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The Hsp70 system is essential for maintaining protein homeostasis and comprises a central Hsp70 and two accessory proteins that belong to the J-domain protein (JDP) and nucleotide exchange factor families. Posttranslational modifications offer a means to tune the activity of the system. We explore how phosphorylation of specific residues of the J-domain of DNAJA2, a class A JDP, regulates Hsc70 activity using biochemical and structural approaches. Among these residues, we find that pseudophosphorylation of Y10 and S51 enhances the holding/folding balance of the Hsp70 system, reducing cochaperone collaboration with Hsc70 while maintaining the holding capacity. Truly phosphorylated J domains corroborate phosphomimetic variant effects. Notably, distinct mechanisms underlie functional impacts of these DNAJA2 variants. Pseudophosphorylation of Y10 induces partial disordering of the J domain, whereas the S51E substitution weakens essential DNAJA2-Hsc70 interactions without a large structural reorganization of the protein. S51 phosphorylation might be class-specific, as all cytosolic class A human JDPs harbor a phosphorylatable residue at this position., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Phosphorus-containing metabolites occupy a prominent position in cell pathways. The phosphorometabolomic approach in human sperm samples will deliver valuable information as new male fertility biomarkers could emerge. This study analyzed, by 31 P-NMR, seminal plasma and whole semen from asthenozoospermic and normozoospermic samples (71% vs. 27% and 45% vs. 17%, total and progressive sperm motility, respectively), and also ejaculates from healthy donors. At least 16 phosphorus-containing metabolites involved in central energy metabolism and phospholipid, nucleotide, and nicotinamide metabolic pathways were assigned and different abundances between the samples with distinct sperm quality was detected. Specifically, higher levels of phosphocholine, glucose-1-phosphate, and to a lesser degree, acetyl phosphate were found in the asthenozoospermic seminal plasma. Notably, the phosphorometabolites implicated in lipid metabolism were highlighted in the seminal plasma, while those associated with carbohydrate metabolism were more abundant in the spermatozoa. Higher levels of phosphocholine, glucose-1-phosphate, and acetyl phosphate in the seminal plasma with poor quality suggest their crucial role in supporting sperm motility through energy metabolic pathways. In the seminal plasma, phosphorometabolites related to lipid metabolism were prominent; however, spermatozoa metabolism is more dependent on carbohydrate-related energy pathways. Understanding the presence and function of sperm phosphorylated metabolites will enhance our knowledge of the metabolic profile of healthy human sperm, improving assessment and differential diagnosis.

There has been an intense focus to uncover the molecular mechanisms by which fasting triggers the adaptive cellular responses in the major organs of the body. Here, we show that in mice, hepatic S-adenosylmethionine (SAMe)-the principal methyl donor-acts as a metabolic sensor of nutrition to fine-tune the catabolic-fasting response by modulating phosphatidylethanolamine N-methyltransferase (PEMT) activity, endoplasmic reticulum-mitochondria contacts, β-oxidation, and ATP production in the liver, together with FGF21-mediated lipolysis and thermogenesis in adipose tissues. Notably, we show that glucagon induces the expression of the hepatic SAMe-synthesizing enzyme methionine adenosyltransferase α1 (MAT1A), which translocates to mitochondria-associated membranes. This leads to the production of this metabolite at these sites, which acts as a brake to prevent excessive β-oxidation and mitochondrial ATP synthesis and thereby endoplasmic reticulum stress and liver injury. This work provides important insights into the previously undescribed function of SAMe as a new arm of the metabolic adaptation to fasting., Competing Interests: Declaration of interests M.L.M.-C. advises for Mitotherapeutix., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuronal K V 7 channels, important regulators of cell excitability, are among the most sensitive proteins to reactive oxygen species. The S2S3 linker of the voltage sensor was reported as a site-mediating redox modulation of the channels. Recent structural insights reveal potential interactions between this linker and the Ca 2+ -binding loop of the third EF-hand of calmodulin (CaM), which embraces an antiparallel fork formed by the C-terminal helices A and B, constituting the calcium responsive domain (CRD). We found that precluding Ca 2+ binding to the EF3 hand, but not to EF1, EF2, or EF4 hands, abolishes oxidation-induced enhancement of K V 7.4 currents. Monitoring FRET (Fluorescence Resonance Energy Transfer) between helices A and B using purified CRDs tagged with fluorescent proteins, we observed that S2S3 peptides cause a reversal of the signal in the presence of Ca 2+ but have no effect in the absence of this cation or if the peptide is oxidized. The capacity of loading EF3 with Ca 2+ is essential for this reversal of the FRET signal, whereas the consequences of obliterating Ca 2+ binding to EF1, EF2, or EF4 are negligible. Furthermore, we show that EF3 is critical for translating Ca 2+ signals to reorient the AB fork. Our data are consistent with the proposal that oxidation of cysteine residues in the S2S3 loop relieves K V 7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is crucial for this signaling., Competing Interests: EN, FJ, AM, JU, AA, CM, GB, CD, OM, NG, AV No competing interests declared, (© 2023, Nuñez et al.)

