128 research outputs found
Molecular analysis of the dimerization and aggregation processes of human alanine:glyoxylate aminotransferase and effect of mutations leading to Primary Hyperoxaluria Type I
No full textPrimary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive disorder characterized by the deposition of insoluble calcium oxalate crystals at first in the kidneys and urinary tract and then in the whole body. PH1 is caused by the deficiency of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT). AGT is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, which converts glyoxylate to glycine, thus preventing glyoxylate oxidation to oxalate and calcium oxalate formation. Only two curative therapeutic approaches are currently available for PH1: the administration of pyridoxine (PN), a precursor of PLP, which is only effective in a minority of patients (25- 35%), and liver transplantation, a very invasive procedure. AGT is encoded by the AGXT gene, which is present in humans as two polymorphic forms: the major allele (encoding AGT-Ma) and the minor allele (encoding AGT-Mi). PH1 is a very heterogeneous disease with respect to the clinical manifestations, the response to treatment and the pathogenic mechanisms. In fact, more than 200 pathogenic mutations have been identified so far and the molecular mechanisms by which missense mutations cause AGT deficiency span from functional, to structural and to subcellular localization defects or to a combination of them. Several lines of evidence at both molecular and cellular level, indicate that many disease-causing missense mutations interfere with AGT dimer stability and/or aggregation propensity. However, neither the dimerization nor the aggregation process of AGT have been analyzed in detail. Therefore, we engineered a mutant form of AGT stable in solution in the monomeric form and studied its biochemical properties and dimerization kinetics. We found that monomeric AGT is able to bind PLP and that the coenzyme stabilizes the dimeric structure. Moreover, the identification of key dimerization hot-spots at the monomer-monomer interface allowed us to unravel the mechanisms at the basis of the aberrant mitochondrial mistargeting of two of the most common PH1-causing variants. We also elucidated the molecular and cellular consequences of the pathogenic mutations R36H, G42E, I56N, G63R and G216R, involving residues located at the dimer interface, and tested their in-vitro responsiveness to the treatment with PN. The latter results allowed us to suggest a possible correlation between the structural defect of a variant and its degree of responsiveness to PN. Finally, by combining bioinformatic and biochemical approaches, we analyzed in detail the tendency of AGT to undergo an electrostatically-driven aggregation. We found that the polymorphic changes typical of the minor allele have opposite effect on the aggregation propensity of the protein, and we predicted the possible effect/s of pathogenic mutations of residues located on the AGT surface. Overall, the results obtained allow not only to better understand PH1 pathogenesis, but also to predict the response of the patients to the available therapies as well as to pave the way for the development of new therapeutic strategies
Opposite effect of polymorphic mutations on the electrostatic aggregation of human alanine:glyoxylate aminotransferase: implications for the pathogenesis of Primary Hyperoxaluria Type I
No full textProtein aggregates formation is the basis of several misfolding diseases, including those displaying loss-of-function pathogenesis. Although aggregation is often attributed to the population of intermediates exposing hydrophobic surfaces, the contribution of electrostatic forces has recently gained attention. Here we combined computational and in vitro studies to investigate the aggregation process of human peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme involved in glyoxylate detoxification. We demonstrated that AGT is susceptible to electrostatic aggregation due to its peculiar surface charge anisotropy, and that PLP binding counteracts the self-association process. The two polymorphic mutations P11L and I340M exert opposite effects. The P11L substitution enhances the aggregation tendency, by probably increasing surface charge anisotropy, while the I340M plays a stabilizing role. In light of these results, we examined the effects of the most common missense mutations leading to Primary Hyperoxaluria Type I (PH1), a rare genetic disorder associated with abnormal calcium oxalate precipitation in the urinary tract. All of them endow AGT with a strong electrostatic aggregation propensity. Moreover, we predicted that pathogenic mutations of surface residues could alter charge distribution, thus inducing aggregation under physiological conditions. A global model describing the AGT aggregation process is provided. Overall, the results indicate that the contribution of electrostatic interactions in determining the fate of proteins as well as the effect of amino acid substitutions should not be underestimated, and provide the basis for the development of new therapeutic strategies for PH1 aimed at increasing AGT stability. This article is protected by copyright. All rights reserved
Correlation between the molecular effects of mutations at the dimer interface of alanine-glyoxylate aminotransferase leading to primary hyperoxaluria type I and the cellular response to vitamin B6
