The complex machinery of human cobalamin metabolism

Vitamin B$_{12}$ (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

Structural basis of fumarate hydratase deficiency

Fumarate hydratase catalyzes the stereospecific hydration across the olefinic double bond in fumarate leading to L-malate. The enzyme is expressed in mitochondrial and cytosolic compartments, and participates in the Krebs cycle in mitochondria, as well as in regulation of cytosolic fumarate levels. Fumarate hydratase deficiency is an autosomal recessive trait presenting as metabolic disorder with severe encephalopathy, seizures and poor neurological outcome. Heterozygous mutations are associated with a predisposition to cutaneous and uterine leiomyomas and to renal cancer. The crystal structure of human fumarate hydratase shows that mutations can be grouped into two distinct classes either affecting structural integrity of the core enzyme architecture, or are localized around the enzyme active site

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 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. Keywords: Cobalamin; Cobamide; MMUT; Methylmalonic aciduria; Methylmalonyl-CoA mutase; Vitamin B(12)

Genetic, structural, and functional analysis of pathogenic variations 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 pathogenic variations 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 pathogenic variations in MCEE. Nine patients were homozygous for the known nonsense variation p.Arg47* (c.139C > T), and one for the novel missense variation p.Ile53Arg (c.158T > G). To understand better the molecular basis of MCEE deficiency, we mapped p.Ile53Arg, and two previously described pathogenic variations p.Lys60Gln and p.Arg143Cys, onto our 1.8 Å 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. We solved the structure of MCEE-Arg143Cys to 1.9 Å and found significant disruption of two important loop structures, potentially impacting surface features as well as the active-site pocket. 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 variation may cause disease. MCEE-Arg143Cys was detectable at comparable levels to wt MCEE, but had slightly altered unfolding kinetics and greatly reduced activity. These studies reveal ten new patients with MCEE deficiency and rationalize misfolding and loss of activity as molecular defects in MCEE-type MMAuria

Snapshots of actin and tubulin folding inside the TRiC chaperonin

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. Tagging of the endogenous type II chaperonin TRiC complex using CRISPR knock-in enables its purification for cryo-EM. A series of structures reveal the fate of substrates and co-chaperones inside the TRiC chamber to uncover its inner workings.Peer reviewe

Oxidation resistance 1 regulates post-translational modifications of peroxiredoxin 2 in the cerebellum

Protein aggregation, oxidative and nitrosative stress are etiological factors common to all major neurodegenerative disorders. Therefore, identifying proteins that function at the crossroads of these essential pathways may provide novel targets for therapy. Oxidation resistance 1 (Oxr1) is a protein proven to be neuroprotective against oxidative stress, although the molecular mechanisms involved remain unclear. Here, we demonstrate that Oxr1 interacts with the multifunctional protein, peroxiredoxin 2 (Prdx2), a potent antioxidant enzyme highly expressed in the brain that can also act as a molecular chaperone. Using a combination of in vitro assays and two animal models, we discovered that expression levels of Oxr1 regulate the degree of oligomerization of Prdx2 and also its post-translational modifications (PTMs), specifically suggesting that Oxr1 acts as a functional switch between the antioxidant and chaperone functions of Prdx2. Furthermore, we showed in the Oxr1 knockout mouse that Prdx2 is aberrantly modified by overoxidation and S-nitrosylation in the cerebellum at the presymptomatic stage; this in-turn affected the oligomerization of Prdx2, potentially impeding its normal functions and contributing to the specific cerebellar neurodegeneration in this mouse model

Novel homozygous variants in PRORP expand the genotypic spectrum of combined oxidative phosphorylation deficiency 54

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

Simplifying the use of prognostic information in traumatic brain injury. Part 2: Graphical Presentation of Probabilities

Objective: Clinical features such as those included in the Glasgow Coma Scale (GCS) score, pupil reactivity, and patient age, as well as CT findings, have clear established relationships with patient outcomes due to neurotrauma. Nevertheless, predictions made from combining these features in probabilistic models have not found a role in clinical practice. In this study, the authors aimed to develop a method of displaying probabilities graphically that would be simple and easy to use, thus improving the usefulness of prognostic information in neurotrauma. This work builds on a companion paper describing the GCS-Pupils score (GCS-P) as a tool for assessing the clinical severity of neurotrauma. Methods: Information about early GCS score, pupil response, patient age, CT findings, late outcome according to the Glasgow Outcome Scale, and mortality were obtained at the individual adult patient level from the CRASH (Corticosteroid Randomisation After Significant Head Injury; n = 9045) and IMPACT (International Mission for Prognosis and Clinical Trials in TBI; n = 6855) databases. These data were combined into a pooled data set for the main analysis. Logistic regression was first used to model the combined association between the GCS-P and patient age and outcome, following which CT findings were added to the models. The proportion of variability in outcomes “explained” by each model was assessed using Nagelkerke’s R2. Results: The authors observed that patient age and GCS-P have an additive effect on outcome. The probability of mortality 6 months after neurotrauma is greater with increasing age, and for all age groups the probability of death is greater with decreasing GCS-P. Conversely, the probability of favorable recovery becomes lower with increasing age and lessens with decreasing GCS-P. The effect of combining the GCS-P with patient age was substantially more informative than the GCS-P, age, GCS score, or pupil reactivity alone. Two-dimensional charts were produced displaying outcome probabilities, as percentages, for 5-year increments in age between 15 and 85 years, and for GCS-Ps ranging from 1 to 15; it is readily seen that the movement toward combinations at the top right of the charts reflects a decreasing likelihood of mortality and an increasing likelihood of favorable outcome. Analysis of CT findings showed that differences in outcome are very similar between patients with or without a hematoma, absent cisterns, or subarachnoid hemorrhage. Taken in combination, there is a gradation in risk that aligns with increasing numbers of any of these abnormalities. This information provides added value over age and GCS-P alone, supporting a simple extension of the earlier prognostic charts by stratifying the original charts in the following 3 CT groupings: none, only 1, and 2 or more CT abnormalities. Conclusions: The important prognostic features in neurotrauma can be brought together to display graphically their combined effects on risks of death or on prospects for independent recovery. This approach can support decision making and improve communication of risk among health care professionals, patients, and their relatives. These charts will not replace clinical judgment, but they will reduce the risk of influences from biases

Structural insights into the MMACHC-MMADHC protein complex involved in vitamin B12 trafficking

Conversion of vitamin B12 (cobalamin, Cbl) into the cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzymes, mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase, respectively. The intracellular proteins MMACHC and MMADHC play important roles in processing and targeting the Cbl cofactor to its destination enzymes, and recent evidence suggests that they may interact while performing these essential trafficking functions. To better understand the molecular basis of this interaction, we have mapped the crucial protein regions required, indicate that Cbl is likely processed by MMACHC prior to interaction with MMADHC, and identify patient mutations on both proteins that interfere with complex formation, via different mechanisms. We further report the crystal structure of the MMADHC C-terminal region at 2.2 Å resolution, revealing a modified nitroreductase fold with surprising homology to MMACHC despite their poor sequence conservation. Because MMADHC demonstrates no known enzymatic activity, we propose it as the first protein known to repurpose the nitroreductase fold solely for protein-protein interaction. Using small angle x-ray scattering, we reveal the MMACHC-MMADHC complex as a 1:1 heterodimer and provide a structural model of this interaction, where the interaction region overlaps with the MMACHC-Cbl binding site. Together, our findings provide novel structural evidence and mechanistic insight into an essential biological process, whereby an intracellular "trafficking chaperone" highly specific for a trace element cofactor functions via protein-protein interaction, which is disrupted by inherited disease mutations