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Genes May 2024LONP1 is the principal AAA+ unfoldase and bulk protease in the mitochondrial matrix, so its deletion causes embryonic lethality. The AAA+ unfoldase CLPX and the... (Review)
Review
Knockout Mouse Studies Show That Mitochondrial CLPP Peptidase and CLPX Unfoldase Act in Matrix Condensates near IMM, as Fast Stress Response in Protein Assemblies for Transcript Processing, Translation, and Heme Production.
LONP1 is the principal AAA+ unfoldase and bulk protease in the mitochondrial matrix, so its deletion causes embryonic lethality. The AAA+ unfoldase CLPX and the peptidase CLPP also act in the matrix, especially during stress periods, but their substrates are poorly defined. Mammalian CLPP deletion triggers infertility, deafness, growth retardation, and cGAS-STING-activated cytosolic innate immunity. CLPX mutations impair heme biosynthesis and heavy metal homeostasis. CLPP and CLPX are conserved from bacteria to humans, despite their secondary role in proteolysis. Based on recent proteomic-metabolomic evidence from knockout mice and patient cells, we propose that CLPP acts on phase-separated ribonucleoprotein granules and CLPX on multi-enzyme condensates as first-aid systems near the inner mitochondrial membrane. Trimming within assemblies, CLPP rescues stalled processes in mitoribosomes, mitochondrial RNA granules and nucleoids, and the D-foci-mediated degradation of toxic double-stranded mtRNA/mtDNA. Unfolding multi-enzyme condensates, CLPX maximizes PLP-dependent delta-transamination and rescues malformed nascent peptides. Overall, their actions occur in granules with multivalent or hydrophobic interactions, separated from the aqueous phase. Thus, the role of CLPXP in the matrix is compartment-selective, as other mitochondrial peptidases: MPPs at precursor import pores, m-AAA and i-AAA at either IMM face, PARL within the IMM, and OMA1/HTRA2 in the intermembrane space.
Topics: Endopeptidase Clp; Animals; Mice; Mitochondria; Mitochondrial Proteins; Mice, Knockout; Heme; Protein Biosynthesis; Humans; Mitochondrial Membranes; Stress, Physiological
PubMed: 38927630
DOI: 10.3390/genes15060694 -
International Journal of Biological... Jun 2024In the current study, two sets of compounds: (E)-1-(2-(4-substitutedphenyl)-2-oxoethyl)-4-((hydroxyimino)methyl)pyridinium derivatives (3a-3e); and...
Identification of potent indolizine derivatives against Mycobacterial tuberculosis: In vitro anti-TB properties, in silico target validation, molecular docking and dynamics studies.
In the current study, two sets of compounds: (E)-1-(2-(4-substitutedphenyl)-2-oxoethyl)-4-((hydroxyimino)methyl)pyridinium derivatives (3a-3e); and (E)-3-(substitutedbenzoyl)-7-((hydroxyimino)methyl)-2-substitutedindolizine-1-carboxylate derivatives (5a-5j), were synthesized and biologically evaluated against two strains of Mycobacterial tuberculosis (ATCC 25177) and multi-drug resistant (MDR) strains. Further, they were also tested in vitro against the mycobacterial InhA enzyme. The in vitro results showed excellent inhibitory activities against both MTB strains and compounds 5a-5j were found to be more potent, and their MIC values ranged from 5 to 16 μg/mL and 16-64 μg/mL against the M. tuberculosis (ATCC 25177) and MDR-TB strains, respectively. Compound 5h with phenyl and 4-fluorobenzoyl groups attached to the 2- and 3-position of the indolizine core was found to be the most active against both strains with MIC values of 5 μg/mL and 16 μg/mL, respectively. On the other hand, the two sets of compounds showed weak to moderate inhibition of InhA enzyme activity that ranged from 5 to 17 % and 10-52 %, respectively, with compound 5f containing 4-fluoro benzoyl group attached to the 3-position of the indolizine core being the most active (52 % inhibition of InhA). Unfortunately, there was no clear correlation between the InhA inhibitory activity and MIC values of the tested compounds, indicating the probability that they might have different modes of action other than InhA inhibition. Therefore, a computational investigation was conducted by employing molecular docking to identify their putative drug target(s) and, consequently, understand their mechanism of action. A panel of 20 essential mycobacterial enzymes was investigated, of which β-ketoacyl acyl carrier protein synthase I (KasA) and pyridoxal-5'-phosphate (PLP)-dependent aminotransferase (BioA) enzymes were revealed as putative targets for compounds 3a-3e and 5a-5j, respectively. Moreover, in silico ADMET predictions showed adequate properties for these compounds, making them promising leads worthy of further optimization.