(1) Background: Human frataxin is an iron binding protein that participates in the biogenesis of iron sulfur clusters and enhances ferrochelatase activity. While frataxin association to other proteins has been extensively characterized up to the structural level, much less is known about the putative capacity of frataxin to interact with functionally related metabolites. In turn, current knowledge about frataxin's capacity to coordinate metal ions is limited to iron (II and III); (2) Methods: here, we used NMR spectroscopy, Molecular Dynamics, and Docking approaches to demonstrate new roles of frataxin; (3) Results: We demonstrate that frataxin also binds Zn 2+ in a structurally similar way to Fe 2+ , but with lower affinity. In turn, both Fe 2+ -loaded and Zn 2+ -loaded frataxins specifically associate to protoporphyrin IX with micromolar affinity, while apo-frataxin does not bind to the porphyrin. Protoporphyrin IX association to metal-loaded frataxin shares the binding epitope with ferrochelatase; and (4) Conclusions: these findings expand the plethora of relevant molecular targets for frataxin and may help to elucidate the yet unknown different roles that this protein exerts in iron regulation and metabolism.

For a long time, conventional medicine has analysed biomolecules to diagnose diseases. Yet, this approach has proven valid only for a limited number of metabolites and often through a bijective relationship with the disease (i.e. glucose relationship with diabetes), ultimately offering incomplete diagnostic value. Nowadays, precision medicine emerges as an option to improve the prevention and/or treatment of numerous pathologies, focusing on the molecular mechanisms, acting in a patient-specific dimension, and leveraging multiple contributing factors such as genetic, environmental, or lifestyle. Metabolomics grasps the required subcellular complexity while being sensitive to all these factors, which results in a most suitable technique for precision medicine. The aim of this chapter is to describe how NMR-based metabolomics can be integrated in the design of a precision medicine strategy, using the Precision Medicine Initiative of the Basque Country (the AKRIBEA project) as a case study. To that end, we will illustrate the procedures to be followed when conducting an NMR-based metabolomics study with a large cohort of individuals, emphasizing the critical points. The chapter will conclude with the discussion of some relevant biomedical applications., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Rare diseases are most often caused by inherited genetic disorders that, after translation, will result in a protein with altered function. Decreased protein stability is the most frequent mechanism associated with a congenital pathogenic missense mutation and it implies the destabilization of the folded conformation in favour of unfolded or misfolded states. In the cellular context and when experimental data is available, a mutant protein with altered thermodynamic stability often also results in impaired homeostasis, with the deleterious accumulation of protein aggregates, metabolites and/or metabolic by-products. In the last decades, a significant effort has enabled the characterization of rare diseases associated to protein stability defects and triggered the development of innovative therapeutic intervention lines, say, the use of pharmacological chaperones to correct the intracellular impaired homeostasis. Here, we review the current knowledge on rare diseases caused by reduced protein stability, paying special attention to the thermodynamic aspects of the protein destabilization, also focusing on some examples where pharmacological chaperones are being tested., (© 2022 Wiley-VCH GmbH.)

