Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ.

Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.

A Map of Protein-Metabolite Interactions Reveals Principles of Chemical Communication.

Metabolite-protein interactions control a variety of cellular processes, thereby playing a major role in maintaining cellular homeostasis. Metabolites comprise the largest fraction of molecules in cells, but our knowledge of the metabolite-protein interactome lags behind our understanding of protein-protein or protein-DNA interactomes. Here, we present a chemoproteomic workflow for the systematic identification of metabolite-protein interactions directly in their native environment. The approach identified a network of known and novel interactions and binding sites in Escherichia coli, and we demonstrated the functional relevance of a number of newly identified interactions. Our data enabled identification of new enzyme-substrate relationships and cases of metabolite-induced remodeling of protein complexes. Our metabolite-protein interactome consists of 1,678 interactions and 7,345 putative binding sites. Our data reveal functional and structural principles of chemical communication, shed light on the prevalence and mechanisms of enzyme promiscuity, and enable extraction of quantitative parameters of metabolite binding on a proteome-wide scale.

Global analysis of aging-related protein structural changes uncovers enzyme-polymerization-based control of longevity.

Aging is associated with progressive phenotypic changes. Virtually all cellular phenotypes are produced by proteins, and their structural alterations can lead to age-related diseases. However, we still lack comprehensive knowledge of proteins undergoing structural-functional changes during cellular aging and their contributions to age-related phenotypes. Here, we conducted proteome-wide analysis of early age-related protein structural changes in budding yeast using limited proteolysis-mass spectrometry (LiP-MS). The results, compiled in online ProtAge catalog, unraveled age-related functional changes in regulators of translation, protein folding, and amino acid metabolism. Mechanistically, we found that folded glutamate synthase Glt1 polymerizes into supramolecular self-assemblies during aging, causing breakdown of cellular amino acid homeostasis. Inhibiting Glt1 polymerization by mutating the polymerization interface restored amino acid levels in aged cells, attenuated mitochondrial dysfunction, and led to lifespan extension. Altogether, this comprehensive map of protein structural changes enables identifying mechanisms of age-related phenotypes and offers opportunities for their reversal.

Systematic identification of structure-specific protein-protein interactions.

The physical interactome of a protein can be altered upon perturbation, modulating cell physiology and contributing to disease. Identifying interactome differences of normal and disease states of proteins could help understand disease mechanisms, but current methods do not pinpoint structure-specific PPIs and interaction interfaces proteome-wide. We used limited proteolysis-mass spectrometry (LiP-MS) to screen for structure-specific PPIs by probing for protease susceptibility changes of proteins in cellular extracts upon treatment with specific structural states of a protein. We first demonstrated that LiP-MS detects well-characterized PPIs, including antibody-target protein interactions and interactions with membrane proteins, and that it pinpoints interfaces, including epitopes. We then applied the approach to study conformation-specific interactors of the Parkinson's disease hallmark protein alpha-synuclein (aSyn). We identified known interactors of aSyn monomer and amyloid fibrils and provide a resource of novel putative conformation-specific aSyn interactors for validation in further studies. We also used our approach on GDP- and GTP-bound forms of two Rab GTPases, showing detection of differential candidate interactors of conformationally similar proteins. This approach is applicable to screen for structure-specific interactomes of any protein, including posttranslationally modified and unmodified, or metabolite-bound and unbound protein states.

Mass Spectrometry Structural Proteomics Enabled by Limited Proteolysis and Cross-Linking.

The exploration of protein structure and function stands at the forefront of life science and represents an ever-expanding focus in the development of proteomics. As mass spectrometry (MS) offers readout of protein conformational changes at both the protein and peptide levels, MS-based structural proteomics is making significant strides in the realms of structural and molecular biology, complementing traditional structural biology techniques. This review focuses on two powerful MS-based techniques for peptide-level readout, namely limited proteolysis-mass spectrometry (LiP-MS) and cross-linking mass spectrometry (XL-MS). First, we discuss the principles, features, and different workflows of these two methods. Subsequently, we delve into the bioinformatics strategies and software tools used for interpreting data associated with these protein conformation readouts and how the data can be integrated with other computational tools. Furthermore, we provide a comprehensive summary of the noteworthy applications of LiP-MS and XL-MS in diverse areas including neurodegenerative diseases, interactome studies, membrane proteins, and artificial intelligence-based structural analysis. Finally, we discuss the factors that modulate protein conformational changes. We also highlight the remaining challenges in understanding the intricacies of protein conformational changes by LiP-MS and XL-MS technologies.

