Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively. Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.
- MeSH
- adenosintrifosfát * metabolismus MeSH
- alosterická regulace MeSH
- elektronová kryomikroskopie MeSH
- fosfofruktokinasa-1 * metabolismus chemie genetika MeSH
- glykolýza MeSH
- játra enzymologie metabolismus MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.
- MeSH
- adenosintrifosfát metabolismus MeSH
- fosfofruktokinasy metabolismus MeSH
- glukoneogeneze * genetika MeSH
- glukosa metabolismus MeSH
- glycerol metabolismus MeSH
- lidé MeSH
- savci MeSH
- transaldolasa metabolismus MeSH
- Trypanosoma brucei brucei * genetika metabolismus MeSH
- uhlík metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.
- MeSH
- adenosindifosfát metabolismus farmakologie MeSH
- adenosintrifosfát metabolismus farmakologie MeSH
- alosterická regulace MeSH
- bakteriální proteiny antagonisté a inhibitory metabolismus MeSH
- enzymová indukce MeSH
- fosfofruktokinasy antagonisté a inhibitory metabolismus MeSH
- fruktosadifosfáty biosyntéza farmakologie MeSH
- fruktosafosfáty metabolismus farmakologie MeSH
- glukoneogeneze MeSH
- glykolýza MeSH
- hexosafosfáty metabolismus MeSH
- izoenzymy antagonisté a inhibitory metabolismus MeSH
- katalýza MeSH
- kinetika MeSH
- kyslík farmakologie MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- Mycobacterium tuberculosis účinky léků enzymologie MeSH
- pyruvátkinasa metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- substrátová specifita MeSH
- zpětná vazba fyziologická MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
OBJECTIVE: Enzymatic fingerprinting of key enzymes of glucose metabolism is a valuable analysis tool in cell physiological phenotyping of plant samples. Yet, a similar approach for mammalian cell line samples is missing. In this study, we applied semi-high throughput enzyme activity assays that were originally designed for plant samples and tested their feasibility in extracts of six frequently used mammalian cell lines (Caco2, HaCaT, C2C12, HEK293, HepG2 and INS-1E). RESULTS: Enzyme activities for aldolase, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, phosphofructokinase could be detected in samples of one or more mammalian cell lines. We characterized effects of sample dilution, assay temperature and repeated freeze-thaw cycles causing potential biases. After careful selection of experimental parameters, the presented semi-high throughput methods could be established as useful tool for physiological phenotyping of cultured mammalian cells.
- MeSH
- aldolasa metabolismus MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- Caco-2 buňky MeSH
- enzymatické testy metody MeSH
- fenotyp MeSH
- fosfofruktokinasy metabolismus MeSH
- fosfoglukomutasa metabolismus MeSH
- fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus MeSH
- glukosa-6-fosfátdehydrogenasa metabolismus MeSH
- glukosa metabolismus MeSH
- glykolýza * MeSH
- HEK293 buňky MeSH
- hexokinasa metabolismus MeSH
- lidé MeSH
- metabolismus sacharidů * MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- pilotní projekty MeSH
- studie proveditelnosti MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PPi-dependent phosphofructokinase (TvPPi-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPPi-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to "short" bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.
- MeSH
- adenosintrifosfát farmakologie MeSH
- difosfáty metabolismus MeSH
- ferredoxiny metabolismus MeSH
- fosfofruktokinasy chemie metabolismus MeSH
- fylogeneze MeSH
- mitochondrie účinky léků metabolismus MeSH
- molekulární sekvence - údaje MeSH
- organely účinky léků metabolismus MeSH
- promotorové oblasti (genetika) genetika MeSH
- Saccharomyces cerevisiae účinky léků metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- transport proteinů účinky léků MeSH
- Trichomonas vaginalis účinky léků enzymologie MeSH
- vodík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