PubMed: 38925196
DOI: 10.1016/j.ijbiomac.2024.133285 -
Journal of Bone and Mineral Research :... Jun 2024
PubMed: 38905292
DOI: 10.1093/jbmr/zjae098 -
Nutrients May 2024Exploring the link between genetic polymorphisms in folate metabolism genes (MTHFR, MTR, and MTRR) and cardiovascular disease (CVD), this study evaluates the effect of B... (Randomized Controlled Trial)
Randomized Controlled Trial
Effect of Methylfolate, Pyridoxal-5'-Phosphate, and Methylcobalamin (Soloways) Supplementation on Homocysteine and Low-Density Lipoprotein Cholesterol Levels in Patients with Methylenetetrahydrofolate Reductase, Methionine Synthase, and Methionine Synthase Reductase Polymorphisms: A Randomized...
Exploring the link between genetic polymorphisms in folate metabolism genes (MTHFR, MTR, and MTRR) and cardiovascular disease (CVD), this study evaluates the effect of B vitamin supplements (methylfolate, pyridoxal-5'-phosphate, and methylcobalamin) on homocysteine and lipid levels, potentially guiding personalized CVD risk management. In a randomized, double-blind, placebo-controlled trial, 54 patients aged 40-75 with elevated homocysteine and moderate LDL-C levels were divided based on MTHFR, MTR, and MTRR genetic polymorphisms. Over six months, they received either a combination of methylfolate, P5P, and methylcobalamin, or a placebo. At the 6 months follow-up, the treatment group demonstrated a significant reduction in homocysteine levels by 30.0% (95% CI: -39.7% to -20.3%) and LDL-C by 7.5% (95% CI: -10.3% to -4.7%), compared to the placebo ( < 0.01 for all). In the subgroup analysis, Homozygous Minor Allele Carriers showed a more significant reduction in homocysteine levels (48.3%, 95% CI: -62.3% to -34.3%, < 0.01) compared to mixed allele carriers (18.6%, 95% CI: -25.6% to -11.6%, < 0.01), with a notable intergroup difference (29.7%, 95% CI: -50.7% to -8.7%, < 0.01). LDL-C levels decreased by 11.8% in homozygous carriers (95% CI: -15.8% to -7.8%, < 0.01) and 4.8% in mixed allele carriers (95% CI: -6.8% to -2.8%, < 0.01), with a significant between-group difference (7.0%, 95% CI: -13.0% to -1.0%, < 0.01). Methylfolate, P5P, and methylcobalamin supplementation tailored to genetic profiles effectively reduced homocysteine and LDL-C levels in patients with specific MTHFR, MTR, and MTRR polymorphisms, particularly with homozygous minor allele polymorphisms.
Topics: Humans; Middle Aged; Homocysteine; Female; Male; Dietary Supplements; Methylenetetrahydrofolate Reductase (NADPH2); Double-Blind Method; 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Cholesterol, LDL; Aged; Vitamin B 12; Pyridoxal Phosphate; Adult; Ferredoxin-NADP Reductase; Tetrahydrofolates; Polymorphism, Genetic; Vitamin B Complex
PubMed: 38892484
DOI: 10.3390/nu16111550 -
Biochemistry Jun 2024The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and...
