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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 -
The Journal of Cell Biology Aug 2017Disturbances in endoplasmic reticulum (ER) homeostasis create a condition termed ER stress. This activates the unfolded protein response (UPR), which alters the...
Disturbances in endoplasmic reticulum (ER) homeostasis create a condition termed ER stress. This activates the unfolded protein response (UPR), which alters the expression of many genes involved in ER quality control. We show here that ER stress causes the aggregation of proteins, most of which are not ER or secretory pathway proteins. Proteomic analysis of the aggregated proteins revealed enrichment for intrinsically aggregation-prone proteins rather than proteins which are affected in a stress-specific manner. Aggregation does not arise because of overwhelming proteasome-mediated degradation but because of a general disruption of cellular protein homeostasis. We further show that overexpression of certain chaperones abrogates protein aggregation and protects a UPR mutant against ER stress conditions. The onset of ER stress is known to correlate with various disease processes, and our data indicate that widespread amorphous and amyloid protein aggregation is an unanticipated outcome of such stress.
Topics: Basic-Leucine Zipper Transcription Factors; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Genotype; Membrane Glycoproteins; Molecular Chaperones; Mutation; Phenotype; Prion Proteins; Protein Aggregates; Protein Aggregation, Pathological; Protein Serine-Threonine Kinases; Proteomics; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Time Factors; Ubiquitin-Protein Ligases; Unfolded Protein Response
PubMed: 28630146
DOI: 10.1083/jcb.201612165 -
Nature Structural & Molecular Biology Jan 2021SWI/SNF chromatin remodelers modify the position and spacing of nucleosomes and, in humans, are linked to cancer. To provide insights into the assembly and regulation of...
SWI/SNF chromatin remodelers modify the position and spacing of nucleosomes and, in humans, are linked to cancer. To provide insights into the assembly and regulation of this protein family, we focused on a subcomplex of the Saccharomyces cerevisiae RSC comprising its ATPase (Sth1), the essential actin-related proteins (ARPs) Arp7 and Arp9 and the ARP-binding protein Rtt102. Cryo-EM and biochemical analyses of this subcomplex shows that ARP binding induces a helical conformation in the helicase-SANT-associated (HSA) domain of Sth1. Surprisingly, the ARP module is rotated 120° relative to the full RSC about a pivot point previously identified as a regulatory hub in Sth1, suggesting that large conformational changes are part of Sth1 regulation and RSC assembly. We also show that a conserved interaction between Sth1 and the nucleosome acidic patch enhances remodeling. As some cancer-associated mutations dysregulate rather than inactivate SWI/SNF remodelers, our insights into RSC complex regulation advance a mechanistic understanding of chromatin remodeling in disease states.
Topics: Amino Acid Sequence; Carrier Proteins; Cell Cycle Proteins; Chromatin; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; Cryoelectron Microscopy; DNA-Binding Proteins; Nuclear Proteins; Nucleosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors
PubMed: 33288924
DOI: 10.1038/s41594-020-00528-8 -
Genetics Aug 2019Recombination between divergent DNA sequences is actively prevented by heteroduplex rejection mechanisms. In baker's yeast, such antirecombination mechanisms can be...
Recombination between divergent DNA sequences is actively prevented by heteroduplex rejection mechanisms. In baker's yeast, such antirecombination mechanisms can be initiated by the recognition of DNA mismatches in heteroduplex DNA by MSH proteins, followed by recruitment of the Sgs1-Top3-Rmi1 helicase-topoisomerase complex to unwind the recombination intermediate. We previously showed that the repair/rejection decision during single-strand annealing recombination is temporally regulated by MSH (utomolog) protein levels and by factors that excise nonhomologous single-stranded tails. These observations, coupled with recent studies indicating that mismatch repair (MMR) factors interact with components of the histone chaperone machinery, encouraged us to explore roles for epigenetic factors and chromatin conformation in regulating the decision to reject repair recombination between divergent DNA substrates. This work involved the use of an inverted repeat recombination assay thought to measure sister chromatid repair during DNA replication. Our observations are consistent with the histone chaperones CAF-1 and Rtt106, and the histone deacetylase Sir2, acting to suppress heteroduplex rejection and the Rpd3, Hst3, and Hst4 deacetylases acting to promote heteroduplex rejection. These observations, and double-mutant analysis, have led to a model in which nucleosomes located at DNA lesions stabilize recombination intermediates and compete with MMR factors that mediate heteroduplex rejection.
Topics: Histone Chaperones; Histone Code; Histone Deacetylases; Homologous Recombination; Molecular Chaperones; Nucleosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Silent Information Regulator Proteins, Saccharomyces cerevisiae; Sirtuin 2
PubMed: 31221666
DOI: 10.1534/genetics.119.302395 -
EMBO Reports Oct 2022Light and ambient high temperature (HT) have opposite effects on seed germination. Light induces seed germination through activating the photoreceptor phytochrome B...
Light and ambient high temperature (HT) have opposite effects on seed germination. Light induces seed germination through activating the photoreceptor phytochrome B (phyB), resulting in the stabilization of the transcription factor HFR1, which in turn sequesters the suppressor PIF1. HT suppresses seed germination and triggers protein S-nitrosylation. Here, we find that HT suppresses seed germination by inducing the S-nitrosylation of HFR1 at C164, resulting in its degradation, the release of PIF1, and the activation of PIF1-targeted SOMNUS (SOM) expression to alter gibberellin (GA) and abscisic acid (ABA) metabolism. Active phyB (phyB ) antagonizes HFR1 S-nitrosylation and degradation by increasing S-nitrosoglutathione reductase (GSNOR) activity. In line with this, substituting cysteine-164 of HFR1 with serine (HFR1 ) abolishes the S-nitrosylation of HFR1 and decreases the HT-induced degradation of HFR1. Taken together, our study suggests that HT and phyB antagonistically modulate the S-nitrosylation level of HFR1 to coordinate seed germination, and provides the possibility to enhance seed thermotolerance through gene-editing of HFR1.
Topics: Abscisic Acid; Arabidopsis; Arabidopsis Proteins; Cysteine; DNA-Binding Proteins; Gene Expression Regulation, Plant; Germination; Gibberellins; Light; Phytochrome; Phytochrome B; Protein S; Seeds; Serine; Temperature; Transcription Factors
PubMed: 36062942
DOI: 10.15252/embr.202154371 -
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