scholarly journals Complexome Profiling—Exploring Mitochondrial Protein Complexes in Health and Disease

2022 ◽  
Vol 9 ◽  
Author(s):  
Alfredo Cabrera-Orefice ◽  
Alisa Potter ◽  
Felix Evers ◽  
Johannes F. Hevler ◽  
Sergio Guerrero-Castillo
Keyword(s):  
Protein Complexes ◽  
Gradient Centrifugation ◽  
Mitochondrial Protein ◽  
Size Exclusion ◽  
Native Proteins ◽  
Mass Spectrometry Identification ◽  
Exclusion Chromatography ◽  
Health And Disease ◽  
The Impact ◽  
Novel Protein

Complexome profiling (CP) is a state-of-the-art approach that combines separation of native proteins by electrophoresis, size exclusion chromatography or density gradient centrifugation with tandem mass spectrometry identification and quantification. Resulting data are computationally clustered to visualize the inventory, abundance and arrangement of multiprotein complexes in a biological sample. Since its formal introduction a decade ago, this method has been mostly applied to explore not only the composition and abundance of mitochondrial oxidative phosphorylation (OXPHOS) complexes in several species but also to identify novel protein interactors involved in their assembly, maintenance and functions. Besides, complexome profiling has been utilized to study the dynamics of OXPHOS complexes, as well as the impact of an increasing number of mutations leading to mitochondrial disorders or rearrangements of the whole mitochondrial complexome. Here, we summarize the major findings obtained by this approach; emphasize its advantages and current limitations; discuss multiple examples on how this tool could be applied to further investigate pathophysiological mechanisms and comment on the latest advances and opportunity areas to keep developing this methodology.

scholarly journals Inhaled Anesthetics Promote Albumin Dimerization through Reciprocal Exchange of Subdomains

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Benjamin J. Pieters ◽  
Eugene E. Fibuch ◽  
Joshua D. Eklund ◽  
Norbert W. Seidler
Keyword(s):  
Fluorescence Emission ◽  
Size Exclusion ◽  
Inhaled Anesthetics ◽  
Protein Protein Interaction ◽  
Reciprocal Exchange ◽  
Proposed Model ◽  
Exclusion Chromatography ◽  
Spectral Shifting ◽  
And Control ◽  
The Impact

Inhaled anesthetics affect protein-protein interaction, but the mechanisms underlying these effects are still poorly understood. We examined the impact of sevoflurane and isoflurane on the dimerization of human serum albumin (HSA), a protein with anesthetic binding sites that are well characterized. Intrinsic fluorescence emission was analyzed for spectral shifting and self-quenching, and control first derivatives (spectral responses to changes in HSA concentration) were compared against those obtained from samples treated with sevoflurane or isoflurane. Sevoflurane increased dimer-dependent self-quenching and both decreased oligomer-dependent spectral shifting, suggesting that inhaled anesthetics promoted HSA dimerization. Size exclusion chromatography and polarization data were consistent with these observations. The data support the proposed model of a reciprocal exchange of subdomains to form an HSA dimer. The open-ended exchange of subdomains, which we propose occuring in HSA oligomers, was inhibited by sevoflurane and isoflurane.

scholarly journals Impact of Botrytis cinerea Contamination on the Characteristics and Foamability of Yeast Macromolecules Released during the Alcoholic Fermentation of a Model Grape Juice

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 472
Author(s):  
Richard Marchal ◽  
Thomas Salmon ◽  
Ramon Gonzalez ◽  
Belinda Kemp ◽  
Céline Vrigneau ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Alcoholic Fermentation ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Grape Juice ◽  
Size Exclusion ◽  
Periodic Acid Schiff ◽  
Exclusion Chromatography ◽  
The Impact

Botrytis cinerea is a fungal pathogen responsible for the decrease in foamability of sparkling wines. The proteolysis of must proteins originating from botrytized grapes is well known, but far less information is available concerning the effect of grape juice contamination by Botrytis. The impact from Botrytis on the biochemical and physico-chemical characteristics of proteins released from Saccharomyces during alcoholic fermentation remains elusive. To address this lack of knowledge, a model grape juice was inoculated with three enological yeasts with or without the Botrytis culture supernatant. Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques (AgNO3 and periodic acid Schiff staining) was used in the study. When Botrytis enzymes were present, a significant degradation of the higher and medium MW molecules released by Saccharomyces was observed during alcoholic fermentation whilst the lower MW fraction increased. For the three yeast strains studied, the results clearly showed a strong decrease in the wine foamability when synthetic musts were inoculated with 5% (v/v) of Botrytis culture due to fungus proteases.

scholarly journals The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization

2008 ◽  
Vol 41 (6) ◽  
pp. 1150-1160 ◽  
Author(s):  
Jichun Ma ◽  
Di Xia
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Quality ◽  
Structural Information ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Blue Native

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.

