Biology subjects

Yue, W. W.

Publications and source records attributed to Yue, W. W..

9 recordsLinked to original sources

bioRxiv2024

Luminescence-based complementation assay to assess target engagement and cell permeability of glycolate oxidase (HAO1) inhibitors.

Glycolate oxidase (HAO1) catalyses the synthesis of glyoxylate, a common metabolic intermediate that causes renal failure if accumulated. HAO1 inhibition is an emerging treatment for primary hyperoxaluria, a rare disorder of glyoxylate metabolism. Here we report the first cell-based measurement of inhibitor uptake and engagement with HAO1, by adapting the cellular thermal shift assay (CETSA) based on Nano luciferase complementation and luminescence readout. By profiling the interaction between HAO1 and four well-characterised inhibitors in intact and lysed HEK293T cells, we showed that our CETSA method differentiates between low-permeability/high-engagement and high-permeability/low-engagement ligands and is able to rank HAO1 inhibitors in line with both recombinant protein methods and previously reported indirect cellular assays. Our methodology addresses the unmet need for a robust, sensitive, and scalable cellular assay to guide HAO1 inhibitor development and, in broader terms, can be rapidly adapted for other targets to simultaneously monitor compound affinity and cellular permeability.

biochemistry↗

bioRxiv2023

Dynamic inter-domain transformations mediate the allosteric regulation of human 5, 10-methylenetetrahydrofolate reductase

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. We determined 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.

biochemistry↗

bioRxiv2023

Architecture and regulation of filamentous humancystathionine beta-synthase

Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life involved in the production of glutathione, cysteine, and hydrogen sulphide1-4. Human CBS appends to its conserved catalytic domain a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerization5-12, however the molecular basis is unknown. 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 molecule of SAM 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. Polymerization stabilises CBS and increases the cooperativity of allosteric activation by SAM. Together our findings elaborate our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders3,13-17.

biochemistry↗

bioRxiv2021

Molecular basis for the regulation of human glycogen synthase by phosphorylation and glucose-6-phosphate

Glycogen synthase (GYS1), in complex with glycogenin (GYG1), is the central enzyme of muscle glycogen biosynthesis, and its inhibition has been proposed as a therapeutic avenue for various glycogen storage diseases (GSDs). GYS1 activity is inhibited by phosphorylation of its N- and C-termini, which can be relieved by allosteric activation of glucose-6-phosphate. However, the structural basis of GYS1 regulation is unclear. Here, we present the first cryo-EM structures of phosphorylated human GYS1 complexed with a minimal interacting region of GYG1 in the inhibited, activated, and catalytically competent states at resolutions of 3.0-4.0 [A]. These structures reveal how phosphorylations of specific N- and C- terminal residues are sensed by different arginine clusters that lock the GYS1 tetramer complex in an inhibited state via inter-subunit interactions. The allosteric activator, glucose-6-phopshate, promotes a conformational change by disrupting these interactions and increases flexibility of GYS1 allowing for a catalytically competent state to occur when bound to the sugar donor UDP-glucose. We also identify an inhibited-like conformation that has not transitioned into the activated state, whereby the locking interaction of phosphorylation with the arginine cluster impedes the subsequent conformational changes due to glucose-6-phosphate binding. Finally, we show that the PP1 phosphatase regulatory subunit PPP1R3C (PTG) is recruited to the GYS1:GYG1 complex through direct interaction with glycogen. Our results address long-standing questions into the mechanism of human glycogen synthase regulation.

biochemistry↗

bioRxiv2020

Naturally occurring cobalamin (B12) analogs can function as cofactors for human methylmalonyl-CoA mutase

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 food 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.

biochemistry↗

bioRxiv2020

Crystal structure and interaction studies of human DHTKD1 provide insight into a mitochondrial megacomplex in lysine catabolism

DHTKD1 is a lesser-studied E1 enzyme belonging to the family of 2-oxoacid dehydrogenases. DHTKD1, in complex with the E2 (dihydrolipoamide succinyltransferase, DLST) and E3 (lipoamide dehydrogenase, DLD) components, is implicated in lysine and tryptophan catabolism by catalysing the oxidative decarboxylation of 2-oxoadipate (2OA) in the mitochondria. Here, we solved the crystal structure of human DHTKD1 at 1.9 [A] resolution in binary complex with the thiamine diphosphate (ThDP) cofactor. Our structure explains the evolutionary divergence of DHTKD1 from the well-characterized homologue 2-oxoglutarate (2OG) dehydrogenase, in its preference for the larger 2OA substrate than 2OG. Inherited DHTKD1 missense mutations cause the lysine metabolic condition 2-aminoadipic and 2-oxoadipic aciduria. Reconstruction of the missense variant proteins reveal their underlying molecular defects, which include protein destabilisation, disruption of protein-protein interactions, and alterations in the protein surface. We further generated a 5.0 [A] reconstruction of the human DLST inner core by single-particle electron microscopy, revealing a 24-mer cubic architecture that serves as a scaffold for assembly of DHTKD1 and DLD. This structural study provides a starting point to develop small molecule DHTKD1 inhibitors for probing mitochondrial energy metabolism.

