RESUMEN
The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3'-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3'-cleavage and 3'-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5' and 3' tRNA-end processing, methylation and 3'-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3'-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria.
Asunto(s)
Mitocondrias , ARN de Transferencia , Ribonucleasa P , ARNt Metiltransferasas , Humanos , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/química , Mitocondrias/metabolismo , Ribonucleasa P/metabolismo , Ribonucleasa P/genética , Ribonucleasa P/química , ARNt Metiltransferasas/metabolismo , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/química , Procesamiento Postranscripcional del ARN , Microscopía por Crioelectrón , ARN Mitocondrial/metabolismo , ARN Mitocondrial/genética , ARN Mitocondrial/química , Metilación , Conformación de Ácido Nucleico , Modelos Moleculares , Precursores del ARN/metabolismo , Precursores del ARN/genéticaRESUMEN
Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders.
Asunto(s)
Cistationina betasintasa , Metionina , Humanos , Cistationina betasintasa/metabolismo , Microscopía por Crioelectrón , S-Adenosilmetionina/metabolismo , Dominio CatalíticoRESUMEN
5,10-methylenetetrahydrofolate reductase (MTHFR) commits folate-derived one-carbon units to generate the methyl-donor S-adenosyl-L-methionine (SAM). Eukaryotic MTHFR appends to the well-conserved catalytic domain (CD) a unique regulatory domain (RD) that confers feedback inhibition by SAM. Here we determine the cryo-electron microscopy structures of human MTHFR bound to SAM and its demethylated product S-adenosyl-L-homocysteine (SAH). In the active state, with the RD bound to a single SAH, the CD is flexible and exposes its active site for catalysis. However, in the inhibited state the RD pocket is remodelled, exposing a second SAM-binding site that was previously occluded. Dual-SAM bound MTHFR demonstrates a substantially rearranged inter-domain linker that reorients the CD, inserts a loop into the active site, positions Tyr404 to bind the cofactor FAD, and blocks substrate access. Our data therefore explain the long-distance regulatory mechanism of MTHFR inhibition, underpinned by the transition between dual-SAM and single-SAH binding in response to cellular methylation status.
Asunto(s)
Metilenotetrahidrofolato Reductasa (NADPH2) , S-Adenosilmetionina , Humanos , Regulación Alostérica , Metilenotetrahidrofolato Reductasa (NADPH2)/química , Microscopía por Crioelectrón , S-Adenosilmetionina/metabolismo , MetilaciónRESUMEN
Biallelic hypomorphic variants in PRORP have been recently described as causing the autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a phenotypic spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy. Here, we report three additional families with homozygous missense PRORP variants with pleiotropic phenotypes. Each missense variant altered a highly conserved residue within the metallonuclease domain. In vitro mitochondrial tRNA processing assays with recombinant TRMT10C, SDR5C1 and PRORP indicated two COXPD54-associated PRORP variants, c.1159A>G (p.Thr387Ala) and c.1241C>T (p.Ala414Val), decreased pre-tRNAIle cleavage, consistent with both variants impacting tRNA processing. No significant decrease in tRNA processing was observed with PRORP c.1093T>C (p.Tyr365His), which was identified in an individual with leukodystrophy. These data provide independent evidence that PRORP variants are associated with COXPD54 and that the assessment of 5' leader mitochondrial tRNA processing is a valuable assay for the functional analysis and clinical interpretation of novel PRORP variants.