The conserved enzyme aminolevulinic acid synthase (ALAS) initiates heme biosynthesis in certain bacteria and eukaryotes by catalyzing the condensation of glycine and succinyl-CoA to yield aminolevulinic acid. In humans, the ALAS isoform responsible for heme production during red blood cell development is the erythroid-specific ALAS2 isoform. Owing to its essential role in erythropoiesis, changes in human ALAS2 (hALAS2) function can lead to two different blood disorders. X-linked sideroblastic anemia results from loss of ALAS2 function, while X-linked protoporphyria results from gain of ALAS2 function. Interestingly, mutations in the ALAS2 C-terminal extension can be implicated in both diseases. Here, we investigate the molecular basis for enzyme dysfunction mediated by two previously reported C-terminal loss-of-function variants, hALAS2 V562A and M567I. We show that the mutations do not result in gross structural perturbations, but the enzyme stability for V562A is decreased. Additionally, we show that enzyme stability moderately increases with the addition of the pyridoxal 5'-phosphate (PLP) cofactor for both variants. The variants display differential binding to PLP and the individual substrates compared to wild-type hALAS2. Although hALAS2 V562A is a more active enzyme , it is less efficient concerning succinyl-CoA binding. In contrast, the M567I mutation significantly alters the cooperativity of substrate binding. In combination with previously reported cell-based studies, our work reveals the molecular basis by which hALAS2 C-terminal mutations negatively affect ALA production necessary for proper heme biosynthesis.
PubMed: 38888931
DOI: 10.1021/acs.biochem.4c00066 -
ELife Jun 2024Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these...
Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5'-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.
Topics: Animals; Mice; Humans; Enzyme Inhibitors; Phosphoric Monoester Hydrolases; Hippocampus; Neurons; Pyridoxal Phosphate; Flavones; Mice, Inbred C57BL
PubMed: 38856179
DOI: 10.7554/eLife.93094 -
RSC Chemical Biology Jun 2024The account attempts to substantiate the hypothesis that, from an evolutionary perspective, the coenzyme couple pyridoxal phosphate and pyridoxamine phosphate preceded... (Review)
Review
The account attempts to substantiate the hypothesis that, from an evolutionary perspective, the coenzyme couple pyridoxal phosphate and pyridoxamine phosphate preceded the coenzyme thiamine pyrophosphate and acted as its less efficient chemical analogue in some form of early metabolism. The analysis combines mechanism-based chemical reactivity with biosynthetic arguments and provides evidence that vestiges of "TPP-like reactivity" are still found for PLP today. From these thoughts, conclusions can be drawn about the key elements of a primordial form of metabolism, which includes the citric acid cycle, amino acid biosynthesis and the pentose phosphate pathway.
PubMed: 38846080
DOI: 10.1039/d4cb00016a -
Physical Chemistry Chemical Physics :... Jun 2024The transsulfuration pathway plays a key role in mammals for maintaining the balance between cysteine and homocysteine, whose concentrations are critical in several...
The transsulfuration pathway plays a key role in mammals for maintaining the balance between cysteine and homocysteine, whose concentrations are critical in several biochemical processes. Human cystathionine β-synthase is a heme-containing, pyridoxal 5'-phosphate (PLP)-dependent enzyme found in this pathway. The heme group does not participate directly in catalysis, but has a regulatory function, whereby CO or NO binding inhibits the PLP-dependent reactions. In this study, we explore the detailed structural changes responsible for inhibition using quantum chemical calculations to validate the experimentally observed bonding patterns associated with heme CO and NO binding and molecular dynamics simulations to explore the medium-range structural changes triggered by gas binding and propagating to the PLP active site, which is more than 20 Å distant from the heme group. Our results support a previously proposed mechanical signaling model, whereby the cysteine decoordination associated with gas ligand binding leads to breaking of a hydrogen bond with an arginine residue on a neighbouring helix. In turn, this leads to a shift in position of the helix, and hence also of the PLP cofactor, ultimately disrupting a key hydrogen bond that stabilizes the PLP in its catalytically active form.
Topics: Cystathionine beta-Synthase; Molecular Dynamics Simulation; Humans; Pyridoxal Phosphate; Gases; Nitric Oxide; Hydrogen Bonding; Carbon Monoxide; Heme; Catalytic Domain; Quantum Theory; Cysteine
PubMed: 38832404
DOI: 10.1039/d4cp01321b -
Cell Death & Disease Jun 2024Vitamin B6 is a water-soluble vitamin which possesses antioxidant properties. Its catalytically active form, pyridoxal 5'-phosphate (PLP), is a crucial cofactor for DNA...
