scholarly journals Deciphering and engineering chromodomain-methyllysine peptide recognition

2018 ◽  
Vol 4 (11) ◽  
pp. eaau1447 ◽  
Author(s):  
Ryan Hard ◽  
Nan Li ◽  
Wei He ◽  
Brian Ross ◽  
Gary C. H. Mo ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Large Scale ◽  
Regulation Of Gene Expression ◽  
Live Cells ◽  
Lysine Methylation ◽  
Dynamic Regulation ◽  
Peptide Recognition ◽  
Protein Functions ◽  
Domain Peptide

Posttranslational modifications (PTMs) play critical roles in regulating protein functions and mediating protein-protein interactions. An important PTM is lysine methylation that orchestrates chromatin modifications and regulates functions of non-histone proteins. Methyllysine peptides are bound by modular domains, of which chromodomains are representative. Here, we conducted the first large-scale study of chromodomains in the human proteome interacting with both histone and non-histone methyllysine peptides. We observed significant degenerate binding between chromodomains and histone peptides, i.e., different histone sites can be recognized by the same set of chromodomains, and different chromodomains can share similar binding profiles to individual histone sites. Such degenerate binding is not dictated by amino acid sequence or PTM motif but rather rooted in the physiochemical properties defined by the PTMs on the histone peptides. This molecular mechanism is confirmed by the accurate prediction of the binding specificity using a computational model that captures the structural and energetic patterns of the domain-peptide interaction. To further illustrate the power and accuracy of our model, we used it to effectively engineer an exceptionally strong H3K9me3-binding chromodomain and to label H3K9me3 in live cells. This study presents a systematic approach to deciphering domain-peptide recognition and reveals a general principle by which histone modifications are interpreted by reader proteins, leading to dynamic regulation of gene expression and other biological processes.

scholarly journals Chemical toolbox for ‘live’ biochemistry to understand enzymatic functions in living systems

2019 ◽  
Author(s):  
Toru Komatsu ◽  
Yasuteru Urano
Keyword(s):  
Environmental Factors ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Live Cell ◽  
Living Systems ◽  
Protein Protein Interactions ◽  
Redox States ◽  
Recent Advances ◽  
Protein Functions ◽  
Research Tools

Abstract In this review, we present an overview of the recent advances in chemical toolboxes that are used to provide insights into ‘live’ protein functions in living systems. Protein functions are mediated by various factors inside of cells, such as protein−protein interactions, posttranslational modifications, and they are also subject to environmental factors such as pH, redox states and crowding conditions. Obtaining a true understanding of protein functions in living systems is therefore a considerably difficult task. Recent advances in research tools have allowed us to consider ‘live’ biochemistry as a valid approach to precisely understand how proteins function in a live cell context.

scholarly journals An ultrasensitive fiveplex activity assay for cellular kinases

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Christian M. Smolko ◽  
Kevin A. Janes
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Large Scale ◽  
Cell Types ◽  
Mitogen Activated Protein Kinase ◽  
Activity Measurement ◽  
Kinase Substrate ◽  
Iκb Kinase ◽  
Mitogen Activated Protein

AbstractProtein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 µg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt–IKK–JNK–MEK–MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.

scholarly journals Protein SUMOylation is crucial for phagocytosis in Entamoeba histolytica trophozoites

2021 ◽  
Author(s):  
Mitzi Díaz-Hernández ◽  
Rosario Javier Reyna ◽  
Izaid Sotto-Ortega ◽  
Guillermina García-Rivera ◽  
Maricela Sarita Montaño ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Covalent Binding ◽  
3D Structure ◽  
Fine Tuning ◽  
Binding Motif ◽  
C Terminus ◽  
Protein Functions ◽  
Target Molecules

