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Journal of the American Chemical Society Jun 2020Proteins form complex biological machineries whose functions in the cell are highly regulated at both the cellular and molecular levels. Cellular regulation of protein... (Review)
Review
Proteins form complex biological machineries whose functions in the cell are highly regulated at both the cellular and molecular levels. Cellular regulation of protein functions involves differential gene expressions, post-translation modifications, and signaling cascades. Molecular regulation, on the other hand, involves tuning an optimal local protein environment for the functional site. Precisely how a protein achieves such an optimal environment around a given functional site is not well understood. Herein, by surveying the literature, we first summarize the various reported strategies used by certain proteins to ensure their correct functioning. We then formulate three key physicochemical factors for regulating a protein's functional site, namely, (i) its immediate interactions, (ii) its solvent accessibility, and (iii) its conformational flexibility. We illustrate how these factors are applied to regulate the functions of free/metal-bound Cys and Zn sites in proteins.
Topics: Humans; Protein Conformation; Proteins
PubMed: 32407086
DOI: 10.1021/jacs.0c02430 -
ACS Chemical Biology Apr 2020In contrast to the myriad approaches available to study protein misfolding and aggregation , relatively few tools are available for the study of these processes in the...
In contrast to the myriad approaches available to study protein misfolding and aggregation , relatively few tools are available for the study of these processes in the cellular context. This is in part due to the complexity of the cellular environment which, for instance, interferes with many spectroscopic approaches. Here, we describe a tripartite fusion approach that can be used to assess protein stability and solubility in the cytosol of . Our biosensors contain tripartite fusions in which a protein of interest is inserted into antibiotic resistance markers. These fusions act to directly link the aggregation susceptibility and stability of the inserted protein to antibiotic resistance. We demonstrate a linear relationship between the thermodynamic stabilities of variants of the model folding protein immunity protein 7 (Im7) fused into the resistance markers and their antibiotic resistance readouts. We also use this system to investigate the properties of the yeast prion proteins Sup35 and Rnq1 and proteins whose aggregation is associated with some of the most prevalent neurodegenerative misfolding disorders, including peptide amyloid beta 1-42 (Aβ42), which is involved in Alzheimer's disease, and protein α-synuclein, which is linked to Parkinson's disease.
Topics: Amyloid beta-Peptides; Biosensing Techniques; Carrier Proteins; Escherichia coli; Escherichia coli Proteins; Peptide Fragments; Peptide Termination Factors; Prions; Protein Folding; Protein Multimerization; Protein Stability; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; alpha-Synuclein
PubMed: 32105441
DOI: 10.1021/acschembio.0c00083 -
Cell Reports Jun 2023Efficient replication of terminal DNA is crucial to maintain telomere stability. In fission yeast, Taz1 and the Stn1-Ten1 (ST) complex play prominent roles in DNA-ends...
Efficient replication of terminal DNA is crucial to maintain telomere stability. In fission yeast, Taz1 and the Stn1-Ten1 (ST) complex play prominent roles in DNA-ends replication. However, their function remains elusive. Here, we have analyzed genome-wide replication and show that ST does not affect genome-wide replication but is crucial for the efficient replication of a subtelomeric region called STE3-2. We further show that, when ST function is compromised, a homologous recombination (HR)-based fork restart mechanism becomes necessary for STE3-2 stability. While both Taz1 and Stn1 bind to STE3-2, we find that the STE3-2 replication function of ST is independent of Taz1 but relies on its association with the shelterin proteins Pot1-Tpz1-Poz1. Finally, we demonstrate that the firing of an origin normally inhibited by Rif1 can circumvent the replication defect of subtelomeres when ST function is compromised. Our results help illuminate why fission yeast telomeres are terminal fragile sites.
Topics: Schizosaccharomyces; Telomere-Binding Proteins; Schizosaccharomyces pombe Proteins; Telomere; Shelterin Complex; DNA Replication; DNA-Binding Proteins
PubMed: 37243596
DOI: 10.1016/j.celrep.2023.112537 -
Experimental Cell Research Nov 2020Epigenetic modifications allow cells to quickly alter their gene expression and adapt to different stresses. In addition to direct chromatin modifications, prion-like... (Review)
Review
Epigenetic modifications allow cells to quickly alter their gene expression and adapt to different stresses. In addition to direct chromatin modifications, prion-like proteins have recently emerged as a system that can sense and adapt the cellular response to stressful conditions. Interestingly, such responses are maintained through prions' self-templating conformations and transmitted to the progeny of the cell that established a prion trait. Alternatively, mnemons are prion-like proteins which conformational switch encodes memories of past events and yet does not propagate to daughter cells. In this review, we explore the biology of the recently described prions found in Saccharomyces cerevisiae including [ESI], [SMAUG], [GAR], [MOT3], [MOD], [LSB] as well as the Whi3 mnemon. The reversibility of the phenotypes they encode allows cells to remove traits which are no longer adaptive under stress relief and chaperones play a fundamental role in all steps of prion-like proteins functions. Thus, the interplay between chaperones and prion-like proteins provides a framework to establish responses to challenging environments.
