scholarly journals Cross-linking mass spectrometry uncovers protein interactions and functional assemblies in synaptic vesicle membranes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sabine Wittig ◽  
Marcelo Ganzella ◽  
Marie Barth ◽  
Susann Kostmann ◽  
Dietmar Riedel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Synaptic Vesicle ◽  
Protein Interactions ◽  
Synaptic Vesicles ◽  
Large Scale ◽  
Cross Linking ◽  
Synaptic Membrane ◽  
Biophysical Parameters ◽  
Vesicle Membrane ◽  
Pass Through

AbstractSynaptic vesicles are storage organelles for neurotransmitters. They pass through a trafficking cycle and fuse with the pre-synaptic membrane when an action potential arrives at the nerve terminal. While molecular components and biophysical parameters of synaptic vesicles have been determined, our knowledge on the protein interactions in their membranes is limited. Here, we apply cross-linking mass spectrometry to study interactions of synaptic vesicle proteins in an unbiased approach without the need for specific antibodies or detergent-solubilisation. Our large-scale analysis delivers a protein network of vesicle sub-populations and functional assemblies including an active and an inactive conformation of the vesicular ATPase complex as well as non-conventional arrangements of the luminal loops of SV2A, Synaptophysin and structurally related proteins. Based on this network, we specifically target Synaptobrevin-2, which connects with many proteins, in different approaches. Our results allow distinction of interactions caused by ‘crowding’ in the vesicle membrane from stable interaction modules.

scholarly journals Synaptic vesicle membrane proteins interact to form a multimeric complex.

1992 ◽  
Vol 116 (3) ◽  
pp. 761-775 ◽  
Author(s):  
M K Bennett ◽  
N Calakos ◽  
T Kreiner ◽  
R H Scheller
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Protein Interactions ◽  
Synaptic Vesicles ◽  
Spatial Organization ◽  
Gel Filtration ◽  
Vesicle Membrane ◽  
Multicomponent Complex ◽  
Membrane Components

Potential interactions between membrane components of rat brain synaptic vesicles were analyzed by detergent solubilization followed by size fractionation or immunoprecipitation. The behavior of six synaptic vesicle membrane proteins as well as a plasma membrane protein was monitored by Western blotting. Solubilization of synaptic vesicle membranes in CHAPS resulted in the recovery of a large protein complex that included SV2, p65, p38, vesicle-associated membrane protein, and the vacuolar proton pump. Solubilization in octylglucoside resulted in the preservation of interactions between SV2, p38, and rab3A, while solubilization of synaptic vesicles with Triton X-100 resulted in two predominant interactions, one involving p65 and SV2, and the other involving p38 and vesicle-associated membrane protein. The multicomponent complex preserved with CHAPS solubilization was partially reconstituted following octylglucoside solubilization and subsequent dialysis against CHAPS. Reduction of the CHAPS concentration by gel filtration chromatography resulted in increased recovery of the multicomponent complex. Examination of the large complex isolated from CHAPS-solubilized vesicles by negative stain EM revealed structures with multiple globular domains, some of which were specifically labeled with gold-conjugated antibodies directed against p65 and SV2. The protein interactions defined in this report are likely to underlie aspects of neurotransmitter secretion, membrane traffic, and the spatial organization of vesicles within the nerve terminal.

scholarly journals EVIDENCE FOR RECYCLING OF SYNAPTIC VESICLE MEMBRANE DURING TRANSMITTER RELEASE AT THE FROG NEUROMUSCULAR JUNCTION

1973 ◽  
Vol 57 (2) ◽  
pp. 315-344 ◽  
Author(s):  
J. E. Heuser ◽  
T. S. Reese
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Motor Nerve ◽  
Coated Vesicles ◽  
Synaptic Membrane ◽  
Nerve Terminals ◽  
Frog Neuromuscular Junction ◽  
Vesicle Membrane ◽  
Intracellular Compartments

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.

