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Cell Reports Jun 2020The formation of insoluble inclusions in the cytosol and nucleus is associated with impaired protein homeostasis and is a hallmark of several neurodegenerative diseases....
The formation of insoluble inclusions in the cytosol and nucleus is associated with impaired protein homeostasis and is a hallmark of several neurodegenerative diseases. Due to the absence of the autophagic machinery, nuclear protein aggregates require a solubilization step preceding degradation by the 26S proteasome. Using yeast, we identify a nuclear protein quality control pathway required for the clearance of protein aggregates. The nuclear J-domain protein Apj1 supports protein disaggregation together with Hsp70 but independent of the canonical disaggregase Hsp104. Disaggregation mediated by Apj1/Hsp70 promotes turnover rather than refolding. A loss of Apj1 activity uncouples disaggregation from proteasomal turnover, resulting in accumulation of toxic soluble protein species. Endogenous substrates of the Apj1/Hsp70 pathway include both nuclear and cytoplasmic proteins, which aggregate inside the nucleus upon proteotoxic stress. These findings demonstrate the coordinated activity of the Apj1/Hsp70 disaggregation system with the 26S proteasome in facilitating the clearance of toxic inclusions inside the nucleus.
Topics: HSP110 Heat-Shock Proteins; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Heat-Shock Proteins; Nuclear Proteins; Proteasome Endopeptidase Complex; Protein Aggregates; Protein Folding; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 32492414
DOI: 10.1016/j.celrep.2020.107680 -
PloS One 2014Elevated levels of erythrocyte-derived microparticles are present in the circulation in medical conditions affecting the red blood cells. Erythrocyte-derived...
Elevated levels of erythrocyte-derived microparticles are present in the circulation in medical conditions affecting the red blood cells. Erythrocyte-derived microparticles expose phosphatidylserine thus providing a suitable surface for procoagulant reactions leading to thrombin formation via the tenase and prothrombinase complexes. Patients with elevated levels of circulating erythrocyte-derived microparticles have increased thrombin generation in vivo. The aim of the present study was to investigate whether erythrocyte-derived microparticles are able to support the anticoagulant reactions of the protein C system. Erythrocyte-derived microparticles were isolated using ultracentrifugation after incubation of freshly prepared erythrocytes with the ionophore A23187 or from outdated erythrocyte concentrates, the different microparticles preparations yielding similar results. According to flow cytometry analysis, the microparticles exposed phoshatidylserine and bound lactadherin, annexin V, and protein S, which is a cofactor to activated protein C. The microparticles were able to assemble the tenase and prothrombinase complexes and to stimulate the formation of thrombin in plasma-based thrombin generation assay both in presence and absence of added tissue factor. The addition of activated protein C in the thrombin generation assay inhibited thrombin generation in a dose-dependent fashion. The anticoagulant effect of activated protein C in the thrombin generation assay was inhibited by a monoclonal antibody that prevents binding of protein S to microparticles and also attenuated by anti-TFPI antibodies. In the presence of erythrocyte-derived microparticles, activated protein C inhibited tenase and prothrombinase by degrading the cofactors FVIIIa and FVa, respectively. Protein S stimulated the Arg306-cleavage in FVa, whereas efficient inhibition of FVIIIa depended on the synergistic cofactor activity of protein S and FV. In summary, the erythrocyte-derived microparticle surface is suitable for the anticoagulant reactions of the protein C system, which may be important to balance the initiation and propagation of coagulation in vivo.
Topics: Annexin A5; Antibodies; Antigens, Surface; Blood Coagulation; Blood Coagulation Tests; Calcimycin; Cell-Derived Microparticles; Cells, Cultured; Erythrocytes; Factor V; Factor VIIIa; Factor Va; Factor Xa; Humans; Lipoproteins; Milk Proteins; Phosphatidylserines; Protein C; Protein S; Thrombin; Thromboplastin; Ultracentrifugation
PubMed: 25136857
DOI: 10.1371/journal.pone.0104200 -
Cell Oct 2016A comprehensive analysis uncovered a set of yeast proteins promoting protein-based inheritance that shares many of the non-Mendelian properties of prions. Lacking any... (Review)
Review
A comprehensive analysis uncovered a set of yeast proteins promoting protein-based inheritance that shares many of the non-Mendelian properties of prions. Lacking any sequence or structural signatures of known prions, these proteins represent a new class of non-amyloid, protein-based epigenetic determinants that can control phenotype without impacting genotype.
Topics: Epigenesis, Genetic; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Heat-Shock Proteins; Heredity; Prions; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 27716500
DOI: 10.1016/j.cell.2016.09.036 -
PloS One 2021Protein sumoylation, especially when catalyzed by the Mms21 SUMO E3 ligase, plays a major role in suppressing duplication-mediated gross chromosomal rearrangements...
