scholarly journals Time-lapsed proteomics reveals a role for the novel protein, SNED1, in modulating ECM composition and protein folding

2022 ◽  
Author(s):  
Fred Lee ◽  
Xinhao Shao ◽  
Yu Gao ◽  
Alexandra Naba
Keyword(s):  
Protein Interactions ◽  
Dynamic Range ◽  
Structural Information ◽  
Limited Proteolysis ◽  
The Novel ◽  
Protein Protein Interactions ◽  
Sequence Coverage ◽  
Post Translational Modifications ◽  
Ecm Proteins ◽  
Broad Dynamic Range

The extracellular matrix (ECM) is a complex and dynamic meshwork of proteins providing structural support to cells. It also provides biochemical signals governing cellular processes including proliferation and migration. Alterations of ECM structure and/or composition has been shown to lead to, or accompany, many pathological processes including cancer and fibrosis. To understand how the ECM contributes to diseases, we first need to obtain a comprehensive characterization of the ECM of tissues and of its changes during disease progression. Over the past decade, mass-spectrometry-based proteomics has become the state-of-the-art method to profile the protein composition of ECMs. However, existing methods do not fully capture the broad dynamic range of protein abundance in the ECM, nor do they permit to achieve the high coverage needed to gain finer biochemical information, including the presence of isoforms or post-translational modifications. In addition, broadly adopted proteomic methods relying on extended trypsin digestion do not provide structural information on ECM proteins, yet, gaining insights into ECM protein structure is critical to better understanding protein functions. Here, we present the optimization of a time-lapsed proteomic method using limited proteolysis of partially denatured samples and the sequential release of peptides to achieve superior sequence coverage as compared to standard ECM proteomic workflow. Exploiting the spatio-temporal resolution of this method, we further demonstrate how 3-dimensional time-lapsed peptide mapping can identify protein regions differentially susceptible to trypsin and can thus identify sites of post-translational modifications, including protein-protein interactions. We further illustrate how this approach can be leveraged to gain insight on the role of the novel ECM protein SNED1 in ECM homeostasis. We found that the expression of SNED1 expression by mouse embryonic fibroblasts results in the alteration of overall ECM composition and the sequence coverage of certain ECM proteins, raising the possibility that SNED1 could modify accessibility to trypsin by engaging in protein-protein interactions.

Protein Interaction Domains and Post-Translational Modifications: Structural Features and Drug Discovery Applications

2020 ◽  
Vol 27 (37) ◽  
pp. 6306-6355 ◽  
Author(s):  
Marian Vincenzi ◽  
Flavia Anna Mercurio ◽  
Marilisa Leone
Keyword(s):  
Drug Discovery ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Structural Information ◽  
Protein Complexes ◽  
Structural Features ◽  
Protein Protein Interactions ◽  
Modular Architecture ◽  
Post Translational Modifications ◽  
Interaction Domains

Background:: Many pathways regarding healthy cells and/or linked to diseases onset and progression depend on large assemblies including multi-protein complexes. Protein-protein interactions may occur through a vast array of modules known as protein interaction domains (PIDs). Objective:: This review concerns with PIDs recognizing post-translationally modified peptide sequences and intends to provide the scientific community with state of art knowledge on their 3D structures, binding topologies and potential applications in the drug discovery field. Method:: Several databases, such as the Pfam (Protein family), the SMART (Simple Modular Architecture Research Tool) and the PDB (Protein Data Bank), were searched to look for different domain families and gain structural information on protein complexes in which particular PIDs are involved. Recent literature on PIDs and related drug discovery campaigns was retrieved through Pubmed and analyzed. Results and Conclusion:: PIDs are rather versatile as concerning their binding preferences. Many of them recognize specifically only determined amino acid stretches with post-translational modifications, a few others are able to interact with several post-translationally modified sequences or with unmodified ones. Many PIDs can be linked to different diseases including cancer. The tremendous amount of available structural data led to the structure-based design of several molecules targeting protein-protein interactions mediated by PIDs, including peptides, peptidomimetics and small compounds. More studies are needed to fully role out, among different families, PIDs that can be considered reliable therapeutic targets, however, attacking PIDs rather than catalytic domains of a particular protein may represent a route to obtain selective inhibitors.

scholarly journals Applications of In-Cell NMR in Structural Biology and Drug Discovery

2019 ◽  
Vol 20 (1) ◽  
pp. 139 ◽  
Author(s):  
CongBao Kang
Keyword(s):  
Drug Discovery ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Structural Information ◽  
Living Cells ◽  
Bacterial Cells ◽  
Protein Protein Interactions ◽  
Nmr Studies ◽  
Post Translational Modifications

