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ELife May 2024Stramenopiles form a clade of diverse eukaryotic organisms, including multicellular algae, the fish and plant pathogenic oomycetes, such as the potato blight , and the...
Stramenopiles form a clade of diverse eukaryotic organisms, including multicellular algae, the fish and plant pathogenic oomycetes, such as the potato blight , and the human intestinal protozoan . In most eukaryotes, glycolysis is a strictly cytosolic metabolic pathway that converts glucose to pyruvate, resulting in the production of NADH and ATP (Adenosine triphosphate). In contrast, stramenopiles have a branched glycolysis in which the enzymes of the pay-off phase are located in both the cytosol and the mitochondrial matrix. Here, we identify a mitochondrial carrier in that can transport glycolytic intermediates, such as dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, across the mitochondrial inner membrane, linking the cytosolic and mitochondrial branches of glycolysis. Comparative analyses with the phylogenetically related human mitochondrial oxoglutarate carrier (SLC25A11) and dicarboxylate carrier (SLC25A10) show that the glycolytic intermediate carrier has lost its ability to transport the canonical substrates malate and oxoglutarate. lacks several key components of oxidative phosphorylation required for the generation of mitochondrial ATP, such as complexes III and IV, ATP synthase, and ADP/ATP carriers. The presence of the glycolytic pay-off phase in the mitochondrial matrix generates ATP, which powers energy-requiring processes, such as macromolecular synthesis, as well as NADH, used by mitochondrial complex I to generate a proton motive force to drive the import of proteins and molecules. Given its unique substrate specificity and central role in carbon and energy metabolism, the carrier for glycolytic intermediates identified here represents a specific drug and pesticide target against stramenopile pathogens, which are of great economic importance.
Topics: Blastocystis; Glycolysis; Humans; Mitochondria; Cytosol; Biological Transport; Protozoan Proteins
PubMed: 38780415
DOI: 10.7554/eLife.94187 -
The Journal of Biological Chemistry Jun 2024Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug...
Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize a phosphorylation site at Ser situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser phosphorylation on the protein's properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.
Topics: Humans; Phosphorylation; Receptors, Antigen, T-Cell; Protein Tyrosine Phosphatase, Non-Receptor Type 22; Signal Transduction; Gain of Function Mutation; T-Lymphocytes; Jurkat Cells; HEK293 Cells
PubMed: 38777143
DOI: 10.1016/j.jbc.2024.107393 -
Current Biology : CB Jun 2024The Mps1 and Aurora B kinases regulate and monitor kinetochore attachment to spindle microtubules during cell division, ultimately ensuring accurate chromosome...
The Mps1 and Aurora B kinases regulate and monitor kinetochore attachment to spindle microtubules during cell division, ultimately ensuring accurate chromosome segregation. In yeast, the critical spindle attachment components are the Ndc80 and Dam1 complexes (Ndc80c and DASH/Dam1c, respectively). Ndc80c is a 600-Å-long heterotetramer that binds microtubules through a globular "head" at one end and centromere-proximal kinetochore components through a globular knob at the other end. Dam1c is a heterodecamer that forms a ring of 16-17 protomers around the shaft of the single kinetochore microtubule in point-centromere yeast. The ring coordinates the approximately eight Ndc80c rods per kinetochore. In published work, we showed that a site on the globular "head" of Ndc80c, including residues from both Ndc80 and Nuf2, binds a bipartite segment in the long C-terminal extension of Dam1. Results reported here show, both by in vitro binding experiments and by crystal structure determination, that the same site binds a conserved segment in the long N-terminal extension of Mps1. It also binds, less tightly, a conserved segment in the N-terminal extension of Ipl1 (yeast Aurora B). Together with results from experiments in yeast cells and from biochemical assays reported in two accompanying papers, the structures and graded affinities identify a communication hub for ensuring uniform bipolar attachment and for signaling anaphase onset.