No full textPrimary hyperoxaluria type I (PH1) is a rare disease caused by the deficit of liver alanine-glyoxylate aminotransferase (AGT). AGT prevents oxalate formation by converting peroxisomal glyoxylate to glycine. When the enzyme is deficient, progressive calcium oxalate stones deposit first in the urinary tract and then at the systemic level. Pyridoxal 5'-phosphate (PLP), the AGT coenzyme, exerts a chaperone role by promoting dimerization, as demonstrated by studies at protein and cellular level. Thus, variants showing a destabilized dimeric structure should, in principle, be responsive to vitamin B6, a precursor of PLP. However, models to predict the extent of responsiveness of each variant are missing. We examined the effects of pathogenic interfacial mutations by combining bioinformatic predictions with molecular and cellular studies on selected variants (R36H, G42E, I56N, G63R, and G216R), in both their holo- (i.e., with bound PLP) and apo- (i.e., without bound PLP) form. We found that all variants displayed structural alterations mainly related to the apoform and consisting of an altered tertiary and quaternary structure. G216R also shows a strongly reduced catalytic efficiency. Moreover, all but G216R respond to vitamin B6, as shown by their increased specific activity and expression level in a cellular disease model. A global analysis of data unraveled a possible inverse correlation between the degree of destabilization/misfolding induced by a mutation and the extent of B6 responsiveness. These results provide a first explanation of factors influencing B6 response in PH1, a model possibly valuable for other rare diseases caused by protein deficits
Interaction of Human Dopa Decarboxylase with L-Dopa: Spectroscopic and Kinetic Studies as a Function of pH
No full textHuman Dopa decarboxylase (hDDC), a pyridoxal 5
-phosphate (PLP) enzyme, displays maxima at 420 and 335 nm and emits
fluorescence at 384 and 504 nm upon excitation at 335nm and at 504nm when excited at 420 nm. Absorbance and fluorescence
titrations of hDDC-bound coenzyme identify a single pKspec of ∼7.2. This pKspec could not represent the ionization of a functional
group on the Schiff base but that of an enzymic residue governing the equilibrium between the low- and the high-pH forms of
the internal aldimine. During the reaction of hDDC with L-Dopa, monitored by stopped-flow spectrophotometry, a 420nm band
attributed to the 4
-N-protonated external aldimine first appears, and its decrease parallels the emergence of a 390 nmpeak, assigned
to the 4
-N-unprotonated external aldimine. The pH profile of the spectral change at 390nm displays a pK of 6.4, a value similar
to that (∼6.3) observed in both cat and cat/K profiles. This suggests that this pK represents the ESH+
→ ES catalytic step. The
assignment of the pKs of 7.9 and 8.3 observed on the basic side of cat and the PLP binding affinity profiles, respectively, is also
analyzed and discussed
Natural and unnatural compounds rescue folding defects of human alanine:glyoxylate aminotransferase leading to Primary Hyperoxaluria Type I
No full textThe functional deficit of alanine:glyoxylate aminotransferase (AGT) in human hepatocytes leads to a rare recessive disorder named primary hyperoxaluria type I (PH1). PH1 is characterized by the progressive accumulation and deposition of calcium oxalate stones in the kidneys and urinary tract, leading to a life-threatening and potentially fatal condition. In the last decades, substantial progresses in the clarification of the molecular pathogenesis of the disease have been made. They resulted in the understanding that many mutations cause AGT deficiency by affecting the folding pathway of the protein leading to a reduced expression level, an increased aggregation propensity, and/or an aberrant mitochondrial localization. Thus, PH1 can be considered a misfolding disease and possibly treated by approaches aimed at counteracting the conformational defects of the variants. In this review, we summarize recent advances in the development of new strategies to identify molecules able to rescue AGT folding and trafficking either by acting as pharmacological chaperones or by preventing the mistargeting of the protein
Editorial: The role of cofactors in protein stability and homeostasis: Focus on human metabolism
No full textBiochemical properties and oxalate-degrading activity of oxalate decarboxylase from Bacillus Subtilis at neutral pH
No full textOxalate decarboxylase (OxDC) from Bacillus subtilis is a Mn-dependent hexameric enzyme that converts oxalate to carbon dioxide and formate. OxDC has greatly attracted the interest of the scientific community, mainly due to its biotechnological and medical applications in particular for the treatment of hyperoxaluria, a group of pathologic conditions caused by oxalate accumulation. The enzyme has an acidic optimum pH, but most of its applications involve processes occurring at neutral pH. Nevertheless, a detailed biochemical characterization of the enzyme at neutral pH is lacking. Here, we compared the structural-functional properties at acidic and neutral pH of wild-type OxDC and of a mutant form, called OxDC-DSSN, bearing four amino acid substitutions in the lid (Ser161-to-Asp, Glu162-to-Ser, Asn163-toSer, and Ser164-to-Asn) that improve the oxalate oxidase activity and almost abolish the decarboxylase activity. We found that both enzymatic forms do not undergo major structural changes as a function of pH, although OxDC-DSSN displays an increased tendency to aggregation, which is counteracted by the presence of an active-site ligand. Notably, OxDC and OxDC-DSSN at pH 7.2 retain 7 and 15% activity, respectively, which is sufficient to degrade oxalate in a cellular model of primary hyperoxaluria type I, a rare inherited disease caused by excessive endogenous oxalate production. The significance of the data in the light of the possible use of OxDC as biological drug is discussed. © 2019 IUBMB Life, 1-11, 2019