Asunto(s)
Pérdida Auditiva Sensorineural , Enfermedades Mitocondriales , Ribonucleasa P , Femenino , Humanos , Genotipo , Pérdida Auditiva Sensorineural/genética , Homocigoto , Enfermedades Mitocondriales/genética , ARN de Transferencia , Ribonucleasa P/genéticaRESUMEN
Vitamin B12 (cobalamin, Cbl) is required as a cofactor by two human enzymes, 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) and methylmalonyl-CoA mutase (MMUT). Within the body, a vast array of transporters, enzymes and chaperones are required for the generation and delivery of these cofactor forms. How they perform these functions is dictated by the structure and interactions of the proteins involved, the molecular bases of which are only now being elucidated. In this review, we highlight recent insights into human Cbl metabolism and address open questions in the field by employing a protein structure and interactome based perspective. We discuss how three very similar proteins-haptocorrin, intrinsic factor and transcobalamin-exploit slight structural differences and unique ligand receptor interactions to effect selective Cbl absorption and internalisation. We describe recent advances in the understanding of how endocytosed Cbl is transported across the lysosomal membrane and the implications of the recently solved ABCD4 structure. We detail how MMACHC and MMADHC cooperate to modify and target cytosolic Cbl to the client enzymes MTR and MMUT using ingenious modifications to an ancient nitroreductase fold, and how MTR and MMUT link with their accessory enzymes to sustainably harness the supernucleophilic potential of Cbl. Finally, we provide an outlook on how future studies may combine structural and interactome based approaches and incorporate knowledge of post-translational modifications to bring further insights.
Asunto(s)
Metilmalonil-CoA Mutasa , Vitamina B 12 , Humanos , Vitamina B 12/metabolismo , Metilmalonil-CoA Mutasa/metabolismo , Transporte Biológico , Chaperonas Moleculares , Transportadoras de Casetes de Unión a ATP/metabolismo , Oxidorreductasas/metabolismoRESUMEN
Mitophagy, the elimination of mitochondria via the autophagy-lysosome pathway, is essential for the maintenance of cellular homeostasis. The best characterised mitophagy pathway is mediated by stabilisation of the protein kinase PINK1 and recruitment of the ubiquitin ligase Parkin to damaged mitochondria. Ubiquitinated mitochondrial surface proteins are recognised by autophagy receptors including NDP52 which initiate the formation of an autophagic vesicle around the mitochondria. Damaged mitochondria also generate reactive oxygen species (ROS) which have been proposed to act as a signal for mitophagy, however the mechanism of ROS sensing is unknown. Here we found that oxidation of NDP52 is essential for the efficient PINK1/Parkin-dependent mitophagy. We identified redox-sensitive cysteine residues involved in disulphide bond formation and oligomerisation of NDP52 on damaged mitochondria. Oligomerisation of NDP52 facilitates the recruitment of autophagy machinery for rapid mitochondrial degradation. We propose that redox sensing by NDP52 allows mitophagy to function as a mechanism of oxidative stress response.
Asunto(s)
Mitofagia , Proteínas Nucleares , Proteínas Quinasas , Humanos , Autofagia , Células HeLa , Mitofagia/fisiología , Oxidación-Reducción , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Nucleares/metabolismoRESUMEN
Glycogen synthase (GYS1) is the central enzyme in muscle glycogen biosynthesis. GYS1 activity is inhibited by phosphorylation of its amino (N) and carboxyl (C) termini, which is relieved by allosteric activation of glucose-6-phosphate (Glc6P). We present cryo-EM structures at 3.0-4.0 Å resolution of phosphorylated human GYS1, in complex with a minimal interacting region of glycogenin, in the inhibited, activated and catalytically competent states. Phosphorylations of specific terminal residues are sensed by different arginine clusters, locking the GYS1 tetramer in an inhibited state via intersubunit interactions. The Glc6P activator promotes conformational change by disrupting these interactions and increases the flexibility of GYS1, such that it is poised to adopt a catalytically competent state when the sugar donor UDP-glucose (UDP-glc) binds. We also identify an inhibited-like conformation that has not transitioned into the activated state, in which the locking interaction of phosphorylation with the arginine cluster impedes subsequent conformational changes due to Glc6P binding. Our results address longstanding questions regarding the mechanism of human GYS1 regulation.
