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Current Genetics Jun 2024The Byr2 kinase of fission yeast Schizosaccharomyces pombe is recruited to the membrane with the assistance of Ras1. Byr2 is also negatively regulated by 14-3-3 proteins...
The Byr2 kinase of fission yeast Schizosaccharomyces pombe is recruited to the membrane with the assistance of Ras1. Byr2 is also negatively regulated by 14-3-3 proteins encoded by rad24 and rad25. We conducted domain and mutational analysis of Byr2 to determine which region is critical for its binding to 14-3-3 proteins. Rad24 and Rad25 bound to both the Ras interaction domain in the N-terminus and to the C-terminal catalytic domain of Byr2. When amino acid residues S87 and T94 of the Ras-interacting domain of Byr2 were mutated to alanine, Rad24 could no longer bind to Byr2. S402, S566, S650, and S654 mutations in the C-terminal domain of Byr2 also abolished its interaction with Rad24 and Rad25. More than three mutations in the C-terminal domain were required to abolish completely its interaction with 14-3-3 protein, suggesting that multiple residues are involved in this interaction. Expression of the N-terminal domain of Byr2 in wild-type cells lowered the mating ratio, because it likely blocked the interaction of Byr2 with Ste4 and Ras1, whereas expression of the catalytic domain of Byr2 increased the mating ratio as a result of freeing from intramolecular regulation by the N-terminal domain of Byr2. The S87A and T94A mutations of Byr2 increased the mating ratio and attenuated inhibition of Byr2 by Rad24; therefore, these two amino acids are critical for its regulation by Rad24. S566 of Byr2 is critical for activity of Byr2 but not for its interaction with 14-3-3 proteins. In this study, we show that 14-3-3 proteins interact with two separate domains in Byr2 as negative regulators.
Topics: 14-3-3 Proteins; Schizosaccharomyces pombe Proteins; Schizosaccharomyces; Protein Binding; Mutation; DNA Mutational Analysis; Protein Domains; Protein Interaction Domains and Motifs; Cell Cycle Proteins; Intracellular Signaling Peptides and Proteins
PubMed: 38913087
DOI: 10.1007/s00294-024-01293-7 -
International Journal of Applied &... 2024Pregnancy is the state of carrying a developing embryo or fetus within a female body. Once pregnancy is established, a range of endocrinological events appear in its...
BACKGROUND
Pregnancy is the state of carrying a developing embryo or fetus within a female body. Once pregnancy is established, a range of endocrinological events appear in its maintenance, finally helping in the successful pregnancy. The complications which are usually observed in pregnancy are gestational diabetes, preeclampsia, preterm labor, and spontaneous pregnancy loss or miscarriage, while 10%-15% of clinically recognized pregnancies terminate into spontaneous miscarriage. Thus, many attempts have been made by different researchers for the diagnosis of high-risk pregnancy on altered protein pattern using placental villous tissue or follicular fluid, but these are difficult to obtain and results of different studies are not constant.
AIM
This study was designed to identify the association (if any) among serum protein(s) electrophoretic pattern and different serum hormones in normal pregnant women (controls) and gestational age-matched women with spontaneous pregnancy loss (cases).
MATERIALS AND METHODS
This study was carried out for 1½ year from October 2018 to March 2020 and included 120 participants (60 normal pregnant women and 60 women with spontaneous pregnancy loss) between 20 and 45 years of age with no mean age difference. The electrophoresis of serum was carried out using slab gel electrophoretic unit and serum thyroid-stimulating hormone (TSH), total tri-iodothyronine (TT), total thyroxine (TT), prolactin, and beta human chorionic gonadotropin (β-hCG) levels were analyzed using TSOSH AIA analyzer at Adesh University, Bathinda.
RESULTS
Significant variations in the expression of proteins with molecular weight around ~150 kDa, ~50 kDa, and ~25 kDa were observed in normal pregnant women and women with spontaneous pregnancy loss. However, the protein band of ~50 kDa was found to be highly expressed in the serum of 1 and 2 trimester women experiencing spontaneous pregnancy loss. Therefore, selected protein band of ~50 kDa was further processed by ECI-mass spectrophotometry QUAD time of flight and 365 different proteins were found, out of these; 34 proteins were found to be unidentified protein products ( ). Further, TT, total proteins, β-hCG, and prolactin level were found to be low, whereas, TSH was found to be high in women experiencing spontaneous pregnancy loss. However, difference in the level of β-hCG in the 1 trimester and TT among normal pregnant women and women with spontaneous pregnancy loss was observed to be statistically insignificant.
CONCLUSION
This study indicated that the evaluation of serum protein variations along with hormonal profile may provide valuable information about high-risk pregnancy. Moreover, the differential expression of proteins in women with spontaneous pregnancy loss can be further explored to develop potential biomarker for the early identification of high-risk pregnancy and appropriate preventive measure.
PubMed: 38912357
DOI: 10.4103/ijabmr.ijabmr_383_23 -
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 -
Cell Jun 2024Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and...
Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.
Topics: Aldehyde Dehydrogenase; Cell Nucleus; Coenzyme A Ligases; Cryoelectron Microscopy; Cytoplasm; Electron Microscope Tomography; Gametogenesis; Meiosis; Mitochondria; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spores, Fungal; Models, Molecular; Protein Structure, Quaternary
PubMed: 38906101
DOI: 10.1016/j.cell.2024.04.026 -
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; Disease Models, Animal; Electroretinography; Eye Proteins; Mice, Inbred C57BL; Mice, Knockout; Myopia; Retina; Retinal Degeneration; Retinol-Binding Proteins; Tomography, Optical Coherence; Male; Female
PubMed: 38904640
DOI: 10.1167/iovs.65.6.32 -
Physical Chemistry Chemical Physics :... Jun 2024Natural deep eutectic solvents (NADESs) comprised of osmolytes are of interest as potential biomolecular (cryo)protectants. However, the way these solvents influence the...
Natural deep eutectic solvents (NADESs) comprised of osmolytes are of interest as potential biomolecular (cryo)protectants. However, the way these solvents influence the structure and dynamics of biomolecules as well as the role of water remains poorly understood. We carried out principal component analysis of various secondary structure elements of ubiquitin in water and a betaine : glycerol : water (1 : 2 : ; = 0, 1, 2, 5, 10, 20, 45) NADES, from molecular dynamics trajectories, to gain insight into the protein dynamics as it undergoes a transition from a highly viscous anhydrous to an aqueous environment. A crossover of the protein's essential dynamics at ∼ 5, induced by solvent-shell coupled fluctuations, is observed, indicating that ubiquitin might (re)fold in the NADES upon water addition at > ∼5. Further, in contrast to water, the anhydrous NADES preserves ubiquitin's essential modes at high temperatures explaining the protein's seemingly enhanced thermal stability.
PubMed: 38904333
DOI: 10.1039/d4cp01773k -
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: Female; Humans; Male; Computer Simulation; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA Mutational Analysis; Genetic Predisposition to Disease; Germ-Line Mutation; Iran; Microsatellite Repeats; Phenotype; Child, Preschool; Infant
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