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Communications Biology Jun 2024Replicative senescence is triggered when telomeres reach critically short length and activate permanent DNA damage checkpoint-dependent cell cycle arrest. Mitochondrial...
Replicative senescence is triggered when telomeres reach critically short length and activate permanent DNA damage checkpoint-dependent cell cycle arrest. Mitochondrial dysfunction and increase in oxidative stress are both features of replicative senescence in mammalian cells. However, how reactive oxygen species levels are controlled during senescence is elusive. Here, we show that reactive oxygen species levels increase in the telomerase-negative cells of Saccharomyces cerevisiae during replicative senescence, and that this coincides with the activation of Hog1, a mammalian p38 MAPK ortholog. Hog1 counteracts increased ROS levels during replicative senescence. While Hog1 deletion accelerates replicative senescence, we found this could stem from a reduced cell viability prior to telomerase inactivation. ROS levels also increase upon telomerase inactivation when Mec1, the yeast ortholog of ATR, is mutated, suggesting that oxidative stress is not simply a consequence of DNA damage checkpoint activation in budding yeast. We speculate that oxidative stress is a conserved hallmark of telomerase-negative eukaryote cells, and that its sources and consequences can be dissected in S. cerevisiae.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Oxidative Stress; Telomerase; Reactive Oxygen Species; Mitogen-Activated Protein Kinases; Intracellular Signaling Peptides and Proteins; Protein Serine-Threonine Kinases; DNA Damage
PubMed: 38909140
DOI: 10.1038/s42003-024-06464-3 -
Biochimica Et Biophysica Acta.... Jun 2024Ubiquinone (UQ) is an essential player in the respiratory electron transfer system. In Saccharomyces cerevisiae strains lacking the ability to synthesize UQ, exogenously...
Ubiquinone (UQ) is an essential player in the respiratory electron transfer system. In Saccharomyces cerevisiae strains lacking the ability to synthesize UQ, exogenously supplied UQs can be taken up and delivered to mitochondria through an unknown mechanism, restoring the growth of UQ-deficient yeast in non-fermentable medium. Since elucidating the mechanism responsible may markedly contribute to therapeutic strategies for patients with UQ deficiency, many attempts have been made to identify the machinery involved in UQ trafficking in the yeast model. However, definite experimental evidence of the direct interaction of UQ with a specific protein(s) has not yet been demonstrated. To gain insight into intracellular UQ trafficking via a chemistry-based strategy, we synthesized a hydrophobic UQ probe (pUQ5), which has a photoreactive diazirine group attached to a five-unit isoprenyl chain and a terminal alkyne to visualize and/or capture the labeled proteins via click chemistry. pUQ5 successfully restored the growth of UQ-deficient S. cerevisiae (Δcoq2) on a non-fermentable carbon source, indicating that this UQ was taken up and delivered to mitochondria, and served as a UQ substrate of respiratory enzymes. Through photoaffinity labeling of the mitochondria isolated from Δcoq2 yeast cells cultured in the presence of pUQ5, we identified many labeled proteins, including voltage-dependent anion channel 1 (VDAC1) and cytochrome c oxidase subunit 3 (Cox3). The physiological relevance of UQ binding to these proteins is discussed.
PubMed: 38906315
DOI: 10.1016/j.bbabio.2024.149147 -
PloS One 2024Homologous recombination is a key process that governs the stability of eukaryotic genomes during DNA replication and repair. Multiple auxiliary factors regulate the...
Homologous recombination is a key process that governs the stability of eukaryotic genomes during DNA replication and repair. Multiple auxiliary factors regulate the choice of homologous recombination pathway in response to different types of replication stress. Using Schizosaccharomyces pombe we have previously suggested the role of DNA translocases Rrp1 and Rrp2, together with Srs2 helicase, in the common synthesis-dependent strand annealing sub-pathway of homologous recombination. Here we show that all three proteins are important for completion of replication after hydroxyurea exposure and provide data comparing the effect of overproduction of Srs2 with Rrp1 and Rrp2. We demonstrate that Srs2 localises to rDNA region and is required for proper replication of rDNA arrays. Upregulation of Srs2 protein levels leads to enhanced replication stress, chromosome instability and viability loss, as previously reported for Rrp1 and Rrp2. Interestingly, our data suggests that dysregulation of Srs2, Rrp1 and Rrp2 protein levels differentially affects checkpoint response: overproduction of Srs2 activates simultaneously DNA damage and replication stress response checkpoints, while cells overproducing Rrp1 mainly launch DNA damage checkpoint. On the other hand, upregulation of Rrp2 primarily leads to replication stress response checkpoint activation. Overall, we propose that Srs2, Rrp1 and Rrp2 have important and at least partially independent functions in the maintenance of distinct difficult to replicate regions of the genome.