Asunto(s)
Glucosa-6-Fosfato , Glucógeno Sintasa , Arginina/metabolismo , Glucosa-6-Fosfato/metabolismo , Glucógeno Sintasa/química , Glucógeno Sintasa/metabolismo , Humanos , Fosforilación , Uridina Difosfato/metabolismoRESUMEN
Urofacial (also called Ochoa) syndrome (UFS) is an autosomal recessive congenital disorder of the urinary bladder featuring voiding dysfunction and a grimace upon smiling. Biallelic variants in HPSE2, coding for the secreted protein heparanase-2, are described in around half of families genetically studied. Hpse2 mutant mice have aberrant bladder nerves. We sought to expand the genotypic spectrum of UFS and make insights into its pathobiology. Sanger sequencing, next generation sequencing and microarray analysis were performed in four previously unreported families with urinary tract disease and grimacing. In one, the proband had kidney failure and was homozygous for the previously described pathogenic variant c.429T>A, p.(Tyr143*). Three other families each carried a different novel HPSE2 variant. One had homozygous triplication of exons 8 and 9; another had homozygous deletion of exon 4; and another carried a novel c.419C>G variant encoding the missense p.Pro140Arg in trans with c.1099-1G>A, a previously reported pathogenic splice variant. Expressing the missense heparanase-2 variant in vitro showed that it was secreted as normal, suggesting that 140Arg has aberrant functionality after secretion. Bladder autonomic neurons emanate from pelvic ganglia where resident neural cell bodies derive from migrating neural crest cells. We demonstrated that, in normal human embryos, neuronal precursors near the developing hindgut and lower urinary tract were positive for both heparanase-2 and leucine rich repeats and immunoglobulin like domains 2 (LRIG2). Indeed, biallelic variants of LRIG2 have been implicated in rare UFS families. The study expands the genotypic spectrum in HPSE2 in UFS and supports a developmental neuronal pathobiology.
RESUMEN
Recombinant protein expression in eukaryotic insect cells is a powerful approach for producing challenging targets. However, due to incompatibility with standard baculoviral platforms and existing low-throughput methodology, the use of the Drosophila melanogaster "S2" cell line lags behind more common insect cell lines such as Sf9 or High-Five™. Due to the advantages of S2 cells, particularly for secreted and secretable proteins, the lack of a simple and parallelizable S2-based platform represents a bottleneck, particularly for biochemical and biophysical laboratories. Therefore, we developed FAS2FURIOUS, a simple and rapid S2 expression pipeline built upon an existing low-throughput commercial platform. FAS2FURIOUS is comparable in effort to simple E. coli systems and allows users to clone and test up to 46 constructs in just 2 weeks. Given the ability of S2 cells to express challenging targets, including receptor ectodomains, secreted glycoproteins, and viral antigens, FAS2FURIOUS represents an attractive orthogonal approach for protein expression in eukaryotic cells.
RESUMEN
Primary hyperoxaluria type I (PH1) is caused by AGXT gene mutations that decrease the functional activity of alanine:glyoxylate aminotransferase. A build-up of the enzyme's substrate, glyoxylate, results in excessive deposition of calcium oxalate crystals in the renal tract, leading to debilitating renal failure. Oxidation of glycolate by glycolate oxidase (or hydroxy acid oxidase 1, HAO1) is a major cellular source of glyoxylate, and siRNA studies have shown phenotypic rescue of PH1 by the knockdown of HAO1, representing a promising inhibitor target. Here, we report the discovery and optimization of six low-molecular-weight fragments, identified by crystallography-based fragment screening, that bind to two different sites on the HAO1 structure: at the active site and an allosteric pocket above the active site. The active site fragments expand known scaffolds for substrate-mimetic inhibitors to include more chemically attractive molecules. The allosteric fragments represent the first report of non-orthosteric inhibition of any hydroxy acid oxidase and hold significant promise for improving inhibitor selectivity. The fragment hits were verified to bind and inhibit HAO1 in solution by fluorescence-based activity assay and surface plasmon resonance. Further optimization cycle by crystallography and biophysical assays have generated two hit compounds of micromolar (44 and 158 µM) potency that do not compete with the substrate and provide attractive starting points for the development of potent and selective HAO1 inhibitors.