scholarly journals Development of Large-scale Cross-linking Mass Spectrometry

2017 ◽  
Vol 17 (6) ◽  
pp. 1055-1066 ◽  
Author(s):  
Helena Maria Barysz ◽  
Johan Malmström
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Function ◽  
Large Scale ◽  
Unmet Need ◽  
Large Data ◽  
Cross Linking ◽  
Data Sets ◽  
Protein Protein Interactions ◽  
Model Complex

Cross-linking mass spectrometry (CLMS) provides distance constraints to study the structure of proteins, multiprotein complexes and protein-protein interactions which are critical for the understanding of protein function. CLMS is an attractive technology to bridge the gap between high-resolution structural biology techniques and proteomic-based interactome studies. However, as outlined in this review there are still several bottlenecks associated with CLMS which limit its application on a proteome-wide level. Specifically, there is an unmet need for comprehensive software that can reliably identify cross-linked peptides from large data sets. In this review we provide supporting information to reason that targeted proteomics of cross-links may provide the required sensitivity to reliably detect and quantify cross-linked peptides and that a reporter ion signature for cross-linked peptides may become a useful approach to increase confidence in the identification process of cross-linked peptides. In addition, the review summarizes the recent advances in CLMS workflows using the analysis of condensin complex in intact chromosomes as a model complex.

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.

Integrated mass spectrometry-based multi-omics for elucidating mechanisms of bacterial virulence

2021 ◽  
Author(s):  
Lok Man ◽  
William P. Klare ◽  
Ashleigh L. Dale ◽  
Joel A. Cain ◽  
Stuart J. Cordwell
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Large Scale ◽  
Lipid A ◽  
Phenotypic Characterization ◽  
Post Translational Modification ◽  
Form Of Life ◽  
Antimicrobial Interventions

Despite being considered the simplest form of life, bacteria remain enigmatic, particularly in light of pathogenesis and evolving antimicrobial resistance. After three decades of genomics, we remain some way from understanding these organisms, and a substantial proportion of genes remain functionally unknown. Methodological advances, principally mass spectrometry (MS), are paving the way for parallel analysis of the proteome, metabolome and lipidome. Each provides a global, complementary assay, in addition to genomics, and the ability to better comprehend how pathogens respond to changes in their internal (e.g. mutation) and external environments consistent with infection-like conditions. Such responses include accessing necessary nutrients for survival in a hostile environment where co-colonizing bacteria and normal flora are acclimated to the prevailing conditions. Multi-omics can be harnessed across temporal and spatial (sub-cellular) dimensions to understand adaptation at the molecular level. Gene deletion libraries, in conjunction with large-scale approaches and evolving bioinformatics integration, will greatly facilitate next-generation vaccines and antimicrobial interventions by highlighting novel targets and pathogen-specific pathways. MS is also central in phenotypic characterization of surface biomolecules such as lipid A, as well as aiding in the determination of protein interactions and complexes. There is increasing evidence that bacteria are capable of widespread post-translational modification, including phosphorylation, glycosylation and acetylation; with each contributing to virulence. This review focuses on the bacterial genotype to phenotype transition and surveys the recent literature showing how the genome can be validated at the proteome, metabolome and lipidome levels to provide an integrated view of organism response to host conditions.

scholarly journals Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b559 in Photosystem II

2017 ◽  
Vol 114 (9) ◽  
pp. 2224-2229 ◽  
Author(s):  
Daniel A. Weisz ◽  
Haijun Liu ◽  
Hao Zhang ◽  
Sundarapandian Thangapandian ◽  
Emad Tajkhorshid ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Photosystem Ii ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Docking ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Cytochrome B559 ◽  
Reaction Center Complex ◽  
Assembly Intermediate