Analysis of Brain Protein Stability Changes in a Mouse Model of Alzheimer's Disease.

The stability of proteins from rates of oxidation (SPROX), thermal proteome profiling (TPP), and limited proteolysis (LiP) techniques were used to profile the stability of ∼2500 proteins in hippocampus tissue cell lysates from 2- and 8-months-old wild-type (C57BL/6J; n = 7) and transgenic (5XFAD; n = 7) mice with five Alzheimer's disease (AD)-linked mutations. Approximately 200-500 protein hits with AD-related stability changes were detected by each technique at each age point. The hit overlap from technique to technique was low, and all of the techniques generated protein hits that were more numerous and largely different from those identified in protein expression level analyses, which were also performed here. The hit proteins identified by each technique were enriched in a number of the same pathways and biological processes, many with known connections to AD. The protein stability hits included 25 high-value conformation biomarkers with AD-related stability changes detected using at least 2 techniques at both age points. Also discovered were subunit- and age-specific AD-related stability changes in the proteasome, which had reduced function at both age points. The different folding stability profiles of the proteasome at the two age points are consistent with a different mechanism for proteasome dysfunction at the early and late stages of AD.

Mapping Conformational Changes in the Saliva Proteome Potentially Associated with Oral Cancer Aggressiveness.

Diverse proteomics-based strategies have been applied to saliva to quantitatively identify diagnostic and prognostic targets for oral cancer. Considering that these targets may be regulated by events that do not imply variation in protein abundance levels, we hypothesized that changes in protein conformation can be associated with diagnosis and prognosis, revealing biological processes and novel targets of clinical relevance. For this, we employed limited proteolysis-mass spectrometry in saliva samples to explore structural alterations, comparing the proteome of healthy control and oral squamous cell carcinoma (OSCC) patients with and without lymph node metastasis. Thirty-six proteins with potential structural rearrangements were associated with clinical patient features including transketolase and its interacting partners. Moreover, N-glycosylated peptides contribute to structural rearrangements of potential diagnostic and prognostic markers. Altogether, this approach utilizes saliva proteins to search for targets for diagnosing and prognosing oral cancer and can guide the discovery of potential regulated sites beyond protein-level abundance.

FLiPPR: A Processor for Limited Proteolysis (LiP) Mass Spectrometry Data Sets Built on FragPipe.

Here, we present FLiPPR, or FragPipe LiP (limited proteolysis) Processor, a tool that facilitates the analysis of data from limited proteolysis mass spectrometry (LiP-MS) experiments following primary search and quantification in FragPipe. LiP-MS has emerged as a method that can provide proteome-wide information on protein structure and has been applied to a range of biological and biophysical questions. Although LiP-MS can be carried out with standard laboratory reagents and mass spectrometers, analyzing the data can be slow and poses unique challenges compared to typical quantitative proteomics workflows. To address this, we leverage FragPipe and then process its output in FLiPPR. FLiPPR formalizes a specific data imputation heuristic that carefully uses missing data in LiP-MS experiments to report on the most significant structural changes. Moreover, FLiPPR introduces a data merging scheme and a protein-centric multiple hypothesis correction scheme, enabling processed LiP-MS data sets to be more robust and less redundant. These improvements strengthen statistical trends when previously published data are reanalyzed with the FragPipe/FLiPPR workflow. We hope that FLiPPR will lower the barrier for more users to adopt LiP-MS, standardize statistical procedures for LiP-MS data analysis, and systematize output to facilitate eventual larger-scale integration of LiP-MS data.