Topics: Adaptation, Physiological; Animals; Carrier Proteins; Epigenesis, Genetic; GPI-Linked Proteins; Genotype; Humans; Molecular Chaperones; Phenotype; Prions; Protein Conformation; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Stress, Physiological; Transcription Factors
PubMed: 32896568
DOI: 10.1016/j.yexcr.2020.112262 -
Nucleic Acids Research May 2021H/ACA Box ribonucleoprotein complexes (RNPs) play a major role in modification of rRNA and snRNA, catalyzing the sequence specific pseudouridylation in eukaryotes and...
H/ACA Box ribonucleoprotein complexes (RNPs) play a major role in modification of rRNA and snRNA, catalyzing the sequence specific pseudouridylation in eukaryotes and archaea. This enzymatic reaction takes place on a substrate RNA recruited via base pairing to an internal loop of the snoRNA. Eukaryotic snoRNPs contain the four proteins Nop10, Cbf5, Gar1 and Nhp2, with Cbf5 as the catalytic subunit. In contrast to archaeal H/ACA RNPs, eukaryotic snoRNPs contain several conserved features in both the snoRNA as well as the protein components. Here, we reconstituted the eukaryotic H/ACA RNP containing snR81 as a guide RNA in vitro and report on the effects of these eukaryote specific features on complex assembly and enzymatic activity. We compare their contribution to pseudouridylation activity for stand-alone hairpins versus the bipartite RNP. Using single molecule FRET spectroscopy, we investigated the role of the different eukaryote-specific proteins and domains on RNA folding and complex assembly, and assessed binding of substrate RNA to the RNP. Interestingly, we found diverging effects for the two hairpins of snR81, suggesting hairpin-specific requirements for folding and RNP formation. Our results for the first time allow assessing interactions between the individual hairpin RNPs in the context of the full, bipartite snoRNP.
Topics: Catalysis; Escherichia coli; Fluorescence Resonance Energy Transfer; Gene Expression; Hydro-Lyases; In Vitro Techniques; Inverted Repeat Sequences; Microtubule-Associated Proteins; Models, Molecular; Nuclear Proteins; Protein Domains; RNA Folding; RNA, Small Nucleolar; RNA-Binding Proteins; Recombinant Proteins; Ribonucleoproteins, Small Nuclear; Ribonucleoproteins, Small Nucleolar; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; RNA, Guide, CRISPR-Cas Systems
PubMed: 33823543
DOI: 10.1093/nar/gkab177 -
Nature Structural & Molecular Biology Jan 2022Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase,...
Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.
Topics: Amino Acid Sequence; Cell Cycle Proteins; Checkpoint Kinase 2; DNA, Fungal; Minichromosome Maintenance Complex Component 4; Molecular Docking Simulation; Nucleotides; Phosphorylation; Protein Multimerization; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity
PubMed: 34963704
DOI: 10.1038/s41594-021-00698-z -
The Journal of Cell Biology Jul 2020In this issue, Choudhary et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.201910177) address the nature of the ER subdomain from which lipid droplets emanate and...
In this issue, Choudhary et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.201910177) address the nature of the ER subdomain from which lipid droplets emanate and how several assembly proteins interact. Their data indicate that seipin/Nem1 marks these sites and provide a detailed working model for assembling the protein complex.
Topics: GTP-Binding Protein gamma Subunits; Lipid Droplets; Nuclear Proteins; Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 32580210
DOI: 10.1083/jcb.202006025 -
Nucleic Acids Research Oct 2022Efficient gene expression requires properly matured mRNAs for functional transcript translation. Several factors including the guard proteins monitor maturation and act...