Photosystem II (PSII), a large pigment protein complex, undergoes rapid turnover under natural conditions. During assembly of PSII, oxidative damage to vulnerable assembly intermediate complexes must be prevented. Psb28, the only cytoplasmic extrinsic protein in PSII, protects the RC47 assembly intermediate of PSII and assists its efficient conversion into functional PSII. Its role is particularly important under stress conditions when PSII damage occurs frequently. Psb28 is not found, however, in any PSII crystal structure, and its structural location has remained unknown. In this study, we used chemical cross-linking combined with mass spectrometry to capture the transient interaction of Psb28 with PSII. We detected three cross-links between Psb28 and the α- and β-subunits of cytochrome b559, an essential component of the PSII reaction-center complex. These distance restraints enable us to position Psb28 on the cytosolic surface of PSII directly above cytochrome b559, in close proximity to the QB site. Protein–protein docking results also support Psb28 binding in this region. Determination of the Psb28 binding site and other biochemical evidence allow us to propose a mechanism by which Psb28 exerts its protective effect on the RC47 intermediate. This study also shows that isotope-encoded cross-linking with the “mass tags” selection criteria allows confident identification of more cross-linked peptides in PSII than has been previously reported. This approach thus holds promise to identify other transient protein–protein interactions in membrane protein complexes.

scholarly journals XlinkCyNET: a Cytoscape application for visualization of protein interaction networks based on cross-linking mass-spectrometry identifications

2020 ◽  
Author(s):  
Diogo Borges Lima ◽  
Ying Zhu ◽  
Fan Liu
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Large Scale ◽  
Interaction Network ◽  
Protein Interaction Networks ◽  
Interaction Networks ◽  
Cross Linking ◽  
Protein Interaction Data ◽  
Interaction Data ◽  
Link Type

ABSTRACTSoftware tools that allow visualization and analysis of protein interaction networks are essential for studies in systems biology. One of the most popular network visualization tools in biology is Cytoscape, which offers a large selection of plugins for interpretation of protein interaction data. Chemical cross-linking coupled to mass spectrometry (XL-MS) is an increasingly important source for such interaction data, but there are currently no Cytoscape tools to analyze XL-MS results. In light of the suitability of Cytoscape platform but also to expand its toolbox, here we introduce XlinkCyNET, an open-source Cytoscape Java plugin for exploring large-scale XL-MS-based protein interaction networks. XlinkCyNET offers rapid and easy visualization of intra and intermolecular cross-links and the locations of protein domains in a rectangular bar style, allowing subdomain-level interrogation of the interaction network. XlinkCyNET is freely available from the Cytoscape app store: http://apps.cytoscape.org/apps/xlinkcynet and at https://www.theliulab.com/software/xlinkcynet.

scholarly journals Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Cross-Linking Mass Spectrometry

2021 ◽  
Author(s):  
Dmitri R. Davydov ◽  
Bikash Dangi ◽  
Guihua Yue ◽  
Bhagwat Prasad ◽  
Viktor G. Zgoda
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Protein Interactions ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Cross Linking ◽  
Cytochrome P450 2E1 ◽  
Protein Protein Interactions ◽  
Chemical Cross Linking

This study aimed on exploration of the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) in the human liver on drug metabolism. Using membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide (BPM) and 4-(N-succinimidylcarboxy)benzophenone (BPS), we explored the array of its protein-protein interactions (proteome) in human liver microsomes (HLM) with chemical cross-linking mass spectrometry (CXMS). Exposure of bait-incorporated HLM samples to light was followed by isolation of the His-tagged bait protein and its cross-linked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the cross-linked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively cross-linked partners of CYP2E1 are cytochromes P450 2A6, 3A4, 2C9, and 4A11. We also detected the conjugates of CYP2E1 with UDP-glucuronosyltransferases (UGTs) 1A6, 1A9, 2B4, 2B15, and 2B17. These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes. Of particular interest is the observation of efficient cross-linking of CYP2E1 with CYP4A11. This enzyme plays a central role in the synthesis of vasoactive eicosanoids and its interactions with alcohol-inducible CYP2E1 may shed light on the mechanisms of alcohol-induced hypertension.

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