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International Journal of Molecular... Nov 2021Most secreted and membrane proteins are targeted to and translocated across the endoplasmic reticulum (ER) membrane through the Sec61 protein-conducting channel.... (Review)
Review
Most secreted and membrane proteins are targeted to and translocated across the endoplasmic reticulum (ER) membrane through the Sec61 protein-conducting channel. Evolutionarily conserved Sec62 and Sec63 associate with the Sec61 channel, forming the Sec complex and mediating translocation of a subset of proteins. For the last three decades, it has been thought that ER protein targeting and translocation occur via two distinct pathways: signal recognition particle (SRP)-dependent co-translational or SRP-independent, Sec62/Sec63 dependent post-translational translocation pathway. However, recent studies have suggested that ER protein targeting and translocation through the Sec translocon are more intricate than previously thought. This review summarizes the current understanding of the molecular functions of Sec62/Sec63 in ER protein translocation.
Topics: Endoplasmic Reticulum; Heat-Shock Proteins; Membrane Transport Proteins; Protein Processing, Post-Translational; Protein Transport; SEC Translocation Channels; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34884562
DOI: 10.3390/ijms222312757 -
Transfusion and Apheresis Science :... Aug 2019Although suspected conceptually in the 60 s, Protein C and Protein S activities in hemostasis were investigated and reported from the mid-80 s, followed by the... (Review)
Review
Although suspected conceptually in the 60 s, Protein C and Protein S activities in hemostasis were investigated and reported from the mid-80 s, followed by the discovery of Thrombomodulin, an endothelial cell membrane associated protein, playing the most important heamostatic role. These 3 proteins act in regulating thrombogenesis and protecting against thrombo-embolic events. When blood is activated, any trace of circulating thrombin is captured by Thrombomodulin in the microcirculation, making thrombin become an anticoagulant through its capacity to activate Protein C to Activated Protein C, which operates as a sentinel in blood coagulation, in the form of a complex with free Protein S, to block any new blood activation site, and more especially circulating activated Factors V and VIII. Protein S not only acts as the Activated Protein C cofactor, but also as the cofactor of Tissue Factor Pathway Inhibitor. In addition, it has some functions in the complement pathway through its binding to C4b-BP. Another capability of activated protein C is to lower fibrinolytic activity, as the Activated Protein C Inhibitor is also known as Plasminogen Activator Inhibitor 3. The Protein C-Protein S system becomes less efficient in the presence of mutated Factor V (Factor V-Leiden or other variants), which is resistant to its inactivating effect. Other pathologies linked to this system concern the development of allo- or auto-antibodies to Protein S or to thrombin, which can generate severe thrombotic complications in affected patients. Some antithrombotic drugs have originated from this regulatory system. Protein C or Protein S concentrates are used for treating deficient patients. Activated Protein C (especially in patients with sepsis) or Thrombomodulin are proposed as antithrombotic medications. Most importantly, congenital or acquired Protein C or Protein S deficiencies are associated with severe recurrent thrombotic events. From the clinical standpoint most of the patients are heterozygous, as homozygosity is almost incompatible with life in the absence of a continuous and efficient treatment. Laboratory investigation of this highly complex system involves many different specialized assays for measuring these 3 proteins' activities, their antigenic content or their genetic sequence. The Protein S in-vitro anticoagulant activity is weak and contrasts with its high antithrombotic role in-vivo, showing that diagnostic assays have not yet succeeded in reproducing all the natural mechanisms for evidencing the anticoagulant role of Protein S. This paradoxal notion is discussed and illustrated in this manuscript as well is a revisit of the major characteristics and pathophysiological functions of the Protein C-Protein S-Thrombomodulin system; the associated pathologies; and the main laboratory tools available for clinical diagnosis. In respect to future perspectives, we also focused on developing more significant and relevant assays, especially for Protein S, thanks to the understanding of its biological roles.