Protein sumoylation, especially when catalyzed by the Mms21 SUMO E3 ligase, plays a major role in suppressing duplication-mediated gross chromosomal rearrangements (dGCRs). How Mms21 targets its substrates in the cell is insufficiently understood. Here, we demonstrate that Esc2, a protein with SUMO-like domains (SLDs), recruits the Ubc9 SUMO conjugating enzyme to specifically facilitate Mms21-dependent sumoylation and suppress dGCRs. The D430R mutation in Esc2 impairs its binding to Ubc9 and causes a synergistic growth defect and accumulation of dGCRs with mutations that delete the Siz1 and Siz2 E3 ligases. By contrast, esc2-D430R does not appreciably affect sensitivity to DNA damage or the dGCRs caused by the catalytically inactive mms21-CH. Moreover, proteome-wide analysis of intracellular sumoylation demonstrates that esc2-D430R specifically down-regulates sumoylation levels of Mms21-preferred targets, including the nucleolar proteins, components of the SMC complexes and the MCM complex that acts as the catalytic core of the replicative DNA helicase. These effects closely resemble those caused by mms21-CH, and are relatively unaffected by deleting Siz1 and Siz2. Thus, by recruiting Ubc9, Esc2 facilitates Mms21-dependent sumoylation to suppress the accumulation of dGCRs independent of Siz1 and Siz2.
Topics: Amino Acid Sequence; Cell Cycle Proteins; DNA Damage; DNA Replication; Down-Regulation; Mutagenesis; Protein Binding; Protein Domains; Protein Stability; Proteomics; SUMO-1 Protein; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sumoylation; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases
PubMed: 33600463
DOI: 10.1371/journal.pone.0247132 -
Protein and Peptide Letters 2018Cysteine S-sulfenylation is a major type of dynamic post-translational modification of the protein that plays an important role in regulating many biological processes... (Review)
Review
BACKGROUND
Cysteine S-sulfenylation is a major type of dynamic post-translational modification of the protein that plays an important role in regulating many biological processes in both of prokaryotic and eukaryotic species. To understand the function of S-sulfenylated proteins, identification of S-sulfenylation sites is an essential step. Due to numerous restrictions of experimental methods, computational prediction of the potential S-sulfenylation sites becomes popular. In this review, we discuss the recent development and challenges in protein S-sulfenylation site prediction from the available datasets, algorithms and accessible services. We also demonstrate the encountered limitation and future perspective of the computational prediction tools.
CONCLUSION
The development of S-sulfenylation site prediction and their application is an emerging field of protein bioinformatics research. Accurate predictors are expected to identify general and species-specific S-sulfenylation sites when more experimental annotation data are available. Combining experimental and computational technologies will definitely accelerate an understanding of protein S-sulfenylation, discovering regulatory networks in living organisms.
Topics: Algorithms; Computational Biology; Computer Simulation; Protein Processing, Post-Translational; Proteins; Sulfenic Acids; Support Vector Machine
PubMed: 30182830
DOI: 10.2174/0929866525666180905110619 -
Methods in Molecular Biology (Clifton,... 2019Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins that are highly conserved and...
Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins that are highly conserved and tightly regulated by a variety of factors. The exocyst complex is one of the multi-subunit tethering complexes and functions in the tethering of the secretory vesicles to the plasma membrane. We have found that the yeast Sec3, a subunit of the exocyst, binds to the t-SNARE protein Sso2 and promotes its interaction with another t-SNARE protein, Sec9. Here, we describe the structural analysis and in vitro membrane fusion assays, by which we found that Sec3 binding leads to a conformational change within Sso2, and facilitates SNARE assembly and the membrane fusion.
Topics: Amino Acid Motifs; Liposomes; Membrane Fusion; Models, Molecular; Mutagenesis; Protein Binding; Qa-SNARE Proteins; Qc-SNARE Proteins; Recombinant Proteins; Saccharomyces cerevisiae Proteins
PubMed: 30317504
DOI: 10.1007/978-1-4939-8760-3_10 -
Journal of Chemical Information and... Feb 2018Water molecules are an important factor in protein-ligand binding. Upon binding of a ligand with a protein's surface, waters can either be displaced by the ligand or may...
Water molecules are an important factor in protein-ligand binding. Upon binding of a ligand with a protein's surface, waters can either be displaced by the ligand or may be conserved and possibly bridge interactions between the protein and ligand. Depending on the specific interactions made by the ligand, displacing waters can yield a gain in binding affinity. The extent to which binding affinity may increase is difficult to predict, as the favorable displacement of a water molecule is dependent on the site-specific interactions made by the water and the potential ligand. Several methods have been developed to predict the location of water sites on a protein's surface, but the majority of methods are not able to take into account both protein dynamics and the interactions made by specific functional groups. Mixed-solvent molecular dynamics (MixMD) is a cosolvent simulation technique that explicitly accounts for the interaction of both water and small molecule probes with a protein's surface, allowing for their direct competition. This method has previously been shown to identify both active and allosteric sites on a protein's surface. Using a test set of eight systems, we have developed a method using MixMD to identify conserved and displaceable water sites. Conserved sites can be determined by an occupancy-based metric to identify sites which are consistently occupied by water even in the presence of probe molecules. Conversely, displaceable water sites can be found by considering the sites which preferentially bind probe molecules. Furthermore, the inclusion of six probe types allows the MixMD method to predict which functional groups are capable of displacing which water sites. The MixMD method consistently identifies sites which are likely to be nondisplaceable and predicts the favorable displacement of water sites that are known to be displaced upon ligand binding.
