the observed morphology changes depend on SPP-mediated cleavage of ER morphogenic proteins, including the SNARE protein syntaxin-18. Thus, we suggest that SPP-mediated protein abundance control by a regulatory branch of ER-associated degradation (ERAD-R) has a role in shaping the early secretory pathway., (© 2019 Avci et al.)