Oligomeric assembly and interactions within the human RuvB-like RuvBL1 and RuvBL2 complexes

2010 ◽  
Vol 429 (1) ◽  
pp. 113-125 ◽  
Author(s):  
Andrew Niewiarowski ◽  
Alison S. Bradley ◽  
Jayesh Gor ◽  
Adam R. McKay ◽  
Stephen J. Perkins ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Structural Model ◽  
Analytical Ultracentrifugation ◽  
Protein Complexes ◽  
Adenine Nucleotides ◽  
Size Exclusion ◽  
Molecular Architecture ◽  
Essential Components ◽  
Exclusion Chromatography

The two closely related eukaryotic AAA+ proteins (ATPases associated with various cellular activities), RuvBL1 (RuvB-like 1) and RuvBL2, are essential components of large multi-protein complexes involved in diverse cellular processes. Although the molecular mechanisms of RuvBL1 and RuvBL2 function remain unknown, oligomerization is likely to be important for their function together or individually, and different oligomeric forms might underpin different functions. Several experimental approaches were used to investigate the molecular architecture of the RuvBL1–RuvBL2 complex and the role of the ATPase-insert domain (domain II) for its assembly and stability. Analytical ultracentrifugation showed that RuvBL1 and RuvBL2 were mainly monomeric and each monomer co-existed with small proportions of dimers, trimers and hexamers. Adenine nucleotides induced hexamerization of RuvBL2, but not RuvBL1. In contrast, the RuvBL1–RuvBL2 complexes contained single- and double-hexamers together with smaller forms. The role of domain II in complex assembly was examined by size-exclusion chromatography using deletion mutants of RuvBL1 and RuvBL2. Significantly, catalytically competent dodecameric RuvBL1–RuvBL2, complexes lacking domain II in one or both proteins could be assembled but the loss of domain II in RuvBL1 destabilized the dodecamer. The composition of the RuvBL1–RuvBL2 complex was analysed by MS. Several species of mixed RuvBL1/2 hexamers with different stoichiometries were seen in the spectra of the RuvBL1–RuvBL2 complex. A number of our results indicate that the architecture of the human RuvBL1–RuvBL2 complex does not fit the recent structural model of the yeast Rvb1–Rvb2 complex.

scholarly journals Impact of Electron Beam Irradiation on Thermoplastic Polyurethanes Unraveled by Thermal Field-Flow Fractionation

2020 ◽  
Author(s):  
Martin Geisler ◽  
Tuhin Subhra Pal ◽  
Kerstin Arnhold ◽  
Mikhail Malanin ◽  
Michael Thomas Müller ◽  
...  
Keyword(s):  
Electron Beam ◽  
Size Exclusion Chromatography ◽  
Electron Beam Irradiation ◽  
Thermal Field ◽  
Size Exclusion ◽  
Field Flow Fractionation ◽  
Beam Irradiation ◽  
Exclusion Chromatography ◽  
Flow Fractionation ◽  
The Impact

The impact of electron beam irradiation on thermoplastic polyurethane material was studied for both an aliphatic and an aromatic polyurethane with equal amount of hard and soft segments. Irradiation doses up to 300 kGy at room temperature and at 100 °C were applied. Changes in chemical structure, molar mass and size were assessed using infrared spectroscopy, differential scanning calorimetry, size exclusion chromatography and thermal field flow fractionation. Material alterations were correlated with trends regarding to degradation, crosslinking or branching changes. Thereby, limits of characteri-zation by size exclusion chromatography are addressed and amended by thermal field-flow fractionation studies. In addition, a thermophoretic analysis has been carried out complementary to the portfolio of analytical methods applied in this work.

scholarly journals Systematic protein complex profiling and differential analysis from co-fractionation mass spectrometry data

2020 ◽  
Author(s):  
Andrea Fossati ◽  
Chen Li ◽  
Peter Sykacek ◽  
Moritz Heusel ◽  
Fabian Frommelt ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Quantification ◽  
Mass Spectrometry Data ◽  
Size Exclusion ◽  
Differential Analysis ◽  
Experimental Conditions ◽  
Systematic Analysis ◽  
Improved Performance ◽  
Novel Protein