Stability-based approaches in chemoproteomics.

Target deconvolution can help understand how compounds exert therapeutic effects and can accelerate drug discovery by helping optimise safety and efficacy, revealing mechanisms of action, anticipate off-target effects and identifying opportunities for therapeutic expansion. Chemoproteomics, a combination of chemical biology with mass spectrometry has transformed target deconvolution. This review discusses modification-free chemoproteomic approaches that leverage the change in protein thermodynamics induced by small molecule ligand binding. Unlike modification-based methods relying on enriching specific protein targets, these approaches offer proteome-wide evaluations, driven by advancements in mass spectrometry sensitivity, increasing proteome coverage and quantitation methods. Advances in methods based on denaturation/precipitation by thermal or chemical denaturation, or by protease degradation are evaluated, emphasising the evolving landscape of chemoproteomics and its potential impact on future drug-development strategies.

Identification of Mortalin as the Main Interactor of Mycalin A, a Poly-Brominated C-15 Acetogenin Sponge Metabolite, by MS-Based Proteomics.

Mycalin A (MA) is a polybrominated C-15 acetogenin isolated from the marine sponge Mycale rotalis. Since this substance displays a strong antiproliferative bioactivity towards some tumour cells, we have now directed our studies towards the elucidation of the MA interactome through functional proteomic approaches, (DARTS and t-LIP-MS). DARTS experiments were performed on Hela cell lysates with the purpose of identifying MA main target protein(s); t-LiP-MS was then applied for an in-depth investigation of the MA-target protein interaction. Both these techniques exploit limited proteolysis coupled with MS analysis. To corroborate LiP data, molecular docking studies were performed on the complexes. Finally, biological and SPR analysis were conducted to explore the effect of the binding. Mortalin (GRP75) was identified as the MA's main interactor. This protein belongs to the Hsp70 family and has garnered significant attention due to its involvement in certain forms of cancer. Specifically, its overexpression in cancer cells appears to hinder the pro-apoptotic function of p53, one of its client proteins, because it becomes sequestered in the cytoplasm. Our research, therefore, has been focused on the possibility that MA might prevent this sequestration, promoting the re-localization of p53 to the nucleus and facilitating the apoptosis of tumor cells.

Chemoproteomics Reveals USP5 (Ubiquitin Carboxyl-Terminal Hydrolase 5) as Promising Target of the Marine Polyketide Gracilioether A.

Mass spectrometry-based chemical proteomic approaches using limited proteolysis have become a powerful tool for the identification and analysis of the interactions between a small molecule (SM) and its protein target(s). Gracilioether A (GeA) is a polyketide isolated from a marine sponge, for which we aimed to trace the interactome using this strategy. DARTS (Drug Affinity Responsive Target Stability) and t-LiP-MS (targeted-Limited Proteolysis-Mass Spectrometry) represented the main techniques used in this study. DARTS was applied on HeLa cell lysate for the identification of the GeA target proteins, and t-LiP-MS was employed to investigate the protein's regions involved in the binding with GeA. The results were complemented through the use of binding studies using Surface Plasmon Resonance (SPR) and in silico molecular docking experiments. Ubiquitin carboxyl-terminal hydrolase 5 (USP5) was identified as a promising target of GeA, and the interaction profile of the USP5-GeA complex was explained. USP5 is an enzyme involved in the pathway of protein metabolism through the disassembly of the polyubiquitin chains on degraded proteins into ubiquitin monomers. This activity is connected to different cellular functions concerning the maintenance of chromatin structure and receptors and the degradation of abnormal proteins and cancerogenic progression. On this basis, this structural information opens the way to following studies focused on the definition of the biological potential of Gracilioether A and the rational development of novel USP5 inhibitors based on a new structural skeleton.

Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model.

