Denaturation and renaturation of the monomeric phosphoglycerate mutase from Schizosaccharomyces pombe.
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ABSTRACT:
SUBMITTER: Johnson CM
PROVIDER: S-EPMC1148153 | biostudies-other | 1987 Jul
REPOSITORIES: biostudies-other
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Project description:The amino acid sequence of the monomeric 2,3-bisphosphoglycerate (BPG)-dependent phosphoglycerate mutase (PGAM) from the fission yeast Schizosaccharomyces pombe has been determined. Amino acid sequencing of proteolytic fragments of the enzyme showed the S. pombe mutase to be similar in sequence to the tetrameric enzyme of baker's yeast (Saccharomyces cerevisiae). An S. pombe cDNA library was screened using a PCR fragment generated from two oligonucleotides complementary to sequences encoding the regions at the two active-site histidine residues. The 0.63 kb cDNA encoded an open reading frame of 210 amino acids. This sequence agreed completely with sequences of peptides derived from the purified protein. The amino acid sequence of S. pombe PGAM is 43% identical with that of S. cerevisiae PGAM and shows an equally high degree of identity with BPG-dependent PGAMs from other sources. However, the sequence of the S. pombe enzyme differs from other BPG-dependent enzymes in three important ways: (i) it does not contain the alanine- and lysine-rich sequence of amino acids at the C-terminus which have been proposed to constitute a flexible tail involved in catalysis; (ii) the sequence spanning residues 122-146 (S. cerevisiae PGAM numbering) is not present in the S. pombe PGAM sequence; in the S. cerevisiae PGAM crystal structure this stretch of sequence has been shown to occur as an extended loop, part of which is involved in inter-subunit interactions; (iii) the amino acid sequence in the region of a second S. cerevisiae inter-subunit contact (residues 74-78) shows radical mutations in the S. pombe enzyme.
| S-EPMC1137876 | biostudies-other
Project description:The renaturation of the tetrameric enzyme phosphoglycerate mutase from baker's yeast after denaturation in guanidinium chloride was studied. Three proteinases (trypsin, chymotrypsin and thermolysin) cause extensive loss of activity of samples taken during the early stages of refolding. As judged by SDS/polyacrylamide-gel electrophoresis, the proteinases cause substantial degradation of the polypeptide chain with no evidence for large quantities of fragments of Mr greater than 6500. These data suggest that the early intermediates in the refolding, especially the folded monomer, possess a number of sites that are susceptible to proteolysis.
| S-EPMC1146886 | biostudies-other
Project description:To investigate the importance of functional regulation of PGAM2 by SUMO conjugation in the context of myogenic differentiation. We performed RNAseq analysis of WT and sumoylation deficient mutant (K176R mutatant) C2C12 cells at different differentiation days, i.e. day 0 and day4, respectively (n=3/group).
2023-01-11 | GSE220249 | GEO
Project description:BackgroundProteostasis refers to the processes that regulate the biogenesis, folding, trafficking, and degradation of proteins. Any alteration in these processes can lead to cell malfunction. Protein synthesis, a key proteostatic process, is highly-regulated at multiple levels to ensure adequate adaptation to environmental and physiological challenges such as different stressors, proteotoxic conditions and aging, among other factors. Because alterations in protein translation can lead to protein misfolding, examining how protein translation is regulated may also help to elucidate in part how proteostasis is controlled. Codon usage bias has been implicated in the fine-tuning of translation rate, as more-frequent codons might be read faster than their less-frequent counterparts. Thus, alterations in codon usage due to synonymous mutations may alter translation kinetics and thereby affect the folding of the nascent polypeptide, without altering its primary structure. To date, it has been difficult to predict the effect of synonymous mutations on protein folding and cellular fitness due to a scarcity of relevant data. Thus, the purpose of this work was to assess the effect of synonymous mutations in discrete regions of the gene that encodes the highly-expressed enzyme 3-phosphoglycerate kinase 1 (pgk1) in the fission yeast Schizosaccharomyces pombe.ResultsBy means of systematic replacement of synonymous codons along pgk1, we found slightly-altered protein folding and activity in a region-specific manner. However, alterations in protein aggregation, heat stress as well as changes in proteasome activity occurred independently of the mutated region. Concomitantly, reduced mRNA levels of the chaperones Hsp9 and Hsp16 were observed.ConclusionTaken together, these data suggest that codon usage bias of the gene encoding this highly-expressed protein is an important regulator of protein function and proteostasis.