Topics: Schizosaccharomyces; Schizosaccharomyces pombe Proteins; DNA Replication; DNA Helicases; DNA Damage; Hydroxyurea; Stress, Physiological; DNA, Ribosomal; Chromosomal Instability
PubMed: 38905307
DOI: 10.1371/journal.pone.0300434 -
Investigative Ophthalmology & Visual... Jun 2024Interphotoreceptor retinoid-binding protein's (IRBP) role in eye growth and its involvement in cell homeostasis remain poorly understood. One hypothesis proposes early...
PURPOSE
Interphotoreceptor retinoid-binding protein's (IRBP) role in eye growth and its involvement in cell homeostasis remain poorly understood. One hypothesis proposes early conditional deletion of the IRBP gene could lead to a myopic response with retinal degeneration, whereas late conditional deletion (after eye size is determined) could cause retinal degeneration without myopia. Here, we sought to understand if prior myopia was required for subsequent retinal degeneration in the absence of IRBP. This study investigates if any cell type or developmental stage is more important in myopia or retinal degeneration.
METHODS
IBRPfl/fl mice were bred with 5 Cre-driver lines: HRGP-Cre, Chx10-Cre, Rho-iCre75, HRGP-Cre Rho-iCre75, and Rx-Cre. Mice were analyzed for IRBP gene expression through digital droplet PCR (ddPCR). Young adult (P30) mice were tested for retinal degeneration and morphology using spectral-domain optical coherence tomography (SD-OCT) and hematoxylin and eosin (H&E) staining. Function was analyzed using electroretinograms (ERGs). Eye sizes and axial lengths were compared through external eye measurements and whole eye biometry.
RESULTS
Across all outcome measures, when bred to IRBPfl/fl, HRGP-Cre and Chx10-Cre lines showed no differences from IRBPfl/fl alone. With the Rho-iCre75 line, small but significant reductions were seen in retinal thickness with SD-OCT imaging and postmortem H&E staining without increased axial length. Both the HRGP-Cre+Rho-iCre75 and the Rx-Cre lines showed significant decreases in retinal thickness and outer nuclear layer cell counts. Using external eye measurements and SD-OCT imaging, both lines showed an increase in eye size. Finally, function in both lines was roughly halved across scotopic, photopic, and flicker ERGs.
CONCLUSIONS
Our studies support hypotheses that for both eye size determination and retinal homeostasis, there are two critical timing windows when IRBP must be expressed in rods or cones to prevent myopia (P7-P12) and degeneration (P21 and later). The rod-specific IRBP knockout (Rho-iCre75) showed significant retinal functional losses without myopia, indicating that the two phenotypes are independent. IRBP is needed for early development of photoreceptors and eye size, whereas Rho-iCre75 IRBPfl/fl knockout results in retinal degeneration without myopia.
Topics: Animals; Mice; Retinal Degeneration; Eye Proteins; Tomography, Optical Coherence; Mice, Knockout; Retinol-Binding Proteins; Electroretinography; Disease Models, Animal; Myopia; Mice, Inbred C57BL; Retina
PubMed: 38904640
DOI: 10.1167/iovs.65.6.32 -
Therapeutic Advances in Respiratory... 2024Cystic fibrosis (CF) is an autosomal recessive disease caused by the inheritance of two mutant cystic fibrosis transmembrane conductance regulator (CFTR) alleles, one...
Identification and structural analysis for the first mutation in the cystic fibrosis transmembrane conductance regulator protein in Iran: case report and developmental insight using microsatellite markers.
