Synthesis and Evaluation of Peptidic Thrombin Inhibitors Bearing Acid-Stable Sulfotyrosine Analogues

2021 ◽  
Author(s):  
Luke James Dowman ◽  
Stijn Agten ◽  
Jorge Ripoll-Rozada ◽  
Bárbara M Calisto ◽  
Pedro José Barbosa Pereira ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Thrombin Inhibitors ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Tyrosine Sulfation ◽  
Acid Stable

Tyrosine sulfation is an important post-translational modification of peptides and proteins which underpins and modulates many protein-protein interactions. In order to overcome the inherent instability of the native modification, we...

scholarly journals New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors

2018 ◽  
Vol 475 (15) ◽  
pp. 2435-2455 ◽  
Author(s):  
Dominic P. Byrne ◽  
Yong Li ◽  
Pawin Ngamlert ◽  
Krithika Ramakrishnan ◽  
Claire E. Eyers ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitors ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Tyrosine Sulfation ◽  
Protein Tyrosine

Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular protein–protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based inhibitors of the Ser/Thr kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro. We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors.

scholarly journals Androgen signaling uses a writer and a reader of ADP-ribosylation to regulate protein complex assembly

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chun-Song Yang ◽  
Kasey Jividen ◽  
Teddy Kamata ◽  
Natalia Dworak ◽  
Luke Oostdyk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Prostate Cancer Cells ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Complex Assembly ◽  
Adp Ribosylation ◽  
Signaling Axis ◽  
Regulate Protein ◽  
Protein Complex Assembly

AbstractAndrogen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity.

scholarly journals The Protein Interactome of Glycolysis in Escherichia coli

Proteomes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 16
Author(s):  
Shomeek Chowdhury ◽  
Stephen Hepper ◽  
Mudassir K. Lodi ◽  
Milton H. Saier ◽  
Peter Uetz
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Allosteric Regulation ◽  
Glycolytic Enzymes ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Interacting Protein ◽  
E Coli ◽  
Protein Interactome ◽  
The Impact

Glycolysis is regulated by numerous mechanisms including allosteric regulation, post-translational modification or protein-protein interactions (PPI). While glycolytic enzymes have been found to interact with hundreds of proteins, the impact of only some of these PPIs on glycolysis is well understood. Here we investigate which of these interactions may affect glycolysis in E. coli and possibly across numerous other bacteria, based on the stoichiometry of interacting protein pairs (from proteomic studies) and their conservation across bacteria. We present a list of 339 protein-protein interactions involving glycolytic enzymes but predict that ~70% of glycolytic interactors are not present in adequate amounts to have a significant impact on glycolysis. Finally, we identify a conserved but uncharacterized subset of interactions that are likely to affect glycolysis and deserve further study.

Tyrosine-O-sulfation is a widespread affinity enhancer among thrombin interactors

2022 ◽  
Author(s):  
Jorge Ripoll-Rozada ◽  
Joshua W. C. Maxwell ◽  
Richard J. Payne ◽  
Pedro José Barbosa Pereira
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Serine Proteinase ◽  
Blood Feeding ◽  
Thrombin Inhibitor ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Human Fibrinogen ◽  
Clotting Cascade ◽  
Bona Fide

Tyrosine-O-sulfation is a common post-translational modification (PTM) of proteins following the cellular secretory pathway. First described in human fibrinogen, tyrosine-O-sulfation has long been associated with the modulation of protein–protein interactions in several physiological processes. A number of relevant interactions for hemostasis are largely dictated by this PTM, many of which involving the serine proteinase thrombin (FIIa), a central player in the blood-clotting cascade. Tyrosine sulfation is not limited to endogenous FIIa ligands and has also been found in hirudin, a well-known and potent thrombin inhibitor from the medicinal leech, Hirudo medicinalis. The discovery of hirudin led to successful clinical application of analogs of leech-inspired molecules, but also unveiled several other natural thrombin-directed anticoagulant molecules, many of which undergo tyrosine-O-sulfation. The presence of this PTM has been shown to enhance the anticoagulant properties of these peptides from a range of blood-feeding organisms, including ticks, mosquitos and flies. Interestingly, some of these molecules display mechanisms of action that mimic those of thrombin's bona fide substrates.

scholarly journals SET7/9-dependent methylation of ARTD1 at K508 stimulates poly-ADP-ribose formation after oxidative stress

Open Biology ◽  
2013 ◽  
Vol 3 (10) ◽  
pp. 120173 ◽  
Author(s):  
Ingrid Kassner ◽  
Anneli Andersson ◽  
Monika Fey ◽  
Martin Tomas ◽  
Elisa Ferrando-May ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Interactions ◽  
Protein Function ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Target Proteins ◽  
Protein Methyltransferase

ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein–protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo . ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo . Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.

