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The Journal of Cell Biology Sep 2023The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The Saccharomyces cerevisiae inner ring nucleoporin Nup170 has been...
The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The Saccharomyces cerevisiae inner ring nucleoporin Nup170 has been implicated in chromatin organization and the maintenance of gene silencing in subtelomeric regions. To gain insight into how Nup170 regulates this process, we used protein-protein interactions, genetic interactions, and transcriptome correlation analyses to identify the Ctf18-RFC complex, an alternative proliferating cell nuclear antigen (PCNA) loader, as a facilitator of the gene regulatory functions of Nup170. The Ctf18-RFC complex is recruited to a subpopulation of NPCs that lack the nuclear basket proteins Mlp1 and Mlp2. In the absence of Nup170, PCNA levels on DNA are reduced, resulting in the loss of silencing of subtelomeric genes. Increasing PCNA levels on DNA by removing Elg1, which is required for PCNA unloading, rescues subtelomeric silencing defects in nup170Δ. The NPC, therefore, mediates subtelomeric gene silencing by regulating PCNA levels on DNA.
Topics: Carrier Proteins; Chromatin; Gene Silencing; Nuclear Pore; Proliferating Cell Nuclear Antigen; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Telomere; DNA, Fungal
PubMed: 37358474
DOI: 10.1083/jcb.202207060 -
Cell Reports Jul 2023Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetics, genomics, and...
Replication forks terminate at TERs and telomeres. Forks that converge or encounter transcription generate topological stress. Combining genetics, genomics, and transmission electron microscopy, we find that Rrm3 and Sen1 helicases assist termination at TERs; Sen1 specifically acts at telomeres. rrm3 and sen1 genetically interact and fail to terminate replication, exhibiting fragility at termination zones (TERs) and telomeres. sen1rrm3 accumulates RNA-DNA hybrids and X-shaped gapped or reversed converging forks at TERs; sen1, but not rrm3, builds up RNA polymerase II (RNPII) at TERs and telomeres. Rrm3 and Sen1 restrain Top1 and Top2 activities, preventing toxic accumulation of positive supercoil at TERs and telomeres. We suggest that Rrm3 and Sen1 coordinate the activities of Top1 and Top2 when forks encounter transcription head on or codirectionally, respectively, thus preventing the slowing down of DNA and RNA polymerases. Hence Rrm3 and Sen1 are indispensable to generate permissive topological conditions for replication termination.
Topics: DNA; DNA Helicases; DNA Replication; DNA Topoisomerases, Type II; RNA Helicases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37405920
DOI: 10.1016/j.celrep.2023.112747 -
The EMBO Journal Dec 2023Protein requirements of eukaryotic cells are ensured by proteostasis, which is mediated by tight control of TORC1 activity. Upon TORC1 inhibition, protein degradation is...
Protein requirements of eukaryotic cells are ensured by proteostasis, which is mediated by tight control of TORC1 activity. Upon TORC1 inhibition, protein degradation is increased and protein synthesis is reduced through inhibition of translation initiation to maintain cell viability. Here, we show that the ribosome-associated complex (RAC)/Ssb chaperone system, composed of the HSP70 chaperone Ssb and its HSP40 co-chaperone Zuo1, is required to maintain proteostasis and cell viability under TORC1 inhibition in Saccharomyces cerevisiae. In the absence of Zuo1, translation does not decrease in response to the loss of TORC1 activity. A functional interaction between Zuo1 and Ssb is required for proper translational control and proteostasis maintenance upon TORC1 inhibition. Furthermore, we have shown that the rapid degradation of eIF4G following TORC1 inhibition is mediated by autophagy and is prevented in zuo1Δ cells, contributing to decreased survival in these conditions. We found that autophagy is defective in zuo1Δ cells, which impedes eIF4G degradation upon TORC1 inhibition. Our findings identify an essential role for RAC/Ssb in regulating translation in response to changes in TORC1 signalling.
Topics: Eukaryotic Initiation Factor-4G; HSP70 Heat-Shock Proteins; Molecular Chaperones; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37984430
DOI: 10.15252/embj.2022113240 -
The Journal of Chemical Physics Oct 2023The application of terahertz radiation has been shown to affect both protein structure and cellular function. As the key to such structural changes lies in the dynamic...
The application of terahertz radiation has been shown to affect both protein structure and cellular function. As the key to such structural changes lies in the dynamic response of a protein, we focus on the susceptibility of the protein's internal dynamics to mechanical stress induced by acoustic pressure waves. We use the open-boundary molecular dynamics method, which allows us to simulate the protein exposed to acoustic waves. By analyzing the dynamic fluctuations of the protein subunits, we demonstrate that the protein is highly susceptible to acoustic waves with frequencies corresponding to those of the internal protein vibrations. This is confirmed by changes in the compactness of the protein structure. As the amplitude of the pressure wave increases, even larger deviations from average positions and larger changes in protein compactness are observed. Furthermore, performing the mode-projection analysis, we show that the breathing-like character of collective modes is enhanced at frequencies corresponding to those used to excite the protein.