Apg2, one of the three cytosolic Hsp110 chaperones in humans, supports reactivation of unordered and ordered protein aggregates by Hsc70 (HspA8). Together with DnaJB1, Apg2 serves to nucleate Hsc70 molecules into sites where productive entropic pulling forces can be developed. During aggregate reactivation, Apg2 performs as a specialized nucleotide exchange factor, but the origin of its specialization is poorly defined. Here we report on the role of the distinctive C-terminal extension present in Apg2 and other metazoan homologs. We found that the first part of this Apg2 subdomain, with propensity to adopt α-helical structure, interacts with the nucleotide binding domain of Hsc70 in a nucleotide-dependent manner, contributing significantly to the stability of the Hsc70:Apg2 complex. Moreover, the second intrinsically disordered segment of Apg2 C-terminal extension plays an important role as a downregulator of nucleotide exchange. An NMR analysis showed that the interaction with Hsc70 nucleotide binding domain modifies the chemical environment of residues located in important functional sites such as the interface between lobe I and II and the nucleotide binding site. Our data indicate that Apg2 C-terminal extension is a fine-tuner of human Hsc70 activity that optimizes the substrate remodeling ability of the chaperone system., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Patients with major forms of acute hepatic porphyria present acute neurological attacks with overproduction of porphobilinogen (PBG) and δ-aminolevulinic acid (ALA). Even if ALA is considered the most likely agent inducing the acute symptoms, the mechanism of its accumulation has not been experimentally demonstrated. In the most frequent form, acute intermittent porphyria (AIP), inherited gene mutations induce a deficiency in PBG deaminase; thus, accumulation of the substrate PBG is biochemically obligated but not that of ALA. A similar scenario is observed in other forms of acute hepatic porphyria (i.e., porphyria variegate, VP) in which PBG deaminase is inhibited by metabolic intermediates. Here, we have investigated the molecular basis of δ-aminolevulinate accumulation using in vitro fluxomics monitored by NMR spectroscopy and other biophysical techniques. Our results show that porphobilinogen, the natural product of δ-aminolevulinate deaminase, effectively inhibits its anabolic enzyme at abnormally low concentrations. Structurally, this high affinity can be explained by the interactions that porphobilinogen generates with the active site, most of them shared with the substrate. Enzymatically, our flux analysis of an altered heme pathway demonstrates that a minimum accumulation of porphobilinogen will immediately trigger the accumulation of δ-aminolevulinate, a long-lasting observation in patients suffering from acute porphyrias.

SARS-CoV-2 infection causes a significant reduction in lipoprotein-bound serum phospholipids give rise to supramolecular phospholipid composite (SPC) signals observed in diffusion and relaxation edited 1 H NMR spectra. To characterize the chemical structural components and compartmental location of SPC and to understand further its possible diagnostic properties, we applied a Statistical HeterospectroscopY in n -dimensions (SHY- n ) approach. This involved statistically linking a series of orthogonal measurements made on the same samples, using independent analytical techniques and instruments, to identify the major individual phospholipid components giving rise to the SPC signals. Thus, an integrated model for SARS-CoV-2 positive and control adults is presented that relates three identified diagnostic subregions of the SPC signal envelope (SPC 1 , SPC 2 , and SPC 3 ) generated using diffusion and relaxation edited (DIRE) NMR spectroscopy to lipoprotein and lipid measurements obtained by in vitro diagnostic NMR spectroscopy and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The SPC signals were then correlated sequentially with (a) total phospholipids in lipoprotein subfractions; (b) apolipoproteins B100, A1, and A2 in different lipoproteins and subcompartments; and (c) MS-measured total serum phosphatidylcholines present in the NMR detection range (i.e., PCs: 16.0,18.2; 18.0,18.1; 18.2,18.2; 16.0,18.1; 16.0,20.4; 18.0,18.2; 18.1,18.2), lysophosphatidylcholines (LPCs: 16.0 and 18.2), and sphingomyelin (SM 22.1). The SPC 3 /SPC 2 ratio correlated strongly ( r = 0.86) with the apolipoprotein B100/A1 ratio, a well-established marker of cardiovascular disease risk that is markedly elevated during acute SARS-CoV-2 infection. These data indicate the considerable potential of using a serum SPC measurement as a metric of cardiovascular risk based on a single NMR experiment. This is of specific interest in relation to understanding the potential for increased cardiovascular risk in COVID-19 patients and risk persistence in post-acute COVID-19 syndrome (PACS).