Topics: Kinetochores; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Microtubules; Phosphorylation; Microtubule-Associated Proteins; Cell Cycle Proteins; Nuclear Proteins
PubMed: 38776904
DOI: 10.1016/j.cub.2024.04.067 -
Current Biology : CB Jun 2024Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents...
Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behavior in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signaling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to promote anaphase onset.
Topics: Saccharomyces cerevisiae Proteins; Kinetochores; Saccharomyces cerevisiae; Protein Serine-Threonine Kinases; Microtubules; Cell Cycle Proteins; Microtubule-Associated Proteins; Nuclear Proteins
PubMed: 38776902
DOI: 10.1016/j.cub.2024.03.062 -
PLoS Genetics May 2024The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here,...
The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinase Dun1. These physical interactions encompass small subpopulations of MCM and RNR, are independent of the major subcellular locations of these two complexes, augment in response to DNA damage and, in the case of the Rnr2 and Rnr4 subunits of RNR, depend on Dun1. Partial disruption of the MCM/RNR interactions impairs the release of Rad52 -but not RPA-from the DNA repair centers despite the lesions are repaired, a phenotype that is associated with hypermutagenesis but not with alterations in the levels of dNTPs. These results suggest that a specifically regulated pool of MCM and RNR complexes plays non-canonical roles in genetic stability preventing persistent Rad52 centers and hypermutagenesis.
Topics: Saccharomyces cerevisiae Proteins; DNA Replication; Saccharomyces cerevisiae; DNA Damage; Cell Cycle Proteins; Rad52 DNA Repair and Recombination Protein; Genomic Instability; Ribonucleotide Reductases; DNA Repair; Protein Serine-Threonine Kinases; DNA Helicases; Minichromosome Maintenance Proteins; Replication Protein A; Ribonucleoside Diphosphate Reductase
PubMed: 38776358
DOI: 10.1371/journal.pgen.1011148 -
PloS One 2024The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5....
The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5. Cells lacking Agp2 are hyper-resistant to polyamine and bleomycin-A5. In these earlier studies, we showed that the protein synthesis inhibitor cycloheximide blocked the uptake of bleomycin-A5 into the cells suggesting that the drug uptake system may require de novo synthesis. However, our recent findings demonstrated that cycloheximide, instead, induced rapid degradation of Agp2, and in the absence of Agp2 cells are resistant to cycloheximide. These observations raised the possibility that the degradation of Agp2 may allow the cell to alter its drug resistance network to combat the toxic effects of cycloheximide. In this study, we show that membrane extracts from agp2Δ mutants accentuated several proteins that were differentially expressed in comparison to the parent. Mass spectrometry analysis of the membrane extracts uncovered the pleiotropic drug efflux pump, Pdr5, involved in the efflux of cycloheximide, as a key protein upregulated in the agp2Δ mutant. Moreover, a global gene expression analysis revealed that 322 genes were differentially affected in the agp2Δ mutant versus the parent, including the prominent PDR5 gene and genes required for mitochondrial function. We further show that Agp2 is associated with the upstream region of the PDR5 gene, leading to the hypothesis that cycloheximide resistance displayed by the agp2Δ mutant is due to the derepression of the PDR5 gene.
Topics: Cycloheximide; Protein Synthesis Inhibitors; Saccharomyces cerevisiae Proteins; ATP-Binding Cassette Transporters; Saccharomyces cerevisiae; Up-Regulation; Drug Resistance, Fungal; Gene Expression Regulation, Fungal
PubMed: 38776347
DOI: 10.1371/journal.pone.0303747 -
Orphanet Journal of Rare Diseases May 2024Marfan syndrome (MFS) is an autosomal dominant connective tissue disease with wide clinical heterogeneity, and mainly caused by pathogenic variants in fibrillin-1 (FBN1).
BACKGROUND
Marfan syndrome (MFS) is an autosomal dominant connective tissue disease with wide clinical heterogeneity, and mainly caused by pathogenic variants in fibrillin-1 (FBN1).