biochemistry↗

bioRxiv2019

Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism

Iron-sulfur clusters (ISC) are essential in all life forms and carry out many crucial cellular functions. The core machinery for de novo ISC biosynthesis, located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreichs ataxia (FRDA). Recently crystal structures depicting the inactive 3- and 4-way sub-complexes of the ISC biosynthesis machinery, lacking the key activator FXN, have been determined. Here, the 3.2 [A] resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates for the first time in any organism the interactions of FXN with the component proteins. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure sheds light on how FXN facilitates ISC production through unlocking the zinc inhibition and stabilizing key loop conformations of NFS1 and ISCU at the protein-protein interfaces, and offers an explanation of how FRDA clinical mutations affect complex formation and FXN activation.

biochemistry↗

bioRxiv2018

Genetic, structural, and functional analysis of mutations causing methylmalonyl-CoA epimerase deficiency

Human methylmalonyl-CoA epimerase (MCEE) catalyzes the interconversion of D-methylmalonyl-CoA and L-methylmalonyl-CoA in propionate catabolism. Autosomal recessive mutations in MCEE reportedly cause methylmalonic aciduria (MMAuria) in eleven patients. We investigated a cohort of 150 individuals suffering from MMAuria of unknown origin, identifying ten new patients with mutations in MCEE. Nine patients were homozygous for the known nonsense mutation p.Arg47* (c.139C>T), and one for the novel missense mutation p.Ile53Arg (c.158T>G). To understand better the molecular basis of MCEE deficiency, we mapped p.Ile53Arg, and two previously described patient mutations p.Lys60Gln and p.Arg143Cys, onto our 1.8 [A] structure of wild-type (wt) human MCEE. This revealed potential dimeric assembly disruption by p.Ile53Arg, but no clear defects from p.Lys60Gln or p.Arg143Cys. Functional analysis of MCEE-Ile53Arg expressed in a bacterial recombinant system as well as patient-derived fibroblasts revealed nearly undetectable soluble protein levels, defective globular protein behavior, and using a newly developed assay, lack of enzymatic activity - consistent with misfolded protein. By contrast, soluble protein levels, unfolding characteristics and activity of MCEE-Lys60Gln were comparable to wt, leaving unclear how this mutation may cause disease. MCEE-Arg143Cys was detectable at comparable levels to wt MCEE, but had slightly altered unfolding kinetics and greatly reduced activity. We solved the structure of MCEE-Arg143Cys to 1.9 [A] and found significant disruption of two important loop structures, potentially impacting surface features as well as the active-site pocket. These studies reveal ten new patients with MCEE deficiency and rationalize misfolding and loss of activity as molecular defects in MCEE-type MMAuria.

biochemistry↗

bioRxiv2017

A homozygous variant in mitochondrial RNase P subunit PRORP is associated with Perrault syndrome characterized by hearing loss and primary ovarian insufficiency

Perrault syndrome is a rare autosomal recessive condition characterised by sensorineural hearing loss in both sexes and primary ovarian insufficiency in 46 XX, females. It is genetically heterogeneous with biallelic variants in six genes identified to date (HSD17B4, HARS2, LARS2, CLPP, C10orf2 and ERAL1). Most genes possessing variants associated with Perrault syndrome are involved in mitochondrial translation. We describe a consanguineous family with three affected individuals homozygous for a novel missense variant c.1454C>T; p.(Ala485Val) in KIAA0391, encoding proteinaceous RNase P (PRORP), the metallonuclease subunit of the mitochondrial RNase P complex, responsible for the 5-end processing of mitochondrial precursor tRNAs. In RNase P activity assays, RNase P complexes containing the PRORP disease variant produced ~35-45% less 5-processed tRNA than wild type PRORP. Consistently, the accumulation of unprocessed polycistronic mitochondrial transcripts was observed in patient dermal fibroblasts, leading to an observable loss of steady-state levels of mitochondrial oxidative phosphorylation components. Expression of wild type KIAA0391 in patient fibroblasts rescued tRNA processing. Immunohistochemistry analyses of the auditory sensory epithelium from postnatal and adult mouse inner ear showed a high level of PRORP in the efferent synapses and nerve fibres of hair cells, indicating a possible mechanism for the sensorineural hearing loss observed in affected individuals. We have identified a variant in an additional gene associated with Perrault syndrome. With the identification of this disease-causing variant in KIAA0391, reduced function of each of the three subunits of mitochondrial RNase P have now been associated with distinct clinical presentations.\n\nAuthor SummaryPerrault syndrome is a rare genetic condition which results in hearing loss in both sexes and ovarian dysfunction in females. Perrault syndrome may also cause neurological symptoms in some patients. Here, we present the features and genetic basis of the condition in three sisters affected by Perrault syndrome. The sisters did not have pathogenic variants in any of the genes previously associated with Perrault syndrome. We identified a change in the gene KIAA0391, encoding PRORP, a subunit of the mitochondrial RNase P complex. Mitochondrial RNase P is a key enzyme in RNA processing in mitochondria. Impaired RNA processing reduces protein production in mitochondria, which we observed in patient cells along with high levels of unprocessed RNA. When we expressed wild type PRORP in patient cells, the RNA processing improved. We also investigated PRORP localisation in the mouse inner ear and found high levels in the synapses and nerve fibers that transmit sound. It may be that disruption of RNA processing in the mitochondria of these cells causes hearing loss in this family.

genetics↗