The Omicron BA.5 variant of SARS-CoV-2 is known for its high transmissibility and its capacity to evade immunity provided by vaccine protection against the (original) Wuhan strain. In our prior research, we successfully produced the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in an E. coli expression system. Extensive biophysical characterization indicated that, even without glycosylation, the RBD maintained native-like conformational and biophysical properties. The current study explores the immunogenicity and neutralization capacity of the E. coli-expressed Omicron BA.5 RBD using a mouse model. Administration of three doses of the RBD without any adjuvant elicited high titer antisera of up to 7.3 × 105 and up to 1.6 × 106 after a booster shot. Immunization with RBD notably enhanced the population of CD44+CD62L+ T cells, indicating the generation of T cell memory. The in vitro assays demonstrated the antisera's protective efficacy through significant inhibition of the interaction between SARS-CoV-2 and its human receptor, ACE2, and through potent neutralization of a pseudovirus. These findings underscore the potential of our E. coli-expressed RBD as a viable vaccine candidate against the Omicron variant of SARS-CoV-2.

Comprehensive morphological study of free radical processes in chronic chorioamnionitis on the background of iron deficiency anemia in pregnancy.

OBJECTIVE: Aim: To establish the features of free radical processes in the endotheliocytes of the chorionic plate of the placenta in chronic chorioamnionitis against the background of iron deficiency anemia of pregnant women using both chemiluminescent and histochemical methods of research. PATIENTS AND METHODS: Materials and Methods: 82 placentas from parturients at 37 - 40 weeks of gestation were studied. Including, for comparison, the placenta during physiological pregnancy and the observation of iron deficiency anemia of pregnant women without inflammation of the placenta. The number of observations in specific study groups is given in the tables. To achieve the objective and solve the tasks set in this study, there were carried out the following histochemical, chemiluminescent, morphometric and statistical methods of material processing. RESULTS: Results: In case of chorionamnionitis against the background of anemia in pregnancy, the R/B ratio (R/B - ratio between amino- (blue) and carboxyl (red) groups of proteins)) in the method with bromophenol blue according to Mikel Calvo was 1.56±0.021, indicators of chemiluminescence of nitroperoxides were 133±4.5, relative optical density units of histochemical staining using the method according to A. Yasuma and T. Ichikawa was - 0.224±0.0015. CONCLUSION: Conclusions: With chronic chorioamnionitis, the intensity of the glow of nitroperoxides, the average indicators of the R/B ratio, and the optical density of histochemical staining for free amino groups of proteins are increased compared to placentas of physiological pregnancy and anemia of pregnant women. Comorbid i anemia of pregnant women causes increasing of the intensity of the glow of nitroperoxides, the average values of the R/B ratio, and the optical density of histochemical staining for free amino groups of proteins comparing to placentas with inflammation without anemia. The key factor in the formation of morphological features of chronic chorioamnionitis with comorbid anemia is the intensification of free radical processes, which is reflected by the increase in the concentration of nitroperoxides in the center of inflammation, with the subsequent intensification of the processes of oxidative modification of proteins, which is followed by the increasing activity of the processes of limited proteolysis.

Sequence Coverage Visualizer: A Web Application for Protein Sequence Coverage 3D Visualization.

Protein structure defines protein function and plays an extremely important role in protein characterization. Recently, two groups of researchers from DeepMind and the Baker lab have independently published protein structure prediction tools that can help us obtain predicted protein structures for the whole human proteome. This enabled us to visualize the entire human proteome using predicted 3D structures for the first time. To help other researchers best utilize these protein structure predictions in proteomics experiments, we present the Sequence Coverage Visualizer (SCV), http://scv.lab.gy, a web application for protein sequence coverage 3D visualization. Here we showed a few possible usages of the SCV, including the labeling of post-translational modifications and isotope labeling experiments. These results highlight the usefulness of such 3D visualization for proteomics experiments and how SCV can turn a regular proteomics experiment (identified peptide list) into structural insights. Furthermore, when used together with limited proteolysis, we demonstrated that SCV can help to compare different protein structures from different sources, including predicted ones and existing PDB entries. We hope our tool can provide help in the process of improving protein structure prediction accuracy. Overall, SCV is a convenient and powerful tool for visualizing proteomics results in 3D.