AbstractDuring phagocytosis, a key event in the virulence of the protozoan Entamoeba histolytica, several molecules in concert contact the target, generate pseudopodia, and internalize and digest the ingested prey. Posttranslational modifications provide proteins the timing and signaling to intervene in these processes. SUMOylation is a posttranslational modification that in several systems grants a fine tuning for protein functions, protein interactions and cellular location, but it has not been studied in E. histolytica. In this paper, we characterized the E. histolytica SUMO gene and its product (EhSUMO) and elucidated the EhSUMO 3D-structure. Furthermore, here we studied the relevance of SUMOylation in phagocytosis, particularly in its association with EhADH (an ALIX family protein) and EhVps32 (a protein of the ESCRT-III complex), both involved in phagocytosis. Our results indicated that EhSUMO has an extended N-terminus that differentiates other SUMO from ubiquitin. It also presents the GG residues at the C-terminus and the ΨKXE/D binding motif, both involved in target protein contact. Additionally, E. histolytica genome possesses the enzymes belonging to the SUMOylation-deSUMOylation machineries. Confocal microscopy assays, using α−EhSUMO antibodies disclosed a remarkable membrane activity with convoluted and changing structures in trophozoites during erythrophagocytosis. SUMOylated proteins appeared in pseudopodia, phagocytic channels, and around the adhered and ingested erythrocytes. Docking analysis predicted interaction of EhSUMO with EhADH, and immunoprecipitation and immunofluorescence assays revealed that the EhADH-EhSUMO association increased during phagocytosis, whereas the EhVps32-EhSUMO interaction appeared stronger since basal conditions. In EhSUMO knocked down trophozoites, the bizarre membranous structures disappeared, and EhSUMO interaction with EhADH and EhVps32 diminished. Our results evidenced the presence of a SUMO gene in E. histolytica and the SUMOylation relevance during phagocytosis.Author’s AbstractPhagocytosis is one of the main functions that Entamoeba histolyitica trophozoites carry out during the invasion to the host. Many proteins are involved in this fascinating event, in which the plasmatic membrane undergoes to multiple and speedy changes. Posttraductional modifications activate proteins in the precise time that they must get involved. SUMOylation, that consists in the non-covalent binding of SUMO protein with target molecules, is one of the main changes suffered by proteins in order to enable them to participate in cellular functions. SUMOylation had not been studied in E. histolytica nor in phagocytosis, and our working hypothesis is that this event is deeply engaged in the ingestion of target molecules and cells. The results of this paper prove the presence of an intronless bona fide EhSUMO gene encoding for a predicted 12.6 kDa protein that is actively involved in phagocytosis. Silencing of the EhSUMO gene affected the rate of phagocytosis and interfered with the EhADH and EhVps32 function, two proteins involved in phagocytosis, strongly supporting the importance of SUMOylation in this event.

scholarly journals Systematic discovery of mutation-directed neo-protein-protein interactions in cancer

2021 ◽  
Author(s):  
Xiulei Mo ◽  
Qiankun Niu ◽  
Andrey A Ivanov ◽  
Yiu Huen Tsang ◽  
Cong Tang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Large Scale ◽  
Braf V600e ◽  
Driver Mutations ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Mutant Variant ◽  
Signaling Axis ◽  
Tumorigenesis And Progression ◽  
Associated Proteins

Comprehensive sequencing of patient tumors reveals numerous genomic mutations across tumor types that enable tumorigenesis and progression. A subset of oncogenic driver mutations results in neomorphic activity where the mutant protein mediates functions not engaged by the parental molecule. Here, we identify prevalent variant-enabled neomorph-protein-protein interactions (neoPPI) with a quantitative High Throughput differential Screening (qHT-dS) platform. Coupling of highly sensitive BRET biosensors with miniaturized co-expression in an ultra-HTS format allows large-scale monitoring of interactions of wild-type and mutant variant counterparts with a library of cancer-associated proteins in live cells. Screening of 13,392 interactions with 1,474,560 data points revealed a landscape of gain-of-interactions encompassing both oncogenic and tumor suppressor mutations. For example, the recurrent BRAF V600E lesion mediates KEAP1 neoPPI, rewiring a BRAFV600E-KEAP1 signaling axis and creating collateral vulnerability to NQO1 substrates, offering a combination therapeutic strategy. Thus, cancer genomic alterations can create neo-interactions, informing variant-directed therapeutic approaches for precision medicine.

scholarly journals Image-based high-throughput mapping of TGF-β-induced phosphocomplexes at a single-cell level

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Peter Lönn ◽  
Rasel A. Al-Amin ◽  
Ehsan Manouchehri Doulabi ◽  
Johan Heldin ◽  
Radiosa Gallini ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Large Scale ◽  
Cell Cycle Progression ◽  
Drug Effects ◽  
Automated System ◽  
Phosphatase Inhibitors ◽  
Dynamic Events

AbstractProtein interactions and posttranslational modifications orchestrate cellular responses to e.g. cytokines and drugs, but it has been difficult to monitor these dynamic events in high-throughput. Here, we describe a semi-automated system for large-scale in situ proximity ligation assays (isPLA), combining isPLA in microtiter wells with automated microscopy and computer-based image analysis. Phosphorylations and interactions are digitally recorded along with subcellular morphological features. We investigated TGF-β-responsive Smad2 linker phosphorylations and complex formations over time and across millions of individual cells, and we relate these events to cell cycle progression and local cell crowding via measurements of DNA content and nuclear size of individual cells, and of their relative positions. We illustrate the suitability of this protocol to screen for drug effects using phosphatase inhibitors. Our approach expands the scope for image-based single cell analyses by combining observations of protein interactions and modifications with morphological details of individual cells at high throughput.

scholarly journals Sperm Proteomics: Road to Male Fertility and Contraception

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Md Saidur Rahman ◽  
June-Sub Lee ◽  
Woo-Sung Kwon ◽  
Myung-Geol Pang
Keyword(s):  
Posttranslational Modifications ◽  
Gel Electrophoresis ◽  
Male Fertility ◽  
Large Scale ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Biomarkers ◽  
Scientific Publications ◽  
Pubmed Database ◽  
Protein Functions ◽  
Specific Proteins