In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein–protein interactions in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of Xenopus laevis or delivered into human cells. Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells. The available protocols and successful examples encourage wide applications of this technique in different fields such as drug discovery. Despite the challenges in this method, progress has been made in recent years. In this review, applications of in-cell NMR are summarized. The successful applications of this method in mammalian and bacterial cells make it feasible to play important roles in drug discovery, especially in the step of target engagement.

scholarly journals The Role of Low Complexity Regions in Protein Interaction Modes: An Illustration in Huntingtin

2021 ◽  
Vol 22 (4) ◽  
pp. 1727
Author(s):  
Kristina Kastano ◽  
Pablo Mier ◽  
Miguel A. Andrade-Navarro
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Structural Information ◽  
Low Complexity ◽  
Protein Interaction Data ◽  
Protein Protein Interactions ◽  
Post Translational Modifications ◽  
Evolutionarily Conserved ◽  
And Function

Low complexity regions (LCRs) are very frequent in protein sequences, generally having a lower propensity to form structured domains and tending to be much less evolutionarily conserved than globular domains. Their higher abundance in eukaryotes and in species with more cellular types agrees with a growing number of reports on their function in protein interactions regulated by post-translational modifications. LCRs facilitate the increase of regulatory and network complexity required with the emergence of organisms with more complex tissue distribution and development. Although the low conservation and structural flexibility of LCRs complicate their study, evolutionary studies of proteins across species have been used to evaluate their significance and function. To investigate how to apply this evolutionary approach to the study of LCR function in protein–protein interactions, we performed a detailed analysis for Huntingtin (HTT), a large protein that is a hub for interaction with hundreds of proteins, has a variety of LCRs, and for which partial structural information (in complex with HAP40) is available. We hypothesize that proteins RASA1, SYN2, and KAT2B may compete with HAP40 for their attachment to the core of HTT using similar LCRs. Our results illustrate how evolution might favor the interplay of LCRs with domains, and the possibility of detecting multiple modes of LCR-mediated protein–protein interactions with a large hub such as HTT when enough protein interaction data is available.

scholarly journals Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Dan Tan ◽  
Qiang Li ◽  
Mei-Jun Zhang ◽  
Chao Liu ◽  
Chengying Ma ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Structural Information ◽  
Affinity Purification ◽  
Protein Protein Interactions ◽  
C Elegans ◽  
Protein Protein Interaction ◽  
Lysine Residues ◽  
Spacer Arm ◽  
Cross Linker ◽  
Protein Nucleic Acid

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.

scholarly journals Activation and assembly of the NADPH oxidase: a structural perspective

2005 ◽  
Vol 386 (3) ◽  
pp. 401-416 ◽  
Author(s):  
Yvonne GROEMPING ◽  
Katrin RITTINGER
Keyword(s):  
Nadph Oxidase ◽  
Protein Interactions ◽  
Structural Information ◽  
Three Dimensional ◽  
Protein Protein Interactions ◽  
Crucial Component ◽  
Enzyme Subunits ◽  
Membrane Components ◽  
Inhibitory Interactions ◽  
Encompassing Model

The NADPH oxidase of professional phagocytes is a crucial component of the innate immune response due to its fundamental role in the production of reactive oxygen species that act as powerful microbicidal agents. The activity of this multi-protein enzyme is dependent on the regulated assembly of the six enzyme subunits at the membrane where oxygen is reduced to superoxide anions. In the resting state, four of the enzyme subunits are maintained in the cytosol, either through auto-inhibitory interactions or through complex formation with accessory proteins that are not part of the active enzyme complex. Multiple inputs are required to disrupt these inhibitory interactions and allow translocation to the membrane and association with the integral membrane components. Protein interaction modules are key regulators of NADPH oxidase assembly, and the protein–protein interactions mediated via these domains have been the target of numerous studies. Many models have been put forward to describe the intricate network of reversible protein interactions that regulate the activity of this enzyme, but an all-encompassing model has so far been elusive. An important step towards an understanding of the molecular basis of NADPH oxidase assembly and activity has been the recent solution of the three-dimensional structures of some of the oxidase components. We will discuss these structures in the present review and attempt to reconcile some of the conflicting models on the basis of the structural information available.

scholarly journals Molecular dynamics shows complex interplay and long-range effects of post-translational modifications in yeast protein interactions

2021 ◽  
Vol 17 (5) ◽  
pp. e1008988
Author(s):  
Nikolina ŠoŠtarić ◽  
Vera van Noort
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Protein Structures ◽  
Cellular Protein ◽  
Free Energy Calculations ◽  
Protein Protein Interactions ◽  
Post Translational Modifications ◽  
Protein Properties ◽  
Dynamics Simulations