Protein Folding Stability Profiling of Colorectal Cancer Chemoresistance Identifies Functionally Relevant Biomarkers.

Reported here is the application of three protein folding stability profiling techniques (including the stability of proteins from rates of oxidation, thermal protein profiling, and limited proteolysis approaches) to identify differentially stabilized proteins in six patient-derived colorectal cancer (CRC) cell lines with different oxaliplatin sensitivities and eight CRC patient-derived xenografts (PDXs) derived from two of the patient derived cell lines with different oxaliplatin sensitivities. Compared to conventional protein expression level analyses, which were also performed here, the stability profiling techniques identified both unique and novel proteins and cellular components that differentiated the sensitive and resistant samples including 36 proteins that were differentially stabilized in at least two techniques in both the cell line and PDX studies of oxaliplatin resistance. These 36 differentially stabilized proteins included 10 proteins previously connected to cancer chemoresistance. Two differentially stabilized proteins, fatty acid synthase and elongation factor 2, were functionally validated in vitro and found to be druggable protein targets with biological functions that can be modulated to improve the efficacy of CRC chemotherapy. These results add to our understanding of CRC oxaliplatin resistance, suggest biomarker candidates for predicting oxaliplatin sensitivity in CRC, and inform new strategies for overcoming chemoresistance in CRC.

Limited proteolysis of mycobacterial DNA-binding protein 1 with an extended, lysine-rich, intrinsically disordered region to unveil posttranslational modifications.

The basic, intrinsically disordered regions of eukaryotic histones and their bacterial counterparts are presumed to act as signaling hubs to regulate the compaction of chromosomes or nucleoids and various DNA processes such as gene expression, recombination, and DNA replication. Posttranslational modifications (PTMs) on these regions are pivotal in regulating chromosomal or nucleoid compaction and DNA processes. However, the low sequence complexity and the presence of short lysine-rich repeats in the regions have hindered the accurate determination of types and locations of PTMs using conventional proteomic procedures. We described a limited proteolysis protocol using trypsin to analyze PTMs on mycobacterial DNA-binding protein 1 (MDP1), a nucleoid-associated protein in mycobacterial species that possesses an extended, lysine-rich, intrinsically disordered region in its C-terminal domain. This limited proteolysis approach successfully revealed significant methylation on many lysine residues in the C-terminal domain of MDP1 purified from Mycobacterium tuberculosis, which was lacking in the corresponding region of recombinant MDP1 expressed in Escherichia coli.

Proteomic tools to study phosphorylation of intrinsically disordered proteins.

INTRODUCTION: Intrinsically disordered proteins (IDPs) represent a family of proteins that lack secondary or tertiary structure. IDPs are hubs in interaction networks, participate in liquid-liquid phase separation processes, and drive the formation of proteinaceous membrane-less organelles. Their unfolded structure makes them particularly prone to post-translational modifications (PTMs) that play key functional modulatory roles. AREAS COVERED: We discuss different analytical approaches to study phosphorylation of IDPs starting from methods for IDP enrichment (strong acid extractions and heat-based pre-fractionation), strategies to enrich and map phosphopeptides/proteins, and mass spectrometry-based tools to study the phosphorylation-dependent conformational alterations of IDPs (limited proteolysis, HDX, chemical cross-linking, covalent labeling, and ion mobility). EXPERT OPINION: There is a growing interest in IDPs and their PTMs since they are involved in several diseases. The intrinsic disorder could be exploited to facilitate purification and synthetic production of IDPs taking full advantage of those structural mass-spectrometry-based methods that can be used to investigate IDPs and their phospho-dependent conformational alterations. The diffusion and implementation of mass spectrometers with ion mobility devices and electron transfer dissociation capabilities could be key-elements for increasing information on IDP biology.

Stability and Resistance to Proteolysis of Enterotoxins SEC and SEL Produced by Staphylococcus epidermidis and Staphylococcus aureus.