METHODS
A Chinese 4-generation MFS pedigree with 16 family members was recruited and exome sequencing (ES) was performed in the proband. Transcript analysis (patient RNA and minigene assays) and in silico structural analysis were used to determine the pathogenicity of the variant. In addition, germline mosaicism in family member (Ι:1) was assessed using quantitative fluorescent polymerase chain reaction (QF-PCR) and short tandem repeat PCR (STR) analyses.
RESULTS
Two cis-compound benign intronic variants of FBN1 (c.3464-4 A > G and c.3464-5G > A) were identified in the proband by ES. As a compound variant, c.3464-5_3464-4delGAinsAG was found to be pathogenic and co-segregated with MFS. RNA studies indicated that aberrant transcripts were found only in patients and mutant-type clones. The variant c.3464-5_3464-4delGAinsAG caused erroneous integration of a 3 bp sequence into intron 28 and resulted in the insertion of one amino acid in the protein sequence (p.Ile1154_Asp1155insAla). Structural analyses suggested that p.Ile1154_Asp1155insAla affected the protein's secondary structure by interfering with one disulfide bond between Cys and Cys and causing the extension of an anti-parallel β sheet in the calcium-binding epidermal growth factor-like (cbEGF)13 domain. In addition, the asymptomatic family member Ι:1 was deduced to be a gonadal mosaic as assessed by inconsistent results of sequencing and STR analysis.
CONCLUSIONS
To our knowledge, FBN1 c.3464-5_3464-4delGAinsAG is the first identified pathogenic intronic indel variant affecting non-canonical splice sites in this gene. Our study reinforces the importance of assessing the pathogenic role of intronic variants at the mRNA level, with structural analysis, and the occurrence of mosaicism.
Topics: Humans; Fibrillin-1; Marfan Syndrome; Female; Male; Pedigree; Mosaicism; Adult; Introns; INDEL Mutation; Middle Aged; Adipokines
PubMed: 38773661
DOI: 10.1186/s13023-024-03139-4 -
Nature Communications May 2024Heterochromatin is generally associated with the nuclear periphery, but how the spatial organization of heterochromatin is regulated to ensure epigenetic silencing...
Heterochromatin is generally associated with the nuclear periphery, but how the spatial organization of heterochromatin is regulated to ensure epigenetic silencing remains unclear. Here we found that Sad1, an inner nuclear membrane SUN-family protein in fission yeast, interacts with histone H2A-H2B but not H3-H4. We solved the crystal structure of the histone binding motif (HBM) of Sad1 in complex with H2A-H2B, revealing the intimate contacts between Sad1 and H2A-H2B. Structure-based mutagenesis studies revealed that the H2A-H2B-binding activity of Sad1 is required for the dynamic distribution of Sad1 throughout the nuclear envelope (NE). The Sad1-H2A-H2B complex mediates tethering telomeres and the mating-type locus to the NE. This complex is also important for heterochromatin silencing. Mechanistically, H2A-H2B enhances the interaction between Sad1 and HDACs, including Clr3 and Sir2, to maintain epigenetic identity of heterochromatin. Interestingly, our results suggest that Sad1 exhibits the histone-enhanced liquid-liquid phase separation property, which helps recruit heterochromatin factors to the NE. Our results uncover an unexpected role of SUN-family proteins in heterochromatin regulation and suggest a nucleosome-independent role of H2A-H2B in regulating Sad1's functionality.
Topics: Heterochromatin; Schizosaccharomyces pombe Proteins; Histones; Schizosaccharomyces; Protein Binding; Telomere; Nuclear Envelope; Cell Cycle Proteins; Histone Deacetylases; Nuclear Proteins; Crystallography, X-Ray
PubMed: 38773107
DOI: 10.1038/s41467-024-48418-7 -
Transplantation Direct Jun 2024Four-factor prothrombin complex concentrate (PCC) is a plasma product that contains factors II, VII, IX, X, protein C, and protein S. PCC can be used off-label to treat...