Applications of Mass Spectrometry in Proteomics

2013 ◽  
Vol 66 (7) ◽  
pp. 721 ◽  
Author(s):  
Izabela Sokolowska ◽  
Armand G. Ngounou Wetie ◽  
Alisa G. Woods ◽  
Costel C. Darie
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Identification ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Cellular Processes ◽  
Physiological Processes ◽  
Protein Functions ◽  
Instruments And Techniques ◽  
Accurate Mass Spectrometry

Characterisation of proteins and whole proteomes can provide a foundation to our understanding of physiological and pathological states and biological diseases or disorders. Constant development of more reliable and accurate mass spectrometry (MS) instruments and techniques has allowed for better identification and quantification of the thousands of proteins involved in basic physiological processes. Therefore, MS-based proteomics has been widely applied to the analysis of biological samples and has greatly contributed to our understanding of protein functions, interactions, and dynamics, advancing our knowledge of cellular processes as well as the physiology and pathology of the human body. This review will discuss current proteomic approaches for protein identification and characterisation, including post-translational modification (PTM) analysis and quantitative proteomics as well as investigation of protein–protein interactions (PPIs).

scholarly journals PathwayMatcher: proteoform-centric network construction enables fine-granularity multi-omics pathway mapping

2018 ◽  
Author(s):  
Luis Francisco Hernández Sánchez ◽  
Bram Burger ◽  
Carlos Horro ◽  
Antonio Fabregat ◽  
Stefan Johansson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Biomedical Data ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Network Construction ◽  
Gene Sets ◽  
Functional Knowledge ◽  
Pathway Analyses ◽  
Different Levels

AbstractBackgroundMapping biomedical data to functional knowledge is an essential task in bioinformatics and can be achieved by querying identifiers, e.g. gene sets, in pathway knowledgebases. However, the isoform and post-translational modification states of proteins are lost when converting input and pathways into gene-centric lists.FindingsBased on the Reactome knowledgebase, we built a network of protein-protein interactions accounting for the documented isoform and modification statuses of proteins. We then implemented a command line application called PathwayMatcher (github.com/PathwayAnalysisPlatform/PathwayMatcher) to query this network. PathwayMatcher supports multiple types of omics data as input, and outputs the possibly affected biochemical reactions, subnetworks, and pathways.ConclusionsPathwayMatcher enables refining the network-representation of pathways by including isoform and post-translational modifications. The specificity of pathway analyses is hence adapted to different levels of granularity and it becomes possible to distinguish interactions between different forms of the same protein.

scholarly journals Assumptions of biological measurements: important considerations when evaluating western blot data

2021 ◽  
Author(s):  
Maxwell DeNies ◽  
Allen Liu ◽  
Santiago Schnell
Keyword(s):  
Cell Signaling ◽  
Western Blotting ◽  
Protein Interactions ◽  
Data Interpretation ◽  
Biological Processes ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Western Blots ◽  
Additional Information ◽  
Experimental Variability

As technological and analytical innovations rapidly advance our ability to reveal increasingly complex biological processes, the importance of understanding the assumptions behind biological measurements and sources of uncertainty are essential for data interpretation. This is particularly important in fields such as cell signaling, as due to its importance for both homeostatic and pathogenic biological processes, a quantitative understanding of the basic mechanisms of these transient events is fundamental to drug development. While developed decades ago, western blotting remains an indispensible research tool to probe cell signaling, protein expression, and protein-protein interactions. While improvements in statistical and methodology reporting have improved data quality, understanding the basic experimental assumptions and visual inspection of western blots provides additional information that is useful when evaluating experimental conclusions. Using agonist-induced receptor post-translational modification as an example we highlight the assumptions of western blotting and showcase how clues from raw western blots can hint at experimental variability that is not captured by statistics and methods that influences quantification. The purpose of this article is not to serve as a detailed review of the technical nuances and caveats of western blotting. Instead using an example we illustrate how experimental assumptions, design, and data normalization can be identified in raw data and influence data interpretation.

scholarly journals Arf•GAPs as Regulators of the Actin Cytoskeleton—An Update

2019 ◽  
Vol 20 (2) ◽  
pp. 442 ◽  
Author(s):  
Christine Tanna ◽  
Louisa Goss ◽  
Calvin Ludwig ◽  
Pei-Wen Chen
Keyword(s):  
Protein Interactions ◽  
Cell Function ◽  
Critical Role ◽  
Focal Adhesions ◽  
Actin Dynamics ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Binding Domains ◽  
Multiple Protein

Arf•GTPase-activating proteins (Arf•GAPs) control the activity of ADP-ribosylation factors (Arfs) by inducing GTP hydrolysis and participate in a diverse array of cellular functions both through mechanisms that are dependent on and independent of their Arf•GAP activity. A number of these functions hinge on the remodeling of actin filaments. Accordingly, some of the effects exerted by Arf•GAPs involve proteins known to engage in regulation of the actin dynamics and architecture, such as Rho family proteins and nonmuscle myosin 2. Circular dorsal ruffles (CDRs), podosomes, invadopodia, lamellipodia, stress fibers and focal adhesions are among the actin-based structures regulated by Arf•GAPs. Arf•GAPs are thus important actors in broad functions like adhesion and motility, as well as the specialized functions of bone resorption, neurite outgrowth, and pathogen internalization by immune cells. Arf•GAPs, with their multiple protein-protein interactions, membrane-binding domains and sites for post-translational modification, are good candidates for linking the changes in actin to the membrane. The findings discussed depict a family of proteins with a critical role in regulating actin dynamics to enable proper cell function.

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