The only staphylococcal enterotoxins produced by Staphylococcus epidermidis include SECepi and SELepi, whereas Staphylococcus aureus produces orthologous SECs and SEL having different sequences. We compared S. epidermidis and S. aureus SECs and SELs in terms of resistance to proteolysis and both, thermal and chemical stability. We show that SECepi and SELepi produced by S. epidermidis have similar resistance to proteolysis if compared with their respective orthologues produced by S. aureus. Studied S. epidermidis and S. aureus SEC variants incubated with pepsin at pH 2.0 were found to be more resistant to proteolysis than SELs. SELs turned out to be more resistant than SECs to proteolysis with trypsin at pH 8.0. SECepi was found to be more resistant to thermal denaturation if compared with its S. aureus orthologues. The S. epidermidis and S. aureus SEC variants were found to have higher thermal stability than SELs. Our data indicate that, due to their high stability, the enterotoxins SECepi and SELepi produced in food by S. epidermidis may pose a food safety risk comparable with that posed by S. aureus enterotoxins.

The differential solvent exposure of N-terminal residues provides "fingerprints" of alpha-synuclein fibrillar polymorphs.

Synucleinopathies are neurodegenerative diseases characterized by the presence of intracellular deposits containing the protein alpha-synuclein (aSYN) within patients' brains. It has been shown that aSYN can form structurally distinct fibrillar assemblies, also termed polymorphs. We previously showed that distinct aSYN polymorphs assembled in vitro, named fibrils, ribbons, and fibrils 91, differentially bind to and seed the aggregation of endogenous aSYN in neuronal cells, which suggests that distinct synucleinopathies may arise from aSYN polymorphs. In order to better understand the differential interactions of aSYN polymorphs with their partner proteins, we mapped aSYN polymorphs surfaces. We used limited proteolysis, hydrogen-deuterium exchange, and differential antibody accessibility to identify amino acids on their surfaces. We showed that the aSYN C-terminal region spanning residues 94 to 140 exhibited similarly high solvent accessibility in these three polymorphs. However, the N-terminal amino acid residues 1 to 38 of fibrils were exposed to the solvent, while only residues 1 to 18 within fibrils 91 were exposed, and no N-terminal residues within ribbons were solvent-exposed. It is likely that these differences in surface accessibility contribute to the differential binding of distinct aSYN polymorphs to partner proteins. We thus posit that the polypeptides exposed on the surface of distinct aSYN fibrillar polymorphs are comparable to fingerprints. Our findings have diagnostic and therapeutic potential, particularly in the prion-like propagation of fibrillar aSYN, as they can facilitate the design of ligands that specifically bind and distinguish between fibrillar polymorphs.

Structural mapping techniques distinguish the surfaces of fibrillar 1N3R and 1N4R human tau.

The rigid core of intracellular tau filaments from Alzheimer's disease (AD), Pick's disease (PiD), and Corticobasal disease (CBD) brains has been shown to differ in their cryo-EM atomic structure. Despite providing critical information on the intimate arrangement of a fraction of htau molecule within the fibrillar scaffold, the cryo-EM studies neither yield a complete picture of tau fibrillar assemblies structure nor contribute insights into the surfaces that define their interactions with numerous cellular components. Here, using proteomic approaches such as proteolysis and molecular covalent painting, we mapped the exposed amino acid stretches at the surface and those constituting the fibrillar core of in vitro-assembled fibrils of human htau containing one N-terminal domain and three (1N3R) or four (1N4R) C-terminal microtubule-binding repeat domains as a result of alternative splicing. Using limited proteolysis, we identified the proteolytic fragments composing the molecular "bar-code" for each type of fibril. Our results are in agreement with structural data reported for filamentous tau from AD, PiD, and CBD cases predigested with the protease pronase. Finally, we report two amino acid stretches, exposed to the solvent in 1N4R not in 1N3R htau, which distinguish the surfaces of these two kinds of fibrils. Our findings open new perspectives for the design of highly specific ligands with diagnostic and therapeutic potential.