Topics: Animals; Blood Coagulation; Humans; Protein C; Protein S; Signal Transduction; Thrombomodulin
PubMed: 31256946
DOI: 10.1016/j.transci.2019.06.008 -
Current Genetics Dec 2019Centromere identity is specified epigenetically by specialized nucleosomes containing the evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast,... (Review)
Review
Centromere identity is specified epigenetically by specialized nucleosomes containing the evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) which is essential for faithful chromosome segregation. However, the mechanisms of epigenetic regulation of Cse4 have not been clearly defined. We have identified two kinases, Cdc5 (Plk1 in humans) and Ipl1 (Aurora B kinase in humans) that phosphorylate Cse4 to prevent chromosomal instability (CIN). Cdc5 associates with Cse4 in mitosis and Cdc5-mediated phosphorylation of Cse4 is coincident with the centromeric enrichment of Cdc5 during metaphase. Defects in Cdc5-mediated Cse4 phosphorylation causes CIN, whereas constitutive association of Cdc5 with Cse4 results in lethality. Cse4 is also a substrate for Ipl1 and phospho-mimetic cse4 mutants suppress growth defects of ipl1 and Ipl1 kinetochore substrate mutants, namely dam1 spc34 and ndc80. Ipl1-mediated phosphorylation of Cse4 regulates kinetochore-microtubule interactions and chromosome biorientation. We propose that collaboration of Cdc5- and Ipl1-mediated phosphorylation of Cse4 modulates kinetochore structure and function, and chromosome biorientation. These findings demonstrate how phosphorylation of Cse4 regulates the integrity of the kinetochore, and acts as an epigenetic marker for mitotic control.
Topics: Aurora Kinases; Cell Cycle Proteins; Centromere; Centromere Protein A; Chromosomal Proteins, Non-Histone; Chromosome Segregation; DNA-Binding Proteins; Kinetochores; Microtubule-Associated Proteins; Mitosis; Phosphorylation; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31119371
DOI: 10.1007/s00294-019-00997-5 -
Open Biology Jan 2022Kinetochore (KTs) are macromolecular protein assemblies that attach sister chromatids to spindle microtubules (MTs) and mediate accurate chromosome segregation during...
Kinetochore (KTs) are macromolecular protein assemblies that attach sister chromatids to spindle microtubules (MTs) and mediate accurate chromosome segregation during mitosis. The outer KT consists of the KMN network, a protein super-complex comprising nl1 (yeast Spc105), is12 (yeast Mtw1), and dc80 (yeast Ndc80), which harbours sites for MT binding. Within the KMN network, Spc105 acts as an interaction hub of components involved in spindle assembly checkpoint (SAC) signalling. It is known that Spc105 forms a complex with KT component Kre28. However, where Kre28 physically localizes in the budding yeast KT is not clear. The exact function of Kre28 at the KT is also unknown. Here, we investigate how Spc105 and Kre28 interact and how they are organized within bioriented yeast KTs using genetics and cell biological experiments. Our microscopy data show that Spc105 and Kre28 localize at the KT with a 1 : 1 stoichiometry. We also show that the Kre28-Spc105 interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the KTs. We created several truncation mutants of kre28 that affect Spc105 loading at the KTs. When over-expressed, these mutants sustain the cell viability, but SAC signalling and KT biorientation are impaired. Therefore, we conclude that Kre28 contributes to chromosome biorientation and high-fidelity segregation at least indirectly by regulating Spc105 localization at the KTs.
Topics: Chromosome Segregation; Kinetochores; Microtubule-Associated Proteins; Microtubules; Mitosis; Nuclear Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spindle Apparatus
PubMed: 35042402
DOI: 10.1098/rsob.210274 -
Nature Structural & Molecular Biology Dec 2023Over half of mitochondrial proteins are imported from the cytosol via the pre-sequence pathway, controlled by the TOM complex in the outer membrane and the TIM23 complex...