| S-EPMC9875302 | biostudies-literature
Project description:Phosphoglycerate mutase 2 (PGAM2) is a critical glycolytic enzyme that is highly expressed in skeletal muscle. In humans, naturally occurring mutations in Phosphoglycerate mutase 2 have been etiologically linked to glycogen storage disease X (GSDX). Phosphoglycerate mutase 2 activity is regulated by several posttranslational modifications such as ubiquitination and acetylation. Here, we report that Phosphoglycerate mutase 2 activity is regulated by sumoylation-a covalent conjugation involved in a wide spectrum of cellular events. We found that Phosphoglycerate mutase 2 contains two primary SUMO acceptor sites, lysine (K)49 and K176, and that the mutation of either K to arginine (R) abolished Phosphoglycerate mutase 2 sumoylation. Given that K176 is more highly evolutionarily conserved across paralogs and orthologs than K49 is, we used the CRISPR-mediated homologous recombination technique in myogenic C2C12 cells to generate homozygous K176R knock-in cells (PGAM2K176R/K176R). Compared with wild-type (WT) C2C12 cells, PGAM2K176R/K176R C2C12 cells exhibited impaired myogenic differentiation, as indicated by decreased differentiation and fusion indexes. Furthermore, the results of glycolytic and mitochondrial stress assays with the XF96 Extracellular Flux analyzer revealed a reduced proton efflux rate (PER), glycolytic PER (glycoPER), extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) in PGAM2K176R/K176R C2C12 cells, both at baseline and in response to stress. Impaired mitochondrial function was also observed in PGAM2K176R/K176R P19 cells, a carcinoma cell line. These findings indicate that the PGAM2-K176R mutation impaired glycolysis and mitochondrial function. Gene ontology term analysis of RNA sequencing data further revealed that several downregulated genes in PGAM2K176R/K176R C2C12 cells were associated with muscle differentiation/development/contraction programs. Finally, PGAM2 with either of two naturally occurring missense mutations linked to GSDX, E89A (conversion of glutamic acid 89 to alanine) or R90W (conversion of arginine 90 to tryptophan), exhibited reduced Phosphoglycerate mutase 2 sumoylation. Thus, sumoylation is an important mechanism that mediates Phosphoglycerate mutase 2 activity and is potentially implicated in Phosphoglycerate mutase 2 mutation-linked disease in humans.
| S-EPMC9795042 | biostudies-literature
Project description:Lower glycolysis involves a series of reversible reactions, which interconvert intermediates that also feed anabolic pathways. 3-phosphoglycerate (3-PG) is an abundant lower glycolytic intermediate that feeds serine biosynthesis via the enzyme phosphoglycerate dehydrogenase, which is genomically amplified in several cancers. Phosphoglycerate mutase 1 (PGAM1) catalyzes the isomerization of 3-PG into the downstream glycolytic intermediate 2-phosphoglycerate (2-PG). PGAM1 needs to be histidine phosphorylated to become catalytically active. We show that the primary PGAM1 histidine phosphate donor is 2,3-bisphosphoglycerate (2,3-BPG), which is made from the glycolytic intermediate 1,3-bisphosphoglycerate (1,3-BPG) by bisphosphoglycerate mutase (BPGM). When BPGM is knocked out, 1,3-BPG can directly phosphorylate PGAM1. In this case, PGAM1 phosphorylation and activity are decreased, but nevertheless sufficient to maintain normal glycolytic flux and cellular growth rate. 3-PG, however, accumulates, leading to increased serine synthesis. Thus, one biological function of BPGM is controlling glycolytic intermediate levels and thereby serine biosynthetic flux.
| S-EPMC5605442 | biostudies-literature
Project description:Dysregulated glycolysis, including the cancerous Warburg effect, is closely involved in pathological mechanisms of diseased states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been known to exert certain physiological impact in vitro, whereas its regulatory role on glycolysis remains unclear. Here, we identified that PGAM plays a key role in regulating glycolysis in cancer cells but not in standard cells. Cancer-prone phenotype by PGAM overexpression in vivo was associated with upregulated glycolytic features. PGAM interacts and cooperates with Chk1 to regulate the enhanced glycolysis in cancer cells, especially under oncogenic Ras expressing conditions. Genetic or chemical interference of the PGAM-Chk1 interaction, with intact PGAM activity, abrogated the maintenance of cancerous enhanced glycolysis. Thus, the nonenzymatic function of PGAM is essential for the Warburg effect that accompanies cancerous proliferation.
| S-EPMC7338839 | biostudies-literature
Project description:Phosphoglycerate mutase 1 (PGAM1) coordinates glycolysis and biosynthesis to promote cancer cell proliferation, and is believed to be a promising target for cancer therapy. Herein, based on the anthraquinone scaffold, we synthesized 31 anthraquinone derivatives and investigated the structure-activity relationship (SAR). The 3-substitient of sulfonamide on the anthraquinone scaffold was essential for maintaining potency and the modifications of the hydroxyl of alizarin would cause a sharp decrease in potency. In the meantime, we determined the co-crystal structure of PGAM1 and one of the anthraquinone inhibitors 9i with IC50 value of 0.27 μM. The co-crystal structure revealed that F22, K100 and R116 of PGAM1 were critical residues for the binding of inhibitors which further validated the SAR. Consistent with the crystal structure, a competitive assay illustrated that compound 9i was a noncompetitive inhibitor. In addition, compound 9i effectively restrained different lung cancer cells proliferation in vitro. Taken together, this work provides reliable guide for future development of PGAM1 inhibitors and compound 9i may act as a new leading compound for further optimization.
| S-EPMC6429356 | biostudies-literature
Project description:Characterize the gpm1 mutant growth on dual substrate of ethanol and glycerol
2011-01-24 | GSE22122 | GEO
Project description:The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, α-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of α-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional α-actinin, we have cloned and characterized each structural domain. Our results show that this a-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional α-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other α-actinins, which may reduce the affinity for actin.
| S-EPMC4824898 | biostudies-literature