Vitamin B6 is a water-soluble vitamin which possesses antioxidant properties. Its catalytically active form, pyridoxal 5'-phosphate (PLP), is a crucial cofactor for DNA and amino acid metabolism. The inverse correlation between vitamin B6 and cancer risk has been observed in several studies, although dietary vitamin B6 intake sometimes failed to confirm this association. However, the molecular link between vitamin B6 and cancer remains elusive. Previous work has shown that vitamin B6 deficiency causes chromosome aberrations (CABs) in Drosophila and human cells, suggesting that genome instability may correlate the lack of this vitamin to cancer. Here we provide evidence in support of this hypothesis. Firstly, we show that PLP deficiency, induced by the PLP antagonists 4-deoxypyridoxine (4DP) or ginkgotoxin (GT), promoted tumorigenesis in eye larval discs transforming benign Ras tumors into aggressive forms. In contrast, PLP supplementation reduced the development of tumors. We also show that low PLP levels, induced by 4DP or by silencing the sgll gene involved in PLP biosynthesis, worsened the tumor phenotype in another Drosophila cancer model generated by concomitantly activating Ras and downregulating Discs-large (Dlg) gene. Moreover, we found that Ras eye discs from larvae reared on 4DP displayed CABs, reactive oxygen species (ROS) and low catalytic activity of serine hydroxymethyltransferase (SHMT), a PLP-dependent enzyme involved in thymidylate (dTMP) biosynthesis, in turn required for DNA replication and repair. Feeding Ras 4DP-fed larvae with PLP or ascorbic acid (AA) plus dTMP, rescued both CABs and tumors. The same effect was produced by overexpressing catalase in Ras Dlg 4DP-fed larvae, thus allowing to establish a relationship between PLP deficiency, CABs, and cancer. Overall, our data provide the first in vivo demonstration that PLP deficiency can impact on cancer by increasing genome instability, which is in turn mediated by ROS and reduced dTMP levels.
Topics: Animals; Vitamin B 6 Deficiency; Drosophila Proteins; Vitamin B 6; Drosophila melanogaster; Drosophila; Pyridoxal Phosphate; Reactive Oxygen Species; Carcinogenesis; ras Proteins; Neoplasms; Larva; Humans
PubMed: 38830901
DOI: 10.1038/s41419-024-06787-3 -
Nature Metabolism Jun 2024Oxygen is critical for all metazoan organisms on the earth and impacts various biological processes in physiological and pathological conditions. While oxygen-sensing...
Oxygen is critical for all metazoan organisms on the earth and impacts various biological processes in physiological and pathological conditions. While oxygen-sensing systems inducing acute hypoxic responses, including the hypoxia-inducible factor pathway, have been identified, those operating in prolonged hypoxia remain to be elucidated. Here we show that pyridoxine 5'-phosphate oxidase (PNPO), which catalyses bioactivation of vitamin B6, serves as an oxygen sensor and regulates lysosomal activity in macrophages. Decreased PNPO activity under prolonged hypoxia reduced an active form of vitamin B6, pyridoxal 5'-phosphate (PLP), and inhibited lysosomal acidification, which in macrophages led to iron dysregulation, TET2 protein loss and delayed resolution of the inflammatory response. Among PLP-dependent metabolism, supersulfide synthesis was suppressed in prolonged hypoxia, resulting in the lysosomal inhibition and consequent proinflammatory phenotypes of macrophages. The PNPO-PLP axis creates a distinct layer of oxygen sensing that gradually shuts down PLP-dependent metabolism in response to prolonged oxygen deprivation.
Topics: Lysosomes; Macrophages; Animals; Mice; Pyridoxal Phosphate; Hypoxia; Cell Hypoxia; Vitamin B 6; Oxygen; Inflammation
PubMed: 38822028
DOI: 10.1038/s42255-024-01053-4