Over half of mitochondrial proteins are imported from the cytosol via the pre-sequence pathway, controlled by the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. The mechanisms through which proteins are translocated via the TOM and TIM23 complexes remain unclear. Here we report the assembly of the active TOM-TIM23 supercomplex of Saccharomyces cerevisiae with translocating polypeptide substrates. Electron cryo-microscopy analyses reveal that the polypeptide substrates pass the TOM complex through the center of a Tom40 subunit, interacting with a glutamine-rich region. Structural and biochemical analyses show that the TIM23 complex contains a heterotrimer of the subunits Tim23, Tim17 and Mgr2. The polypeptide substrates are shielded from lipids by Mgr2 and Tim17, which creates a translocation pathway characterized by a negatively charged entrance and a central hydrophobic region. These findings reveal an unexpected pre-sequence pathway through the TOM-TIM23 supercomplex spanning the double membranes of mitochondria.
Topics: Membrane Transport Proteins; Mitochondrial Precursor Protein Import Complex Proteins; Carrier Proteins; Mitochondrial Membrane Transport Proteins; Saccharomyces cerevisiae Proteins; Protein Transport; Mitochondria; Saccharomyces cerevisiae; Mitochondrial Proteins; Peptides; Membrane Proteins
PubMed: 37696957
DOI: 10.1038/s41594-023-01103-7 -
Cells Feb 2024proliferates by budding, which includes the formation of a cytoplasmic protrusion called the 'bud', into which DNA, RNA, proteins, organelles, and other materials are... (Review)
Review
proliferates by budding, which includes the formation of a cytoplasmic protrusion called the 'bud', into which DNA, RNA, proteins, organelles, and other materials are transported. The transport of organelles into the growing bud must be strictly regulated for the proper inheritance of organelles by daughter cells. In yeast, the RING-type E3 ubiquitin ligases, Dma1 and Dma2, are involved in the proper inheritance of mitochondria, vacuoles, and presumably peroxisomes. These organelles are transported along actin filaments toward the tip of the growing bud by the myosin motor protein, Myo2. During organelle transport, organelle-specific adaptor proteins, namely Mmr1, Vac17, and Inp2 for mitochondria, vacuoles, and peroxisomes, respectively, bridge the organelles and myosin. After reaching the bud, the adaptor proteins are ubiquitinated by the E3 ubiquitin ligases and degraded by the proteasome. Targeted degradation of the adaptor proteins is necessary to unload vacuoles, mitochondria, and peroxisomes from the actin-myosin machinery. Impairment of the ubiquitination of adaptor proteins results in the failure of organelle release from myosin, which, in turn, leads to abnormal dynamics, morphology, and function of the inherited organelles, indicating the significance of proper organelle unloading from myosin. Herein, we summarize the role and regulation of E3 ubiquitin ligases during organelle inheritance in yeast.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin-Protein Ligases; Peroxisomes; Myosins; Ubiquitins; Cell Cycle Proteins; Mitochondrial Proteins
PubMed: 38391905
DOI: 10.3390/cells13040292 -
Current Opinion in Genetics &... Dec 2021DNA double-strand breaks (DSBs) can be repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). HR is initiated by nucleolytic degradation of the... (Review)
Review
DNA double-strand breaks (DSBs) can be repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). HR is initiated by nucleolytic degradation of the DSB ends in a process termed resection. The Mre11-Rad50-Xrs2/NBS1 (MRX/N) complex is a multifunctional enzyme that, aided by the Sae2/CtIP protein, promotes DSB resection and maintains the DSB ends tethered to each other to facilitate their re-ligation. Furthermore, it activates the protein kinase Tel1/ATM, which initiates DSB signaling. In Saccharomyces cerevisiae, these MRX functions are inhibited by the Rif2 protein, which is enriched at telomeres and protects telomeric DNA from being sensed and processed as a DSB. The present review focuses on recent data showing that Sae2 and Rif2 regulate MRX functions in opposite manners by interacting with Rad50 and influencing ATP-dependent Mre11-Rad50 conformational changes. As Sae2 is enriched at DSBs whereas Rif2 is predominantly present at telomeres, the relative abundance of these two MRX regulators can provide an effective mechanism to activate or inactivate MRX depending on the nature of chromosome ends.