Cystic fibrosis (CF) is an autosomal recessive disease caused by the inheritance of two mutant cystic fibrosis transmembrane conductance regulator (CFTR) alleles, one from each parent. Autosomal recessive disorders are rarely associated with germline mutations or mosaicism. Here, we propose a case of paternal germline mutation causing CF. The subject also had an identifiable maternal mutant allele. We identified the compound heterozygous variants in the proband through Sanger sequencing, and studies predicted functional effects on the protein. Also, short tandem repeat markers revealed the nature of the mutation. The maternal mutation in the CFTR gene was c.1000C > T. The mutation was c.178G > A, p.Glu60Lys. This mutation is located in the lasso motif of the CFTR protein and, according to structural analysis, disrupts the interaction of the lasso motif and R-domain, thus influencing protein function. This first reported case of mutation in Asia has notable implications for molecular diagnostics, genetic counseling, and understanding the genetic etiology of recessive disorders in the Iranian population.
Topics: Humans; Cystic Fibrosis Transmembrane Conductance Regulator; Cystic Fibrosis; Iran; Microsatellite Repeats; Female; Male; Germ-Line Mutation; Computer Simulation; Genetic Predisposition to Disease; Phenotype; DNA Mutational Analysis
PubMed: 38904297
DOI: 10.1177/17534666241253990 -
Biochimie Jun 2024Proteins are essential molecular actors in every cellular process. From their synthesis to their degradation, they are subject to continuous quality control mechanisms...
Proteins are essential molecular actors in every cellular process. From their synthesis to their degradation, they are subject to continuous quality control mechanisms to ensure that they fulfil cellular needs in proper and timely fashion. Proteostasis is a key process allowing cells or organisms to maintain an appropriate but dynamic equilibrium of their proteome (the ensemble of all their proteins). It relies on multiple mechanisms that together control the level, fate and function of individual proteins, and ensure elimination of abnormal ones. The proteostasis network is essential for development and adaptation to environmental changes or challenges. Its dysfunctions can lead to accumulation of deleterious proteins or, conversely, to excessive degradation of beneficial ones, and are implicated in many diseases such as cancers, neurodegeneration, or developmental and aging disorders. Manipulating this network to control abundance of selected target proteins is therefore a strategy with enormous therapeutic or biotechnological potential. The ProteoCure COST Action gathers more than 350 researchers and their teams (31 countries represented) from the academic, clinical, and industrial sectors, who share the conviction that our understanding of proteostasis is mature enough to develop novel and highly specific therapies based on selective tunning of protein levels. Towards this objective, the Action organizes community-building activities to foster synergies among its participants and reinforce training of the next generation of European researchers. Its ambition is to function as a knowledge-based network and a creative exchange hub on normal and pathologic proteostasis, focusing on developing innovative tools modulating the level of specific protein(s).
PubMed: 38901793
DOI: 10.1016/j.biochi.2024.06.004 -
Redox Biology Jun 2024Morphine, a typical opiate, is widely used for controlling pain but can lead to various side effects with long-term use, including addiction, analgesic tolerance, and...
Morphine, a typical opiate, is widely used for controlling pain but can lead to various side effects with long-term use, including addiction, analgesic tolerance, and hyperalgesia. At present, however, the mechanisms underlying the development of morphine analgesic tolerance are not fully understood. This tolerance is influenced by various opioid receptor and kinase protein modifications, such as phosphorylation and ubiquitination. Here, we established a murine morphine tolerance model to investigate whether and how S-nitrosoglutathione reductase (GSNOR) is involved in morphine tolerance. Repeated administration of morphine resulted in the down-regulation of GSNOR, which increased excessive total protein S-nitrosation in the prefrontal cortex. Knockout or chemical inhibition of GSNOR promoted the development of morphine analgesic tolerance and neuron-specific overexpression of GSNOR alleviated morphine analgesic tolerance. Mechanistically, GSNOR deficiency enhanced S-nitrosation of cellular protein kinase alpha (PKCα) at the Cys78 and Cys132 sites, leading to inhibition of PKCα kinase activity, which ultimately promoted the development of morphine analgesic tolerance. Our study highlighted the significant role of GSNOR as a key regulator of PKCα S-nitrosation and its involvement in morphine analgesic tolerance, thus providing a potential therapeutic target for morphine tolerance.