RESUMEN
The integrity of a cell's proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.
Asunto(s)
Actinas , Tubulina (Proteína) , Actinas/metabolismo , Chaperonina con TCP-1/metabolismo , Chaperoninas/química , Chaperoninas/metabolismo , Humanos , Masculino , Péptidos , Pliegue de Proteína , Tubulina (Proteína)/metabolismoRESUMEN
Human mitochondrial RNase P (mt-RNase P) is responsible for 5' end processing of mitochondrial precursor tRNAs, a vital step in mitochondrial RNA maturation, and is comprised of three protein subunits: TRMT10C, SDR5C1 (HSD10), and PRORP. Pathogenic variants in TRMT10C and SDR5C1 are associated with distinct recessive or x-linked infantile onset disorders, resulting from defects in mitochondrial RNA processing. We report four unrelated families with multisystem disease associated with bi-allelic variants in PRORP, the metallonuclease subunit of mt-RNase P. Affected individuals presented with variable phenotypes comprising sensorineural hearing loss, primary ovarian insufficiency, developmental delay, and brain white matter changes. Fibroblasts from affected individuals in two families demonstrated decreased steady state levels of PRORP, an accumulation of unprocessed mitochondrial transcripts, and decreased steady state levels of mitochondrial-encoded proteins, which were rescued by introduction of the wild-type PRORP cDNA. In mt-tRNA processing assays performed with recombinant mt-RNase P proteins, the disease-associated variants resulted in diminished mitochondrial tRNA processing. Identification of disease-causing variants in PRORP indicates that pathogenic variants in all three subunits of mt-RNase P can cause mitochondrial dysfunction, each with distinct pleiotropic clinical presentations.
Asunto(s)
Alelos , Pleiotropía Genética , Mitocondrias/enzimología , ARN Mitocondrial/genética , ARN de Transferencia/genética , Ribonucleasa P/genética , Adulto , Femenino , Humanos , Masculino , LinajeRESUMEN
Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2Apro), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2Apro result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2Apro of genetic groups A and B hinder 2Apro as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2Apro cleave the substrate GAB1 at different sites, the 2Apro from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2Apro structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2Apro-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2Apro as a potential target for broad-spectrum anti-enteroviral inhibitors.
Asunto(s)
Antivirales/química , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Rhinovirus/enzimología , Proteínas Virales/química , Proteínas Virales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Cisteína Endopeptidasas/genética , Enterovirus/química , Enterovirus/enzimología , Factor 4G Eucariótico de Iniciación/metabolismo , Variación Genética , Células HeLa , Humanos , Conformación Proteica , Rhinovirus/química , Rhinovirus/genética , Especificidad por Sustrato , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genéticaRESUMEN
Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called "lid loop" is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS "activation" loop, formerly known as the "gating" loop) renders a highly active protein in stable tetrameric form. We conclude that the "activation" loop, a known target for GLS inhibition, may also be a drug target for GLS2.
Asunto(s)
Activación Enzimática , Glutaminasa/química , Hígado/enzimología , Sustitución de Aminoácidos , Catálisis , Glutaminasa/genética , Glutaminasa/metabolismo , Humanos , Mutación Missense , Estructura Cuaternaria de Proteína , Relación Estructura-ActividadRESUMEN
Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP-competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.