AbstractProtein complexes, macro-molecular assemblies of two or more proteins, play vital roles in numerous cellular activities and collectively determine the cellular state. Despite the availability of a range of methods for analysing protein complexes, systematic analysis of complexes under multiple conditions has remained challenging. Approaches based on biochemical fractionation of intact, native complexes and correlation of protein profiles have shown promise, for instance in the combination of size exclusion chromatography (SEC) with accurate protein quantification by SWATH/DIA-MS. However, most approaches for interpreting co-fractionation datasets to yield complex composition, abundance and rearrangements between samples depend heavily on prior evidence. We introduce PCprophet, a computational framework to identify novel protein complexes from SEC-SWATH-MS data and to characterize their changes across different experimental conditions. We demonstrate accurate prediction of protein complexes (AUC >0.99 and accuracy around 97%) via five-fold cross-validation on SEC-SWATH-MS data, show improved performance over state-of-the-art approaches on multiple annotated co-fractionation datasets, and describe a Bayesian approach to analyse altered protein-protein interactions across conditions. PCprophet is a generic computational tool consisting of modules for data pre-processing, hypothesis generation, machine-learning prediction, post-prediction processing, and differential analysis. It can be applied to any co-fractionation MS dataset, independent of separation or quantitative LC-MS workflow employed, and to support the detection and quantitative tracking of novel protein complexes and their physiological dynamics.

scholarly journals The GTPase Rab27b regulates the release, autophagic clearance, and toxicity of α-synuclein

2020 ◽  
Vol 295 (23) ◽  
pp. 8005-8016 ◽  
Author(s):  
Rachel Underwood ◽  
Bing Wang ◽  
Christine Carico ◽  
Robert H. Whitaker ◽  
William J. Placzek ◽  
...  
Keyword(s):  
Lewy Bodies ◽  
Neuronal Model ◽  
Primary Component ◽  
Size Exclusion ◽  
Autophagic Flux ◽  
Protein Levels ◽  
Exclusion Chromatography ◽  
Cell Release ◽  
The Impact

α-Synuclein (αsyn) is the primary component of proteinaceous aggregates termed Lewy bodies that pathologically define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). αsyn is hypothesized to spread through the brain in a prion-like fashion by misfolded protein forming a template for aggregation of endogenous αsyn. The cell-to-cell release and uptake of αsyn are considered important processes for its prion-like spread. Rab27b is one of several GTPases essential to the endosomal-lysosomal pathway and is implicated in protein secretion and clearance, but its role in αsyn spread has yet to be characterized. In this study, we used a paracrine αsyn in vitro neuronal model to test the impact of Rab27b on αsyn release, clearance, and toxicity. shRNA-mediated knockdown (KD) of Rab27b increased αsyn-mediated paracrine toxicity. Rab27b reduced αsyn release primarily through nonexosomal pathways, but the αsyn released after Rab27b KD was of higher-molecular-weight species, as determined by size-exclusion chromatography. Rab27b KD increased intracellular levels of insoluble αsyn and led to an accumulation of endogenous light chain 3 (LC3)-positive puncta. Rab27b KD also decreased LC3 turnover after treatment with an autophagosome-lysosome fusion inhibitor, chloroquine, indicating that Rab27b KD induces a defect in autophagic flux. Rab27b protein levels were increased in brain lysates obtained from postmortem tissues of individuals with PD and DLB compared with healthy controls. These data indicate a role for Rab27b in the release, clearance, and toxicity of αsyn and, ultimately, in the pathogenesis of synucleinopathies.

scholarly journals The interaction of the mitochondrial protein importer TOMM34 with HSP70 is regulated by TOMM34 phosphorylation and binding to 14-3-3 adaptors

2020 ◽  
Vol 295 (27) ◽  
pp. 8928-8944 ◽  
Author(s):  
Filip Trcka ◽  
Michal Durech ◽  
Pavla Vankova ◽  
Veronika Vandova ◽  
Oliver Simoncik ◽  
...  
Keyword(s):  
Structural Changes ◽  
Protein Transport ◽  
Mitochondrial Protein ◽  
Inhibitory Effect ◽  
Size Exclusion ◽  
Tetratricopeptide Repeat ◽  
Interaction Interface ◽  
Exclusion Chromatography ◽  
Hydrogen Deuterium

Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90–mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser93 and Ser160, located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro. In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.

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