Proton nuclear magnetic resonance (NMR) N -acetyl signals (Glyc) from glycoproteins and supramolecular phospholipids composite peak (SPC) from phospholipid quaternary nitrogen methyls in subcompartments of lipoprotein particles) can give important systemic metabolic information, but their absolute quantification is compromised by overlap with interfering resonances from lipoprotein lipids themselves. We present a J-Edited DIffusional (JEDI) proton NMR spectroscopic approach to selectively augment signals from the inflammatory marker peaks Glyc and SPCs in blood serum NMR spectra, which enables direct integration of peaks associated with molecules found in specific compartments. We explore a range of pulse sequences that allow editing based on peak J-modulation, translational diffusion, and T 2 relaxation time and validate them for untreated blood serum samples from SARS-CoV-2 infected patients ( n = 116) as well as samples from healthy controls and pregnant women with physiological inflammation and hyperlipidemia ( n = 631). The data show that JEDI is an improved approach to selectively investigate inflammatory signals in serum and may have widespread diagnostic applicability to disease states associated with systemic inflammation.

The human ERG (hERG) K + channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain., Competing Interests: The authors declare no competing interest.

Background and Aims: The liver plays a central role in all metabolic processes in the body. However, precise characterization of liver metabolism is often obscured by its inherent complexity. Phosphorylated metabolites occupy a prominent position in all anabolic and catabolic pathways. Here, we develop a 31 P nuclear magnetic resonance (NMR)-based method to study the liver "phosphorome" through the simultaneous identification and quantification of multiple hydrophilic and hydrophobic phosphorylated metabolites., Approach and Results: We applied this technique to define the metabolic landscape in livers from a mouse model of the rare disease disorder congenital erythropoietic porphyria (CEP) as well as two well-known murine models of nonalcoholic steatohepatitis: one genetic, methionine adenosyltransferase 1A knockout mice, and the other dietary, mice fed a high-fat choline-deficient diet. We report alterations in the concentrations of phosphorylated metabolites that are readouts of the balance between glycolysis, gluconeogenesis, the pentose phosphate pathway, the tricarboxylic acid cycle, and oxidative phosphorylation and of phospholipid metabolism and apoptosis. Moreover, these changes correlate with the main histological features: steatosis, apoptosis, iron deposits, and fibrosis. Strikingly, treatment with the repurposed drug ciclopirox improves the phosphoromic profile of CEP mice, an effect that was mirrored by the normalization of liver histology., Conclusions: In conclusion, these findings indicate that NMR-based phosphoromics may be used to unravel metabolic phenotypes of liver injury and to identify the mechanism of drug action., (© 2021 by the American Association for the Study of Liver Diseases.)

Congenital erythropoietic porphyria (CEP), also known as Günther's disease, results from a deficient activity in the fourth enzyme, uroporphyrinogen III synthase (UROIIIS), of the heme pathway. Ciclopirox (CPX) is an off-label drug, topically prescribed as an antifungal. It has been recently shown that it also acts as a pharmacological chaperone in CEP, presenting a specific activity in deleterious mutations in UROIIIS. Despite CPX is active at subtoxic concentrations, acute gastrointestinal (GI) toxicity was found due to the precipitation in the stomach of the active compound and subsequent accumulation in the intestine. To increase its systemic availability, we carried out pharmacokinetic (PK) and pharmacodynamic (PD) studies using alternative formulations for CPX. Such strategy effectively suppressed GI toxicity in WT mice and in a mouse model of the CEP disease ( UROIIIS P248Q/P248Q ). In terms of activity, phosphorylation of CPX yielded good results in CEP cellular models but showed limited activity when administered to the CEP mouse model. These results highlight the need of a proper formulation for pharmacological chaperones used in the treatment of rare diseases.