Topics: Allosteric Site; Amyloid Precursor Protein Secretases; Cell Cycle Proteins; HSP90 Heat-Shock Proteins; Humans; Ligands; Molecular Dynamics Simulation; Neuraminidase; Nuclear Proteins; Pepsin A; Protein Binding; Proteins; Reproducibility of Results; Solvents; Tetrahydrofolate Dehydrogenase; Thrombin; Transcription Factors; Water; beta-Lactamases
PubMed: 29286658
DOI: 10.1021/acs.jcim.7b00268 -
Molecular Biology of the Cell May 2021Rab family GTPases are key organizers of membrane trafficking and function as markers of organelle identity. Accordingly, Rab GTPases often occupy specific membrane...
Rab family GTPases are key organizers of membrane trafficking and function as markers of organelle identity. Accordingly, Rab GTPases often occupy specific membrane domains, and mechanisms exist to prevent the inappropriate mixing of distinct Rab domains. The yeast Golgi complex can be divided into two broad Rab domains: Ypt1 (Rab1) and Ypt6 (Rab6) are present at the early/medial Golgi and sharply transition to Ypt31/32 (Rab11) at the late Golgi/-Golgi network (TGN). This Rab conversion has been attributed to GTPase-activating protein (GAP) cascades in which Ypt31/32 recruits the Rab-GAPs Gyp1 and Gyp6 to inactivate Ypt1 and Ypt6, respectively. Here we report that Rab transition at the TGN involves additional layers of regulation. We provide new evidence confirming the TRAPPII complex as an important regulator of Ypt6 inactivation and uncover an unexpected role of the Arf1 GTPase in recruiting Gyp1 to drive Ypt1 inactivation at the TGN. Given its established role in directly recruiting TRAPPII to the TGN, Arf1 is therefore a master regulator of Rab conversion on maturing Golgi compartments.
Topics: ADP-Ribosylation Factor 1; GTPase-Activating Proteins; Golgi Apparatus; Monomeric GTP-Binding Proteins; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins; rab GTP-Binding Proteins; trans-Golgi Network
PubMed: 33788577
DOI: 10.1091/mbc.E20-10-0664 -
Current Biology : CB Feb 2017Stimulated by our 2015 Current Biology paper [1], Zambon et al. reinvestigated how three myosin isoforms participate in the formation and constriction of the contractile...
Stimulated by our 2015 Current Biology paper [1], Zambon et al. reinvestigated how three myosin isoforms participate in the formation and constriction of the contractile ring in fission yeast. Our paper presented evidence that these myosin isoforms have distinct roles: "Conventional myosin-II Myo2 is crucial to ring assembly, unconventional myosin-II Myp2 is most important for ring constriction, and type V myosin Myo51 aids the other two myosins." Zambon et al. used different markers to reexamine the contributions of the three myosins to cytokinesis and concluded "that Myo2p is the major motor involved in ring contraction in S. pombe." Here, we show that most of the differences observed by Zambon et al. can be attributed to their use of the Rlc1p-3GFP marker, which genetically interacts with myo2-E1.
Topics: Constriction; Myosin Heavy Chains; Myosin Type II; Myosins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 28171751
DOI: 10.1016/j.cub.2016.12.025 -
International Journal of Molecular... Apr 2023Living organisms on the surface biosphere are periodically yet consistently exposed to light. The adaptive or protective evolution caused by this source of energy has... (Review)
Review
Living organisms on the surface biosphere are periodically yet consistently exposed to light. The adaptive or protective evolution caused by this source of energy has led to the biological systems present in a large variety of organisms, including fungi. Among fungi, yeasts have developed essential protective responses against the deleterious effects of light. Stress generated by light exposure is propagated through the synthesis of hydrogen peroxide and mediated by regulatory factors that are also involved in the response to other stressors. These have included Msn2/4, Crz1, Yap1, and Mga2, thus suggesting that light stress is a common factor in the yeast environmental response.
Topics: DNA-Binding Proteins; Saccharomyces cerevisiae Proteins; Transcription Factors; Saccharomyces cerevisiae; Yeasts; Membrane Proteins
PubMed: 37108091
DOI: 10.3390/ijms24086929