Topics: DNA; DNA Repair; DNA-Binding Proteins; Endodeoxyribonucleases; Endonucleases; Exodeoxyribonucleases; Intracellular Signaling Peptides and Proteins; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Telomere-Binding Proteins
PubMed: 34311383
DOI: 10.1016/j.gde.2021.07.001 -
International Journal of Molecular... Oct 2020S-glutathionylation, the post-translational modification forming mixed disulfides between protein reactive thiols and glutathione, regulates redox-based signaling events... (Review)
Review
S-glutathionylation, the post-translational modification forming mixed disulfides between protein reactive thiols and glutathione, regulates redox-based signaling events in the cell and serves as a protective mechanism against oxidative damage. S-glutathionylation alters protein function, interactions, and localization across physiological processes, and its aberrant function is implicated in various human diseases. In this review, we discuss the current understanding of the molecular mechanisms of S-glutathionylation and describe the changing levels of expression of S-glutathionylation in the context of aging, cancer, cardiovascular, and liver diseases.
Topics: Animals; Glutathione; Humans; Oxidation-Reduction; Oxidative Stress; Protein Processing, Post-Translational; Proteins; Signal Transduction
PubMed: 33143095
DOI: 10.3390/ijms21218113 -
Proteins Jan 2022Anaerobic ammonium-oxidizing (anammox) bacteria express a distinct acyl carrier protein implicated in the biosynthesis of the highly unusual "ladderane" lipids these...
Anaerobic ammonium-oxidizing (anammox) bacteria express a distinct acyl carrier protein implicated in the biosynthesis of the highly unusual "ladderane" lipids these organisms produce. This "anammox-specific" ACP, or amxACP, shows several unique features such as a conserved FF motif and an unusual sequence in the functionally important helix III. Investigation of the protein's structure and dynamics, both in the crystal by ensemble refinement and by MD simulations, reveals that helix III adopts a rare six-residue-long 3 -helical conformation that confers a large degree of conformational and positional variability on this part of the protein. This way of introducing structural flexibility by using the inherent properties of 3 -helices appears unique among ACPs. Moreover, the structure suggests a role for the FF motif in shielding the thioester linkage between the protein's prosthetic group and its acyl cargo from hydrolysis.
Topics: Acyl Carrier Protein; Amino Acid Motifs; Anaerobic Ammonia Oxidation; Bacterial Proteins; Lipid Metabolism; Molecular Dynamics Simulation; Planctomycetes
PubMed: 34310758
DOI: 10.1002/prot.26187 -
Nature Communications Dec 2021Numerous chromatin remodeling enzymes position nucleosomes in eukaryotic cells. Aside from these factors, transcription, DNA sequence, and statistical positioning of...
Numerous chromatin remodeling enzymes position nucleosomes in eukaryotic cells. Aside from these factors, transcription, DNA sequence, and statistical positioning of nucleosomes also shape the nucleosome landscape. The precise contributions of these processes remain unclear due to their functional redundancy in vivo. By incisive genome engineering, we radically decreased their redundancy in Saccharomyces cerevisiae. The transcriptional machinery strongly disrupts evenly spaced nucleosomes. Proper nucleosome density and DNA sequence are critical for their biogenesis. The INO80 remodeling complex helps space nucleosomes in vivo and positions the first nucleosome over genes in an H2A.Z-independent fashion. INO80 requires its Arp8 subunit but unexpectedly not the Nhp10 module for spacing. Cells with irregularly spaced nucleosomes suffer from genotoxic stress including DNA damage, recombination and transpositions. We derive a model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome.
Topics: Chromatin; Chromatin Assembly and Disassembly; DNA; DNA Damage; Engineering; Epigenomics; Eukaryotic Cells; High Mobility Group Proteins; Histones; Microfilament Proteins; Nucleosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors
PubMed: 34853297
DOI: 10.1038/s41467-021-27285-6