Topics: Proteins; Motion; Acoustics; Vibration; Molecular Dynamics Simulation
PubMed: 37782253
DOI: 10.1063/5.0163801 -
Current Protocols Oct 2023Clickable glutathione is a glutathione-derived chemical probe designed to identify and analyze protein S-glutathionylation, a major cysteine oxidation in redox...
Clickable glutathione is a glutathione-derived chemical probe designed to identify and analyze protein S-glutathionylation, a major cysteine oxidation in redox signaling. An engineered glutathione synthetase mutant (GS M4) is used to synthesize clickable glutathione in cells or in vitro, which affords utility via click chemistry to detect, identify, and quantify glutathionylation on individual or global proteins in biochemical and mass spectrometric analyses. The clickable glutathione approach is valuable for the unequivocal identification of glutathionylated cysteines, among many reversible cysteine oxoforms, via the direct enrichment and detection of glutathionylated proteins or peptides. Clickable glutathione, in combination with GS M4, has demonstrated utility in the mass-spectrometry-based discovery and profiling of new proteins and cysteines for glutathionylation in cell lines in response to physiologic and oxidative stress. The approach is versatile and applicable to validating the glutathionylation of proteins and cysteines in other biochemical analysis beside mass spectrometry. Here, we describe the applications of clickable glutathione and provide detailed protocols for the identification, profiling, and detection of glutathionylated proteins and cysteines. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of glutathionylated cysteine in individual proteins in vitro Basic Protocol 2: Proteomic identification and quantification of glutathionylation Basic Protocol 3: Biochemical validation of glutathionylation in cells.
Topics: Cysteine; Proteomics; Protein Processing, Post-Translational; Glutathione; Proteins; Glutathione Synthase
PubMed: 37818879
DOI: 10.1002/cpz1.907 -
DNA Repair Jul 2024The ATP-dependent molecular chaperone Cdc48 (in yeast) and its human counterpart p97 (also known as VCP), are essential for a variety of cellular processes, including... (Review)
Review
The ATP-dependent molecular chaperone Cdc48 (in yeast) and its human counterpart p97 (also known as VCP), are essential for a variety of cellular processes, including the removal of DNA-protein crosslinks (DPCs) from the DNA. Growing evidence demonstrates in the last years that Cdc48/p97 is pivotal in targeting ubiquitinated and SUMOylated substrates on chromatin, thereby supporting the DNA damage response. Along with its cofactors, notably Ufd1-Npl4, Cdc48/p97 has emerged as a central player in the unfolding and processing of DPCs. This review introduces the detailed structure, mechanism and cellular functions of Cdc48/p97 with an emphasis on the current knowledge of DNA-protein crosslink repair pathways across several organisms. The review concludes by discussing the potential therapeutic relevance of targeting p97 in DPC repair.
Topics: Valosin Containing Protein; DNA Repair; Humans; Saccharomyces cerevisiae Proteins; DNA; Saccharomyces cerevisiae; DNA Damage; Cell Cycle Proteins; Nuclear Proteins; Adenosine Triphosphatases; DNA-Binding Proteins; Animals; Intracellular Signaling Peptides and Proteins
PubMed: 38744091
DOI: 10.1016/j.dnarep.2024.103691 -
Science (New York, N.Y.) Dec 2023Kinetochores couple chromosomes to the mitotic spindle to segregate the genome during cell division. An error correction mechanism drives the turnover of...
Kinetochores couple chromosomes to the mitotic spindle to segregate the genome during cell division. An error correction mechanism drives the turnover of kinetochore-microtubule attachments until biorientation is achieved. The structural basis for how kinetochore-mediated chromosome segregation is accomplished and regulated remains an outstanding question. In this work, we describe the cryo-electron microscopy structure of the budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules. Complex assembly occurs through multiple interfaces, and a staple within Dam1 aids ring assembly. Perturbation of key interfaces suppresses yeast viability. Force-rupture assays indicated that this is a consequence of impaired kinetochore-microtubule attachment. The presence of error correction phosphorylation sites at Ndc80-Dam1 ring complex interfaces and the Dam1 staple explains how kinetochore-microtubule attachments are destabilized and reset.
Topics: Cell Cycle Proteins; Chromosome Segregation; Cryoelectron Microscopy; Kinetochores; Microtubule-Associated Proteins; Microtubules; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Protein Conformation
PubMed: 38060647
DOI: 10.1126/science.adj8736 -
Nucleic Acids Research Sep 2023In meiosis, Dmc1 recombinase and the general recombinase Rad51 are responsible for pairing homologous chromosomes and exchanging strands. Fission yeast...