Protein oligomerization processes are widespread and of crucial importance to understand degenerative diseases and healthy regulatory pathways. One particular case is the homo-oligomerization of folded domains involving domain swapping, often found as a part of the protein homeostasis in the crowded cytosol, composed of a complex mixture of cosolutes. Here, we have investigated the effect of a plethora of cosolutes of very diverse nature on the kinetics of a protein dimerization by domain swapping. In the absence of cosolutes, our system exhibits slow interconversion rates, with the reaction reaching the equilibrium within the average protein homeostasis timescale (24-48 h). In the presence of crowders, though, the oligomerization reaction in the same time frame will, depending on the protein's initial oligomeric state, either reach a pure equilibrium state or get kinetically trapped into an apparent equilibrium. Specifically, when the reaction is initiated from a large excess of dimer, it becomes unsensitive to the effect of cosolutes and reaches the same equilibrium populations as in the absence of cosolute. Conversely, when the reaction starts from a large excess of monomer, the reaction during the homeostatic timescale occurs under kinetic control, and it is exquisitely sensitive to the presence and nature of the cosolute. In this scenario (the most habitual case in intracellular oligomerization processes), the effect of cosolutes on the intermediate conformation and diffusion-mediated encounters will dictate how the cellular milieu affects the domain-swapping reaction., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Fumarylacetoacetate hydrolase (FAH) is the fifth enzyme in the tyrosine catabolism pathway. A deficiency in human FAH leads to hereditary tyrosinemia type I (HT1), an autosomal recessive disorder that results in the accumulation of toxic metabolites such as succinylacetone, maleylacetoacetate, and fumarylacetoacetate in the liver and kidney, among other tissues. The disease is severe and, when untreated, it can lead to death. A low tyrosine diet combined with the herbicidal nitisinone constitutes the only available therapy, but this treatment is not devoid of secondary effects and long-term complications. In this study, we targeted FAH for the first-time to discover new chemical modulators that act as pharmacological chaperones, directly associating with this enzyme. After screening several thousand compounds and subsequent chemical redesign, we found a set of reversible inhibitors that associate with FAH close to the active site and stabilize the (active) dimeric species, as demonstrated by NMR spectroscopy. Importantly, the inhibitors are also able to partially restore the normal phenotype in a newly developed cellular model of HT1.

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder of the heme biosynthetic pathway that is characterized by uroporphyrinogen III synthase (UROS) deficiency and the accumulation of non-physiological isomer I porphyrins. These phototoxic metabolites predominantly produced by the erythron result in ineffective erythropoiesis, chronic hemolysis and splenomegaly, but they also disseminate in tissues causing bullous photosensitivity to UV light and skin fragility that may progress to scarring with photo mutilation. Therapeutic management is currently limited to supportive care and bone marrow transplantation is reserved for the most severe cases. We describe here a 26-year-old women previously diagnosed with CEP harbouring two novel UROS gene mutations whose pathogenic mechanism was investigated by extensive molecular analysis. Clinical features included disabling hypertrichosis and skin photosensitivity without hemolysis. The first and rate-limiting 5-aminolevulinate synthase 2 (ALAS2) enzyme controls heme synthesis and porphyrin production in erythroid cells, while iron availability modulates its expression through a post-transcriptional mechanism. We performed iterative phlebotomies over 26 months to induce iron depletion in the patient and investigated the effectiveness and tolerance of this cost-effective approach. We observed a progressive decrease in plasma ferritin and urinary porphyrins upon treatment without inducing anemia. The patient reported improved quality of life and photosensitivity. Our data confirm recent reports highlighting the benefit of iron restriction on the disease phenotype through a reduction in porphyrin accumulation. This new strategy may represent an efficient and well-tolerated treatment for CEP patients with skin involvement and limited hematological component if iron restriction is carefully monitored., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The interaction of human galectin-1 with a variety of oligosaccharides, from di-(N-acetyllactosamine) to tetra-saccharides (blood B type-II antigen) has been scrutinized by using a combined approach of different NMR experiments, molecular dynamics (MD) simulations, and isothermal titration calorimetry. Ligand- and receptor-based NMR experiments assisted by computational methods allowed proposing three-dimensional structures for the different complexes, which explained the lack of enthalpy gain when increasing the chemical complexity of the glycan. Interestingly, and independently of the glycan ligand, the entropy term does not oppose the binding event, a rather unusual feature for protein-sugar interactions. CLEANEX-PM and relaxation dispersion experiments revealed that sugar binding affected residues far from the binding site and described significant changes in the dynamics of the protein. In particular, motions in the microsecond-millisecond timescale in residues at the protein dimer interface were identified in the presence of high affinity ligands. The dynamic process was further explored by extensive MD simulations, which provided additional support for the existence of allostery in glycan recognition by human galectin-1., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

