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The Methods Employed in Mass Spectrometric Analysis of Posttranslational Modifications (PTMs) and Protein-Protein Interactions (PPIs).
Yakubu, RR, Nieves, E, Weiss, LM
Advances in experimental medicine and biology. 2019;:169-198
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Abstract
Mass Spectrometry (MS) has revolutionized the way we study biomolecules, especially proteins, their interactions and posttranslational modifications (PTM). As such MS has established itself as the leading tool for the analysis of PTMs mainly because this approach is highly sensitive, amenable to high throughput and is capable of assigning PTMs to specific sites in the amino acid sequence of proteins and peptides. Along with the advances in MS methodology there have been improvements in biochemical, genetic and cell biological approaches to mapping the interactome which are discussed with consideration for both the practical and technical considerations of these techniques. The interactome of a species is generally understood to represent the sum of all potential protein-protein interactions. There are still a number of barriers to the elucidation of the human interactome or any other species as physical contact between protein pairs that occur by selective molecular docking in a particular spatiotemporal biological context are not easily captured and measured.PTMs massively increase the complexity of organismal proteomes and play a role in almost all aspects of cell biology, allowing for fine-tuning of protein structure, function and localization. There are an estimated 300 PTMS with a predicted 5% of the eukaryotic genome coding for enzymes involved in protein modification, however we have not yet been able to reliably map PTM proteomes due to limitations in sample preparation, analytical techniques, data analysis, and the substoichiometric and transient nature of some PTMs. Improvements in proteomic and mass spectrometry methods, as well as sample preparation, have been exploited in a large number of proteome-wide surveys of PTMs in many different organisms. Here we focus on previously published global PTM proteome studies in the Apicomplexan parasites T. gondii and P. falciparum which offer numerous insights into the abundance and function of each of the studied PTM in the Apicomplexa. Integration of these datasets provide a more complete picture of the relative importance of PTM and crosstalk between them and how together PTM globally change the cellular biology of the Apicomplexan protozoa. A multitude of techniques used to investigate PTMs, mostly techniques in MS-based proteomics, are discussed for their ability to uncover relevant biological function.
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Protein-protein interactions: scoring schemes and binding affinity.
Gromiha, MM, Yugandhar, K, Jemimah, S
Current opinion in structural biology. 2017;:31-38
Abstract
Protein-protein interactions mediate several cellular functions, which can be understood from the information obtained using the three-dimensional structures of protein-protein complexes and binding affinity data. This review focuses on computational aspects of predicting the best native-like complex structure and binding affinities. The first part covers the prediction of protein-protein complex structures and the advantages of conformational searching and scoring functions in protein-protein docking. The second part is devoted to various aspects of protein-protein interaction thermodynamics, such as databases for binding affinities and other thermodynamic parameters, computational methods to predict the binding affinity using either the three-dimensional structures of complexes or amino acid sequences, and change in binding affinities of the complexes upon mutations. We provide the latest developments on protein-protein docking and binding affinity studies along with a list of available computational resources for understanding protein-protein interactions.
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Advances in protein complex analysis by chemical cross-linking coupled with mass spectrometry (CXMS) and bioinformatics.
Tran, BQ, Goodlett, DR, Goo, YA
Biochimica et biophysica acta. 2016;(1):123-9
Abstract
For the analysis of protein-protein interactions and protein conformations, cross-linking coupled with mass spectrometry (CXMS) has become an essential tool in recent years. A variety of cross-linking reagents are used to covalently link interacting amino acids to identify protein-binding partners. The spatial proximity of cross-linked amino acid residues is used to elucidate structural models of protein complexes. The main challenges for mapping protein-protein interaction are low stoichiometry and low frequency of cross-linked peptides relative to unmodified linear peptides as well as accurate and efficient matches to corresponding peptide sequences with low false discovery rates for identifying the site of cross-link. We evaluate the current state of chemical cross-linking and mass spectrometry applications with the special emphasis on the recent development of informatics data processing and analysis tools that help complexity of interpreting CXMS data. This article is part of a Special Issue entitled:Physiological Enzymology and Protein Functions.
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Identifying novel protein interactions: Proteomic methods, optimisation approaches and data analysis pipelines.
Carneiro, DG, Clarke, T, Davies, CC, Bailey, D
Methods (San Diego, Calif.). 2016;:46-54
Abstract
The technological revolution in high-throughput nucleic acid and protein analysis in the last 15 years has launched the field of 'omics' and led to great advances in our understanding of cell biology. Consequently the study of the cellular proteome and protein dynamics, in particular interactomics, has been a matter of intense investigation, specifically the determination and description of complex protein interaction networks in the cell, not only with other proteins but also with RNA and DNA. The analysis of these interactions, beginning with their identification and ultimately resulting in structural level examination, is one of the cornerstones of modern biological science underpinning basic research and impacting on applied biology, biomedicine and drug discovery. In this review we summarise a selection of emerging and established techniques currently being applied in this field with a particular focus on affinity-based purification systems and their optimisation, including tandem affinity purification (TAP) tagging, isolation of proteins on nascent DNA (IPOND) and RNA-protein immunoprecipitation in tandem (RIPiT). The recent application of quantitative proteomics to improve stringency and specificity is also discussed, including the use of metabolic labelling by stable isotope labelling by amino acids in cell culture (SILAC), localization of organelle proteins by isotope tagging (LOPIT) and proximity-dependent biotin identification (BioID). Finally, we describe a range of software resources that can be applied to interactomics, both to handle raw data and also to scrutinise its broader biological context. In this section we focus especially on open-access online interactomic databases such as Reactome and IntAct.