PubMed: 38901102
DOI: 10.1016/j.redox.2024.103239 -
Journal of Inorganic Biochemistry Jun 2024Over the last 50 years resonance Raman spectroscopy has become an invaluable tool for the exploration of chromophores in biological macromolecules. Among them, heme... (Review)
Review
Over the last 50 years resonance Raman spectroscopy has become an invaluable tool for the exploration of chromophores in biological macromolecules. Among them, heme proteins and metal complexes have attracted considerable attention. This interest results from the fact that resonance Raman spectroscopy probes the vibrational dynamics of these chromophores without direct interference from the surrounding. However, the indirect influence via through-bond and through-space chromophore-protein interactions can be conveniently probed and analyzed. This review article illustrates this point by focusing on class 1 cytochrome c, a comparatively simple heme protein generally known as electron carrier in mitochondria. The article demonstrates how through selective excitation of resonance Raman active modes information about the ligation, the redox state and the spin state of the heme iron can be obtained from band positions in the Raman spectra. The investigation of intensities and depolarization ratios emerged as tools for the analysis of in-plane and out-of-plane deformations of the heme macrocycle. The article further shows how resonance Raman spectroscopy was used to characterize partially unfolded states of oxidized cytochrome c. Finally, it describes its use for exploring structural changes due to the protein's binding to anionic surfaces like cardiolipin containing membranes.
PubMed: 38901065
DOI: 10.1016/j.jinorgbio.2024.112641 -
BioRxiv : the Preprint Server For... Jun 2024Proteins are dynamic systems whose structural preferences determine their function. Unfortunately, building atomically detailed models of protein structural ensembles...
Proteins are dynamic systems whose structural preferences determine their function. Unfortunately, building atomically detailed models of protein structural ensembles remains challenging, limiting our understanding of the relationships between sequence, structure, and function. Combining single molecule Förster resonance energy transfer (smFRET) experiments with molecular dynamics simulations could provide experimentally grounded, all-atom models of a protein's structural ensemble. However, agreement between the two techniques is often insufficient to achieve this goal. Here, we explore whether accounting for important experimental details like averaging across structures sampled during a given smFRET measurement is responsible for this apparent discrepancy. We present an approach to account for this time-averaging by leveraging the kinetic information available from Markov state models of a protein's dynamics. This allows us to accurately assess which timescales are averaged during an experiment. We find this approach significantly improves agreement between simulations and experiments in proteins with varying degrees of dynamics, including the well-ordered protein T4 lysozyme, the partially disordered protein apolipoprotein E (ApoE), and a disordered amyloid protein (Aβ40). We find evidence for hidden states that are not apparent in smFRET experiments because of time averaging with other structures, akin to states in fast exchange in NMR, and evaluate different force fields. Finally, we show how remaining discrepancies between computations and experiments can be used to guide additional simulations and build structural models for states that were previously unaccounted for. We expect our approach will enable combining simulations and experiments to understand the link between sequence, structure, and function in many settings.
PubMed: 38895430
DOI: 10.1101/2024.06.03.597137 -
BioRxiv : the Preprint Server For... Jun 2024Knowledge of the structures formed by proteins and small ligands is of fundamental importance for understanding molecular principles of chemotherapy and for designing...
Knowledge of the structures formed by proteins and small ligands is of fundamental importance for understanding molecular principles of chemotherapy and for designing new and more effective drugs. Due to the still high costs and to the several limitations of experimental techniques, it is most often desirable to predict these ligand-protein complexes , particularly when screening for new putative drugs from databases of millions of compounds. While virtual screening based on molecular docking is widely used for this purpose, it generally fails in mimicking binding events associated with large conformational changes in the protein, particularly when the latter involve multiple domains. In this work, we describe a new methodology aimed at generating bound-like conformations of very flexible and allosteric proteins bearing multiple binding sites. Validation was performed on the enzyme adenylate kinase (ADK), a paradigmatic example of proteins that undergo very large conformational changes upon ligand binding. By only exploiting the unbound structure and the putative binding sites of the protein, we generated a significant fraction of bound-like structures, which employed in ensemble-docking calculations allowed to find native-like poses of substrates, inhibitors, and catalytically incompetent binders. Our protocol provides a general framework for the generation of bound-like conformations of flexible proteins that are suitable to host different ligands, demonstrating high sensitivity to the fine chemical details that regulate protein's activity. We foresee applications in virtual screening for difficult targets, prediction of the impact of amino acid mutations on structure and dynamics, and protein engineering.
PubMed: 38895346
DOI: 10.1101/2024.06.02.597018