Spermatozoa are highly specialized cells that can be easily obtained and purified. Mature spermatozoa are transcriptionally and translationally inactive and incapable of protein synthesis. In addition, spermatozoa contain relatively higher amounts of membrane proteins compared to other cells; therefore, they are very suitable for proteomic studies. Recently, the application of proteomic approaches such as the two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential in-gel electrophoresis has identified several sperm-specific proteins. These findings have provided a further understanding of protein functions involved in different sperm processes as well as of the differentiation of normal state from an abnormal one. In addition, studies on the sperm proteome have demonstrated the importance of spermatozoal posttranslational modifications and their ability to induce physiological changes responsible for fertilization. Large-scale proteomic studies to identify hundreds to thousands of sperm proteins will ultimately result in the development of novel biomarkers that may help to detect fertility, the state of complete contraception, and beyond. Eventually, these protein biomarkers will allow for a better diagnosis of sperm dysfunctions and aid in drug development. This paper reviews the recent scientific publications available from the PubMed database to address sperm proteomics and its potential application to characterize male fertility and contraception.

scholarly journals An ultrasensitive fiveplex activity assay for cellular kinases

2019 ◽  
Author(s):  
Christian M. Smolko ◽  
Kevin A. Janes
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Large Scale ◽  
Cell Types ◽  
Mitogen Activated Protein Kinase ◽  
Activity Measurement ◽  
Kinase Substrate ◽  
Iκb Kinase ◽  
Mitogen Activated Protein

ABSTRACTProtein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 μg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt–IKK–JNK–MEK–MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.

scholarly journals Histone arginine methylations: their roles in chromatin dynamics and transcriptional regulation

2009 ◽  
Vol 29 (2) ◽  
pp. 131-141 ◽  
Author(s):  
Michael Litt ◽  
Yi Qiu ◽  
Suming Huang
Keyword(s):  
Transcriptional Repression ◽  
Regulation Of Gene Expression ◽  
Arginine Methylation ◽  
Nuclear Proteins ◽  
Lysine Methylation ◽  
Histone Tails ◽  
Dynamic Regulation ◽  
Chromatin Dynamics ◽  
Arginine Residues ◽  
Histone Arginine Methylation

PRMTs (protein arginine N-methyltransferases) specifically modify the arginine residues of key cellular and nuclear proteins as well as histone substrates. Like lysine methylation, transcriptional repression or activation is dependent upon the site and type of arginine methylation on histone tails. Recent discoveries imply that histone arginine methylation is an important modulator of dynamic chromatin regulation and transcriptional controls. However, under the shadow of lysine methylation, the roles of histone arginine methylation have been under-explored. The present review focuses on the roles of histone arginine methylation in the regulation of gene expression, and the interplays between histone arginine methylation, histone acetylation, lysine methylation and chromatin remodelling factors. In addition, we discuss the dynamic regulation of arginine methylation by arginine demethylases, and how dysregulation of PRMTs and their activities are linked to human diseases such as cancer.

scholarly journals Optimized cross-linking mass spectrometry for in situ interaction proteomics

2018 ◽  
Author(s):  
Zheng Ser ◽  
Paolo Cifani ◽  
Alex Kentsis
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Large Scale ◽  
Electron Transfer Dissociation ◽  
Live Cells ◽  
Cross Linking ◽  
Native Protein ◽  
Wide Range

AbstractRecent development of mass spectrometer cleavable protein cross-linkers and algorithms for their spectral identification now permits large-scale cross-linking mass spectrometry (XL-MS). Here, we optimized the use of cleavable disuccinimidyl sulfoxide (DSSO) cross-linker for labeling native protein complexes in live human cells. We applied a generalized linear mixture model to calibrate cross-link peptide-spectra matching (CSM) scores to control the sensitivity and specificity of large-scale XL-MS. Using specific CSM score thresholds to control the false discovery rate, we found that higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) can both be effective for large-scale XL-MS protein interaction mapping. We found that the density and coverage of protein-protein interaction maps can be significantly improved through the use of multiple proteases. In addition, the use of sample-specific search databases can be used to improve the specificity of cross-linked peptide spectral matching. Application of this approach to human chromatin labeled in live cells recapitulated known and revealed new protein interactions of nucleosomes and other chromatin-associated complexes in situ. This optimized approach for mapping native protein interactions should be useful for a wide range of biological problems.

Target-Templated de novo Design of Macrocyclic D-/L-Peptides: Inhibitors of the PD-1/PD-L1 Interaction

2020 ◽  
Author(s):  
Salvador Guardiola ◽  
Monica Varese ◽  
Xavier Roig ◽  
Jesús Garcia ◽  
Ernest Giralt
Keyword(s):  
Protein Interactions ◽  
Cyclic Peptides ◽  
General Framework ◽  
Large Scale ◽  
De Novo ◽  
Inhibitory Effect ◽  
Original Text ◽  
Protein Protein Interactions ◽  
Retraction Notice ◽  
Pharmaceutical Properties

<p>NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.</p><p><br></p><p>------------------------------------------------------------------------</p><p><br></p><p>Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-<i>i</i>3 and PD-<i>i</i>6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our <i>de novo </i>design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.<i></i></p>

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