In meiosis, Dmc1 recombinase and the general recombinase Rad51 are responsible for pairing homologous chromosomes and exchanging strands. Fission yeast (Schizosaccharomyces pombe) Swi5-Sfr1 and Hop2-Mnd1 stimulate Dmc1-driven recombination, but the stimulation mechanism is unclear. Using single-molecule fluorescence resonance energy transfer (smFRET) and tethered particle motion (TPM) experiments, we showed that Hop2-Mnd1 and Swi5-Sfr1 individually enhance Dmc1 filament assembly on single-stranded DNA (ssDNA) and adding both proteins together allows further stimulation. FRET analysis showed that Hop2-Mnd1 enhances the binding rate of Dmc1 while Swi5-Sfr1 specifically reduces the dissociation rate during the nucleation, about 2-fold. In the presence of Hop2-Mnd1, the nucleation time of Dmc1 filaments shortens, and doubling the ss/double-stranded DNA (ss/dsDNA) junctions of DNA substrates reduces the nucleation times in half. Order of addition experiments confirmed that Hop2-Mnd1 binds on DNA to recruit and stimulate Dmc1 nucleation at the ss/dsDNA junction. Our studies directly support the molecular basis of how Hop2-Mnd1 and Swi5-Sfr1 act on different steps during the Dmc1 filament assembly. DNA binding of these accessory proteins and nucleation preferences of recombinases thus dictate how their regulation can take place.
Topics: Cell Cycle Proteins; DNA; DNA, Single-Stranded; Meiosis; Rad51 Recombinase; Recombinases; Schizosaccharomyces
PubMed: 37395447
DOI: 10.1093/nar/gkad561 -
Seminars in Liver Disease Feb 2024TAM (TYRO3, AXL, and MERTK) protein tyrosine kinase membrane receptors and their vitamin K-dependent ligands GAS6 and protein S (PROS) are well-known players in tumor...
TAM (TYRO3, AXL, and MERTK) protein tyrosine kinase membrane receptors and their vitamin K-dependent ligands GAS6 and protein S (PROS) are well-known players in tumor biology and autoimmune diseases. In contrast, TAM regulation of fibrogenesis and the inflammation mechanisms underlying metabolic dysfunction-associated steatohepatitis (MASH), cirrhosis, and, ultimately, liver cancer has recently been revealed. GAS6 and PROS binding to phosphatidylserine exposed in outer membranes of apoptotic cells links TAMs, particularly MERTK, with hepatocellular damage. In addition, AXL and MERTK regulate the development of liver fibrosis and inflammation in chronic liver diseases. Acute hepatic injury is also mediated by the TAM system, as recent data regarding acetaminophen toxicity and acute-on-chronic liver failure have uncovered. Soluble TAM-related proteins, mainly released from activated macrophages and hepatic stellate cells after hepatic deterioration, are proposed as early serum markers for disease progression. In conclusion, the TAM system is becoming an interesting pharmacological target in liver pathology and a focus of future biomedical research in this field.
Topics: Humans; Axl Receptor Tyrosine Kinase; c-Mer Tyrosine Kinase; Inflammation; Liver Cirrhosis; Receptor Protein-Tyrosine Kinases
PubMed: 38395061
DOI: 10.1055/a-2275-0408 -
Computers in Biology and Medicine Jun 2024Predicting three-dimensional (3D) protein structures has been challenging for decades. The emergence of AlphaFold2 (AF2), a deep learning-based machine learning method... (Review)
Review
Predicting three-dimensional (3D) protein structures has been challenging for decades. The emergence of AlphaFold2 (AF2), a deep learning-based machine learning method developed by DeepMind, became a game changer in the protein folding community. AF2 can predict a protein's three-dimensional structure with high confidence based on its amino acid sequence. Accurate prediction of protein structures can dramatically accelerate our understanding of biological mechanisms and provide a solid foundation for reliable drug design. Although AF2 breaks through the barriers in predicting protein structures, many rooms remain to be further studied. This review provides a brief historical overview of the development of protein structure prediction, covering template-based, template-free, and machine learning-based methods. In addition to reviewing the potential benefits (Pros) and considerations (Cons) of using AF2, this review summarizes the diverse applications, including protein structure predictions, dynamic changes, point mutation, integration of language model and experimental data, protein complex, and protein-peptide interaction. It underscores recent advancements in efficiency, reliability, and broad application of AF2. This comprehensive review offers valuable insights into the applications of AF2 and AF2-inspired AI methods in structural biology and its potential for clinically significant drug target discovery.
Topics: Proteins; Humans; Protein Folding; Deep Learning; Protein Conformation; Models, Molecular; Computational Biology; Machine Learning
PubMed: 38761500
DOI: 10.1016/j.compbiomed.2024.108620