Post-translational modifications (PTMs) play a vital, yet often overlooked role in the living cells through modulation of protein properties, such as localization and affinity towards their interactors, thereby enabling quick adaptation to changing environmental conditions. We have previously benchmarked a computational framework for the prediction of PTMs’ effects on the stability of protein-protein interactions, which has molecular dynamics simulations followed by free energy calculations at its core. In the present work, we apply this framework to publicly available data on Saccharomyces cerevisiae protein structures and PTM sites, identified in both normal and stress conditions. We predict proteome-wide effects of acetylations and phosphorylations on protein-protein interactions and find that acetylations more frequently have locally stabilizing roles in protein interactions, while the opposite is true for phosphorylations. However, the overall impact of PTMs on protein-protein interactions is more complex than a simple sum of local changes caused by the introduction of PTMs and adds to our understanding of PTM cross-talk. We further use the obtained data to calculate the conformational changes brought about by PTMs. Finally, conservation of the analyzed PTM residues in orthologues shows that some predictions for yeast proteins will be mirrored to other organisms, including human. This work, therefore, contributes to our overall understanding of the modulation of the cellular protein interaction networks in yeast and beyond.

scholarly journals Expanding the potential genes of inborn errors of immunity through protein interactions

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Humza A. Khan ◽  
Manish J. Butte
Keyword(s):  
Genetic Variation ◽  
Protein Interactions ◽  
Immune Cell ◽  
Genetic Disorders ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Inborn Errors ◽  
Post Translational Modifications ◽  
New Genes ◽  
Inborn Errors Of Immunity

Abstract Background Inborn errors of immunity (IEI) are a group of genetic disorders that impair the immune system, with over 400 genes described so far, and hundreds more to be discovered. To facilitate the search for new genes, we need a way to prioritize among all the genes in the genome those most likely to play an important role in immunity. Results Here we identify a new list of genes by linking known IEI genes to new ones by using open-source databases of protein-protein interactions, post-translational modifications, and transcriptional regulation. We analyze this new set of 2,530 IEI-related genes for their tolerance of genetic variation and by their expression levels in various immune cell types. Conclusions By merging genes derived from protein interactions of known IEI genes with transcriptional data, we offer a new list of candidate genes that may play a role in as-yet undiscovered IEIs.

scholarly journals Integrating Structural Information to Study the Dynamics of Protein-Protein Interactions in Cells

Structure ◽  
2018 ◽  
Vol 26 (10) ◽  
pp. 1414-1424.e3 ◽  
Author(s):  
Bo Wang ◽  
Zhong-Ru Xie ◽  
Jiawen Chen ◽  
Yinghao Wu
Keyword(s):  
Protein Interactions ◽  
Structural Information ◽  
Protein Protein Interactions ◽  
In Cells

scholarly journals Aspartic Acid Isomerization Characterized by High Definition Mass Spectrometry Significantly Alters the Bioactivity of a Novel Toxin from Poecilotheria

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 207 ◽  
Author(s):  
Stephen R. Johnson ◽  
Hillary G. Rikli
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Ion Mobility ◽  
Protein Protein Interactions ◽  
High Definition ◽  
Vast Number ◽  
Post Translational Modifications ◽  
C Terminus ◽  
Acid Isomerization

Research in toxinology has created a pharmacological paradox. With an estimated 220,000 venomous animals worldwide, the study of peptidyl toxins provides a vast number of effector molecules. However, due to the complexity of the protein-protein interactions, there are fewer than ten venom-derived molecules on the market. Structural characterization and identification of post-translational modifications are essential to develop biological lead structures into pharmaceuticals. Utilizing advancements in mass spectrometry, we have created a high definition approach that fuses conventional high-resolution MS-MS with ion mobility spectrometry (HDMSE) to elucidate these primary structure characteristics. We investigated venom from ten species of “tiger” spider (Genus: Poecilotheria) and discovered they contain isobaric conformers originating from non-enzymatic Asp isomerization. One conformer pair conserved in five of ten species examined, denominated PcaTX-1a and PcaTX-1b, was found to be a 36-residue peptide with a cysteine knot, an amidated C-terminus, and isoAsp33Asp substitution. Although the isomerization of Asp has been implicated in many pathologies, this is the first characterization of Asp isomerization in a toxin and demonstrates the isomerized product’s diminished physiological effects. This study establishes the value of a HDMSE approach to toxin screening and characterization.

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