COVID-19 is a systemic infection that exerts significant impact on the metabolism. Yet, there is little information on how SARS-CoV-2 affects metabolism. Using NMR spectroscopy, we measured the metabolomic and lipidomic serum profile from 263 (training cohort) + 135 (validation cohort) symptomatic patients hospitalized after positive PCR testing for SARS-CoV-2 infection. We also established the profiles of 280 persons collected before the coronavirus pandemic started. Principal-component analysis discriminated both cohorts, highlighting the impact that the infection has on overall metabolism. The lipidomic analysis unraveled a pathogenic redistribution of the lipoprotein particle size and composition to increase the atherosclerotic risk. In turn, metabolomic analysis reveals abnormally high levels of ketone bodies (acetoacetic acid, 3-hydroxybutyric acid, and acetone) and 2-hydroxybutyric acid, a readout of hepatic glutathione synthesis and marker of oxidative stress. Our results are consistent with a model in which SARS-CoV-2 infection induces liver damage associated with dyslipidemia and oxidative stress., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo -α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope ( E 3 ) conformation. Kinetic isotope effects ( k cat / K M ) for anomeric- 2 H and anomeric- 13 C support an oxocarbenium ion-like transition state, and that for C2- 18 O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

More than 100 mutations in the gene encoding fumarylacetoacetate hydrolase (FAH) cause hereditary tyrosinemia type I (HT1), a metabolic disorder characterized by elevated blood levels of tyrosine. Some of these mutations are known to decrease FAH catalytic activity, but the mechanisms of FAH mutation-induced pathogenicity remain poorly understood. Here, using diffusion ordered NMR spectroscopy, cryo-EM, and CD analyses, along with site-directed mutagenesis, enzymatic assays, and molecular dynamics simulations, we investigated the putative role of thermodynamic and kinetic stability in WT FAH and a representative set of 19 missense mutations identified in individuals with HT1. We found that at physiological temperatures and concentrations, WT FAH is in equilibrium between a catalytically active dimer and a monomeric species, with the latter being inactive and prone to oligomerization and aggregation. We also found that the majority of the deleterious mutations reduce the kinetic stability of the enzyme and always accelerate the FAH aggregation pathway. Depending mainly on the position of the amino acid in the structure, pathogenic mutations either reduced the dimer population or decreased the energy barrier that separates the monomer from the aggregate. The mechanistic insights reported here pave the way for the development of pharmacological chaperones that target FAH to tackle the severe disease HT1., (© 2019 Macias et al.)

Polyglutamine (polyQ) tracts are regions of low sequence complexity frequently found in transcription factors. Tract length often correlates with transcriptional activity and expansion beyond specific thresholds in certain human proteins is the cause of polyQ disorders. To study the structural basis of the association between tract length, transcriptional activity and disease, we addressed how the conformation of the polyQ tract of the androgen receptor, associated with spinobulbar muscular atrophy (SBMA), depends on its length. Here we report that this sequence folds into a helical structure stabilized by unconventional hydrogen bonds between glutamine side chains and main chain carbonyl groups, and that its helicity directly correlates with tract length. These unusual hydrogen bonds are bifurcate with the conventional hydrogen bonds stabilizing α-helices. Our findings suggest a plausible rationale for the association between polyQ tract length and androgen receptor transcriptional activity and have implications for establishing the mechanistic basis of SBMA.