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Inhibidores Enzimáticos/uso terapéutico , Galactoquinasa/antagonistas & inhibidores , Galactosemias/tratamiento farmacológico , Cristalografía por Rayos X , Galactoquinasa/química , Humanos , Conformación ProteicaRESUMEN
Succinyl-CoA:3-oxoacid coenzyme A transferase deficiency (SCOTD) is a rare autosomal recessive disorder of ketone body utilization caused by mutations in OXCT1. We performed a systematic literature search and evaluated clinical, biochemical and genetic data on 34 previously published and 10 novel patients with SCOTD. Structural mapping and in silico analysis of protein variants is also presented. All patients presented with severe ketoacidotic episodes. Age at first symptoms ranged from 36 h to 3 years (median 7 months). About 70% of patients manifested in the first year of life, approximately one quarter already within the neonatal period. Two patients died, while the remainder (95%) were alive at the time of the report. Almost all the surviving patients (92%) showed normal psychomotor development and no neurologic abnormalities. A total of 29 missense mutations are reported. Analysis of the published crystal structure of the human SCOT enzyme, paired with both sequence-based and structure-based methods to predict variant pathogenicity, provides insight into the biochemical consequences of the reported variants. Pathogenic variants cluster in SCOT protein regions that affect certain structures of the protein. The described pathogenic variants can be viewed in an interactive map of the SCOT protein at https://michelanglo.sgc.ox.ac.uk/r/oxct. This comprehensive data analysis provides a systematic overview of all cases of SCOTD published to date. Although SCOTD is a rather benign disorder with often favourable outcome, metabolic crises can be life-threatening or even fatal. As the diagnosis can only be made by enzyme studies or mutation analyses, SCOTD may be underdiagnosed.
Asunto(s)
Acidosis , Encefalopatías Metabólicas Innatas , Coenzima A Transferasas/deficiencia , Mutación Missense , Trastornos del Neurodesarrollo , Acidosis/enzimología , Acidosis/genética , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/genética , Coenzima A Transferasas/química , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Cristalografía por Rayos X , Humanos , Cuerpos Cetónicos/química , Cuerpos Cetónicos/genética , Cuerpos Cetónicos/metabolismo , Trastornos del Neurodesarrollo/enzimología , Trastornos del Neurodesarrollo/genética , Dominios ProteicosRESUMEN
The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme's active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Metilenotetrahidrofolato Reductasa (NADPH2)/antagonistas & inhibidores , Metilenotetrahidrofolato Reductasa (NADPH2)/química , S-Adenosilmetionina/química , Regulación Alostérica , Humanos , Dominios ProteicosRESUMEN
Cobalamin, commonly known as vitamin B12, is an essential micronutrient for humans because of its role as an enzyme cofactor. Cobalamin is one of over a dozen structurally related compounds - cobamides - that are found in certain foods and are produced by microorganisms in the human gut. Very little is known about how different cobamides affect B12-dependent metabolism in human cells. Here, we test in vitro how diverse cobamide cofactors affect the function of methylmalonyl-CoA mutase (MMUT), one of two cobalamin-dependent enzymes in humans. We find that, although cobalamin is the most effective cofactor for MMUT, multiple cobamides support MMUT function with differences in binding affinity (Kd), binding kinetics (kon), and concentration dependence during catalysis (KM, app). Additionally, we find that six disease-associated MMUT variants that cause cobalamin-responsive impairments in enzymatic activity also respond to other cobamides, with the extent of catalytic rescue dependent on the identity of the cobamide. Our studies challenge the exclusive focus on cobalamin in the context of human physiology, indicate that diverse cobamides can support the function of a human enzyme, and suggest future directions that will improve our understanding of the roles of different cobamides in human biology.
Asunto(s)
Coenzimas/química , Metilmalonil-CoA Mutasa/química , Vitamina B 12/química , Coenzimas/metabolismo , Humanos , Cinética , Metilmalonil-CoA Mutasa/metabolismo , Vitamina B 12/metabolismoRESUMEN
A versatile and Lipinski-compliant DNA-encoded library (DEL), comprising 366 600 glutamic acid derivatives coupled to oligonucleotides serving as amplifiable identification barcodes is designed, constructed, and characterized. The GB-DEL library, constructed in single-stranded DNA format, allows de novo identification of specific binders against several pharmaceutically relevant proteins. Moreover, hybridization of the single-stranded DEL with a set of known protein ligands of low to medium affinity coupled to a complementary DNA strand results in self-assembled selectable chemical structures, leading to the identification of affinity-matured compounds.