BACKGROUND
Four-factor prothrombin complex concentrate (PCC) is a plasma product that contains factors II, VII, IX, X, protein C, and protein S. PCC can be used off-label to treat coagulopathy during orthotopic liver transplantation (OLT). However, its use comes with safety concerns regarding thrombosis. The purpose of our study is to determine the safety of PCC in OLT.
METHODS
We conducted a retrospective cohort study of patients who received 4-factor PCC during OLT at our institution from January 1, 2018, to May 1, 2022, with a 1:1 match of 83 patients who received PCC and 83 patients who did not. We evaluated 30-d mortality, 1-y mortality, prevalence of thrombotic complications (portal vein thrombosis, deep venous thrombosis, myocardial infarction, and pulmonary embolus), and postoperative intensive care (ICU) length of stay (LOS).
RESULTS
There was no significant difference in 30-d mortality (odds ratio [OR] 5; 95% confidence interval [CI], 0.58-42.8; = 0.14), 1-y mortality (OR 3; 95% CI, 0.61-14.86; = 0.18), or ICU LOS (OR -13.8; 95% CI, -39.2 to 11.6; = 0.29). There was no increased incidence of thrombotic complications among patients receiving PCC 90 d after surgery, including portal vein thrombosis (OR 1.5; 95% CI, 0.42-5.32; = 0.53), pulmonary embolus (OR 1; 95% CI, 0.14-7.1; = 0.99), deep venous thrombosis (OR 0.67; 95% CI, 0.11-3.99; = 0.66), and myocardial infarction (OR 1.67; 95% CI, 0.4-6.97; = 0.48).
CONCLUSIONS
Although there was a statistically insignificant increase in mortality after PCC administration during OLT, we did not see a significant increase in perioperative complications, including thrombotic events and increased ICU LOS.
PubMed: 38769975
DOI: 10.1097/TXD.0000000000001637 -
Journal of Biotechnology Jul 2024Nutrient signaling pathways play a pivotal role in regulating the balance among metabolism, growth and stress response depending on the available food supply. They are...
Nutrient signaling pathways play a pivotal role in regulating the balance among metabolism, growth and stress response depending on the available food supply. They are key factors for the biotechnological success of the yeast Saccharomyces cerevisiae during food-producing fermentations. One such pathway is Retrograde Response, which controls the alpha-ketoglutarate supply required for the synthesis of amino acids like glutamate and lysine. Repressor MKS1 is linked with the TORC1 complex and negatively regulates this pathway. Deleting MKS1 from a variety of industrial strains causes glycerol to increase during winemaking, brewing and baking. This increase is accompanied by a reduction in ethanol production during grape juice fermentation in four commercial wine strains. Interestingly, this does not lead volatile acidity to increase because acetic acid levels actually lower. Aeration during winemaking usually increases acetic acid levels, but this effect reduces in the MKS1 mutant. Despite the improvement in the metabolites of oenological interest, it comes at a cost given that the mutant shows slower fermentation kinetics when grown in grape juice, malt and laboratory media and using glucose, sucrose and maltose as carbon sources. The deletion of RTG2, an activator of Retrograde Response that acts as an antagonist of MKS1, also results in a defect in wine fermentation speed. These findings suggest that the deregulation of this pathway causes a fitness defect. Therefore, manipulating repressor MKS1 is a promising approach to modulate yeast metabolism and to produce low-ethanol drinks.
Topics: Glycerol; Saccharomyces cerevisiae; Ethanol; Fermentation; Wine; Saccharomyces cerevisiae Proteins; Up-Regulation; Repressor Proteins; Gene Expression Regulation, Fungal; Transaminases
PubMed: 38768686
DOI: 10.1016/j.jbiotec.2024.05.007