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Protein-protein interactions of the LIM-only protein FHL2 and functional implication of the interactions relevant in cardiovascular disease.
Tran, MK, Kurakula, K, Koenis, DS, de Vries, CJ
Biochimica et biophysica acta. 2016;(2):219-28
Abstract
FHL2 belongs to the LIM-domain only proteins and contains four and a half LIM domains, each of which are composed of two zinc finger structures. FHL2 exhibits specific interaction with proteins exhibiting diverse functions, including transmembrane receptors, transcription factors and transcription co-regulators, enzymes, and structural proteins. The function of these proteins is regulated by FHL2, which modulates intracellular signal transduction pathways involved in a plethora of cellular tasks. The present review summarizes the current knowledge on the protein interactome of FHL2 and provides an overview of the functional implication of these interactions in apoptosis, migration, and regulation of nuclear receptor function. FHL2 was originally identified in the heart and there is extensive literature available on the role of FHL2 in the cardiovascular system, which is also summarized in this review.
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6.
The PP1 binding code: a molecular-lego strategy that governs specificity.
Heroes, E, Lesage, B, Görnemann, J, Beullens, M, Van Meervelt, L, Bollen, M
The FEBS journal. 2013;(2):584-95
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Abstract
Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo.
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Marked by association: techniques for proximity-dependent labeling of proteins in eukaryotic cells.
Roux, KJ
Cellular and molecular life sciences : CMLS. 2013;(19):3657-64
Abstract
Various methods have been established for the purpose of identifying and characterizing protein-protein interactions (PPIs). This diverse toolbox provides researchers with options to overcome challenges specific to the nature of the proteins under investigation. Among these techniques is a category based on proximity-dependent labeling of proteins in living cells. These can be further partitioned into either hypothesis-based or unbiased screening methods, each with its own advantages and limitations. Approaches in which proteins of interest are fused to either modifying enzymes or receptor sequences allow for hypothesis-based testing of protein proximity. Protein crosslinking and BioID (proximity-dependent biotin identification) permit unbiased screening of protein proximity for a protein of interest. Here, we evaluate these approaches and their applications in living eukaryotic cells.
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Evolution of protein structures and interactions from the perspective of residue contact networks.
Zhang, X, Perica, T, Teichmann, SA
Current opinion in structural biology. 2013;(6):954-63
Abstract
Here we review mechanisms of protein evolution leading to structural changes in protein complexes. These mechanisms include mutations directly within protein interfaces, as well as the effects of mutations that propagate from distant regions of the protein. We also discuss the constraints protein complex structures impose on sequence evolution. We interpret, wherever possible, these mechanisms using amino acid residue contact networks. Many insights into protein evolution come from studies of monomers, and these results facilitate our understanding of evolution of protein complexes. Finally, we highlight the potential of formalizing a phylogenetic framework to integrate residue evolution, structure evolution, and to quantify changes in residue contact networks in protein families.
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Prediction and design of outer membrane protein-protein interactions.
Nanda, V, Hsieh, D, Davis, A
Methods in molecular biology (Clifton, N.J.). 2013;:183-96
Abstract
Protein-protein interactions (PPI) play central roles in biological processes, motivating us to understand the structural basis underlying affinity and specificity. In this chapter, we focus on biochemical and computational design strategies of assessing and detecting PPIs of β-barrel outer membrane proteins (OMPs). A few case studies are presented highlighting biochemical techniques used to dissect the energetics of oligomerization and determine amino acids forming the key interactions of the PPI sites. Current computational strategies for detecting/predicting PPIs are introduced, and examples of computational and rational engineering strategies applied to OMPs are presented.
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A versatile lentiviral expression system to identify mammalian protein-protein interactions.
Mak, AB, Moffat, J
Methods (San Diego, Calif.). 2012;(4):409-16
Abstract
Protein-protein interactions (PPIs) are central to our understanding of protein function, biological processes and signaling pathways. Affinity purification coupled with mass spectrometry (AP-MS) is a powerful approach for detecting PPIs and protein complexes and relies on the purification of bait proteins using bait-specific binding reagents. These binding reagents may recognize bait proteins directly or affinity tags that are fused to bait proteins. A limitation of the latter approach is that expression of affinity tagged baits is largely constrained to engineered or unnatural cell lines, which results in the AP-MS identification of PPIs that may not accurately reflect those seen in nature. Therefore, generating cell lines stably expressing affinity tagged bait proteins in a broad range of cell types and cell lines is important for identifying PPIs that are dependent on different contexts. To facilitate the identification of PPIs across many mammalian cell types, we developed the mammalian affinity purification and lentiviral expression (MAPLE) system. MAPLE uses recombinant lentiviral technology to stably and efficiently express affinity tagged complementary DNA (cDNA) in mammalian cells, including cells that are difficult to transfect and non-dividing cells. The MAPLE vectors contain a versatile affinity (VA) tag for multi-step protein purification schemes and subcellular localization studies. In this methods article, we present a step-by-step overview of the MAPLE system workflow.