Effects of interface mutations on the dimerization of alanine glyoxylate aminotransferase and implications in the mistargeting of the pathogenic variants F152I and I244T
No full textIn this work the dimerization process of the minor allelic form of human alanine glyoxylate aminotransferase, a pyridoxal 5'-phosphate enzyme, was investigated. Bioinformatic analyses followed by site-directed mutagenesis, size exclusion chromatography and catalytic activity experiments allowed us to identify Arg118, Phe238 and Phe240 as interfacial residues not essential for transaminase activity but important for dimer-monomer dissociation. The apo and the holo forms of the triple mutant R118A-Mi/F238S-Mi/F240S-Mi display a dimer-monomer equilibrium dissociation constant value at least ̴ 260- and 31-fold larger, respectively, than the corresponding ones of AGT-Mi. In the presence of PLP, the apomonomer of the triple mutant undergoes a biphasic process: the fast phase represents the formation of an inactive PLP-bound monomer, while the slow phase depicts the monomer-monomer association that parallels the regain of transaminase activity. The latter events occur with a rate constant of ̴ 0.02 μM(-1)min(-1). In the absence of PLP, the apomonomer is also able to dimerize but with a rate constant value ̴ 2700-fold lower. Thereafter, the possible interference with the dimerization process of AGT-Mi exerted by the mutated residues in the I244T-Mi and F152I-Mi variants associated with Primary Hyperoxaluria type 1 was investigated by molecular dynamics simulations. On the basis of the present and previous studies, a model for the dimerization process of AGT-Mi, I244T-Mi and F152I-Mi, which outlines the structural defects responsible for the complete or partial mistargeting of the pathogenic variants, was proposed and discussed
Folding defects leading to primary hyperoxaluria
No full textProtein misfolding is becoming one of the main mechanisms underlying inherited enzymatic deficits. This review is focused on primary hyperoxalurias, a group of disorders of glyoxylate detoxification associated with massive calcium oxalate deposition mainly in the kidneys. The most common and severe form, primary hyperoxaluria Type I, is due to the deficit of liver peroxisomal alanine/glyoxylate aminotransferase (AGT). Various studies performed in the last decade clearly evidence that many pathogenic missense mutations prevent the AGT correct folding, leading to various downstream effects including aggregation, increased degradation or mistargeting to mitochondria. Primary hyperoxaluria Type II and primary hyperoxaluria Type III are due to the deficit of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) and 4-hydroxy-2-oxoglutarate aldolase (HOGA1), respectively. Although the molecular features of pathogenic variants of GRHPR and HOGA1 have not been investigated in detail, the data available suggest that some of them display folding defects. Thus, primary hyperoxalurias can be ranked among protein misfolding disorders, because in most cases the enzymatic deficit is due to the inability of each enzyme to reach its native and functional conformation. It follows that molecules able to improve the folding yield of the enzymes involved in each disease form could represent new therapeutic strategies
A comprehensive picture of the mutations associated with aromatic amino acid decarboxylase deficiency: from molecular mechanisms to therapy implications
No full textDopa decarboxylase (DDC), or aromatic amino acid decarboxylase (AADC), is a pyridoxal 5'-phosphate enzyme responsible for the production of the neurotransmitters dopamine and serotonin. Deficit of this enzyme causes AADC deficiency, an inherited neurometabolic disorder. To date, 18 missense homozygous mutations have been identified through genetic screening in ∼80 patients. However, little is known about the mechanism(s) by which mutations cause disease. Here we investigated the impact of these pathogenic mutations and of an artificial one on the conformation and the activity of wild-type DDC by a combined approach of bioinformatic, spectroscopic and kinetic analyses. All mutations reduce the kcat value, and, except the mutation R347Q, alter the tertiary structure, as revealed by an increased hydrophobic surface and a decreased near-UV circular dichroism signal. The integrated analysis of the structural and functional consequences of each mutation strongly suggests that the reason underlying the pathogenicity of the majority of disease-causing mutations is the incorrect apo-holo conversion. In fact, the most remarkable effects are seen upon mutation of residues His70, His72, Tyr79, Phe80, Pro81, Arg462 and Arg447 mapping to or directly interacting with loop1, a structural key element involved in the apo-holo switch. Instead, different mechanisms are responsible for the pathogenicity of R347Q, a mere catalytic mutation, and of L38P and A110Q mutations causing structural-functional defects. These are due to local perturbation transmitted to the active site, as predicted by molecular dynamic analyses. Overall, the results not only give comprehensive molecular insights into AADC deficiency, but also provide an experimental framework to suggest appropriate therapeutic treatments
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