Efficient gene expression requires properly matured mRNAs for functional transcript translation. Several factors including the guard proteins monitor maturation and act as nuclear retention factors for unprocessed pre-mRNAs. Here we show that the guard protein Npl3 monitors 5'-capping. In its absence, uncapped transcripts resist degradation, because the Rat1-Rai1 5'-end degradation factors are not efficiently recruited to these faulty transcripts. Importantly, in npl3Δ, these improperly capped transcripts escape this quality control checkpoint and leak into the cytoplasm. Our data suggest a model in which Npl3 associates with the Rai1 bound pre-mRNAs. In case the transcript was properly capped and is thus CBC (cap binding complex) bound, Rai1 dissociates from Npl3 allowing the export factor Mex67 to interact with this guard protein and support nuclear export. In case Npl3 does not detect proper capping through CBC attachment, Rai1 binding persists and Rat1 can join this 5'-complex to degrade the faulty transcript.
Topics: Nuclear Proteins; RNA Precursors; RNA-Binding Proteins; Saccharomyces cerevisiae Proteins; RNA Caps
PubMed: 36305816
DOI: 10.1093/nar/gkac952 -
The Journal of Biological Chemistry Jul 2021Pentatricopeptide repeat (PPR) proteins are a large family of proteins that act primarily at different posttranscriptional steps of organellar gene expression. We have...
Pentatricopeptide repeat (PPR) proteins are a large family of proteins that act primarily at different posttranscriptional steps of organellar gene expression. We have previously found that the Schizosaccharomyces pombe PPR protein mpal10 interacts with mitochondrial translational activator Mpa1, and both are essential for mitochondrial protein synthesis. However, it is unclear how these two proteins function in mitochondrial protein synthesis in S. pombe. In this study, we further investigated the role of Ppr10 and Mpa1 in mitochondrial protein synthesis. Mitochondrial translational initiation requires two initiation factors, Mti2 and Mti3, which bind to the small subunit of the mitochondrial ribosome (mt-SSU) during the formation of the mitochondrial translational initiation complex. Using sucrose gradient sedimentation analysis, we found that disruption of ppr10, mpa1, or the PPR motifs in Ppr10 impairs the association of Mti2 and Mti3 with the mt-SSU, suggesting that both Ppr10 and Mpa1 may be required for the interaction of Mti2 and Mti3 with the mt-SSU during the assembly of mitochondrial translational initiation complex. Loss of Ppr10 perturbs the association of mitochondrially encoded cytochrome b (cob1) and cytochrome c oxidase subunit 1 (cox1) mRNAs with assembled mitochondrial ribosomes. Proteomic analysis revealed that a fraction of Ppr10 and Mpa1 copurified with a subset of mitoribosomal proteins. The PPR motifs of Ppr10 are necessary for its interaction with Mpa1 and that disruption of these PPR motifs impairs mitochondrial protein synthesis. Our results suggest that Ppr10 and Mpa1 function together to mediate mitochondrial translational initiation.
Topics: Binding Sites; Carrier Proteins; Eukaryotic Initiation Factors; Mitochondria; Mitochondrial Proteins; Mitochondrial Ribosomes; Peptide Chain Initiation, Translational; Protein Binding; RNA, Messenger; RNA-Binding Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 34119521
DOI: 10.1016/j.jbc.2021.100869 -
Life Science Alliance Oct 2022Yeast use the G-protein-coupled receptor signaling pathway to detect and track the mating pheromone. The G-protein-coupled receptor pathway is inhibited by the regulator...
Yeast use the G-protein-coupled receptor signaling pathway to detect and track the mating pheromone. The G-protein-coupled receptor pathway is inhibited by the regulator of G-protein signaling (RGS) Sst2 which induces Gα GTPase activity and inactivation of downstream signaling. G-protein signaling activates the MAPK Fus3, which phosphorylates the RGS; however, the role of this modification is unknown. We found that pheromone-induced RGS phosphorylation peaks early; the phospho-state of RGS controls its localization and influences MAPK spatial distribution. Surprisingly, phosphorylation of the RGS promotes completion of cytokinesis before pheromone-induced growth. Completion of cytokinesis in the presence of pheromone is promoted by the kelch-repeat protein, Kel1 and antagonized by the formin Bni1. We found that RGS complexes with Kel1 and prefers the unphosphorylatable RGS mutant. We also found overexpression of unphosphorylatable RGS exacerbates cytokinetic defects, whereas they are rescued by overexpression of Kel1. These data lead us to a model where Kel1 promotes completion of cytokinesis before pheromone-induced polarity but is inhibited by unphosphorylated RGS binding.
Topics: Cytokinesis; GTP-Binding Proteins; GTPase-Activating Proteins; Microfilament Proteins; Mitogen-Activated Protein Kinases; Pheromones; Phosphorylation; RGS Proteins; Receptors, G-Protein-Coupled; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35985794
DOI: 10.26508/lsa.202101245