Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting.

Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5'-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins.

Biochemical properties and oxalate-degrading activity of oxalate decarboxylase from bacillus subtilis at neutral pH.

Oxalate 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.

Insight into the specificity and severity of pathogenic mechanisms associated with missense mutations through experimental and structural perturbation analyses.

Most pathogenic missense mutations cause specific molecular phenotypes through protein destabilization. However, how protein destabilization is manifested as a given molecular phenotype is not well understood. We develop here a structural and energetic approach to describe mutational effects on specific traits such as function, regulation, stability, subcellular targeting or aggregation propensity. This approach is tested using large-scale experimental and structural perturbation analyses in over thirty mutations in three different proteins (cancer-associated NQO1, transthyretin related with amyloidosis and AGT linked to primary hyperoxaluria type I) and comprising five very common pathogenic mechanisms (loss-of-function and gain-of-toxic function aggregation, enzyme inactivation, protein mistargeting and accelerated degradation). Our results revealed that the magnitude of destabilizing effects and, particularly, their propagation through the structure to promote disease-associated conformational states largely determine the severity and molecular mechanisms of disease-associated missense mutations. Modulation of the structural perturbation at a mutated site is also shown to cause switches between different molecular phenotypes. When very common disease-associated missense mutations were investigated, we also found that they were not among the most deleterious possible missense mutations at those sites, and required additional contributions from codon bias and effects of CpG sites to explain their high frequency in patients. Our work sheds light on the molecular basis of pathogenic mechanisms and genotype-phenotype relationships, with implications for discriminating between pathogenic and neutral changes within human genome variability from whole genome sequencing studies.

Molecular basis of primary hyperoxaluria: clues to innovative treatments.

Primary hyperoxalurias (PHs) are rare inherited disorders of liver glyoxylate metabolism, characterized by the abnormal production of endogenous oxalate, a metabolic end-product that is eliminated by urine. The main symptoms are related to the precipitation of calcium oxalate crystals in the urinary tract with progressive renal damage and, in the most severe form named Primary Hyperoxaluria Type I (PH1), to systemic oxalosis. The therapies currently available for PH are either poorly effective, because they address the symptoms and not the causes of the disease, or highly invasive. In the last years, advances in our understanding of the molecular bases of PH have paved the way for the development of new therapeutic strategies. They include (i) substrate-reduction therapies based on small-molecule inhibitors or the RNA interference technology, (ii) gene therapy, (iii) enzyme administration approaches, (iv) colonization with oxalate-degrading intestinal microorganisms, and, in PH1, (v) design of pharmacological chaperones. This paper reviews the basic principles of these new therapeutic strategies and what is currently known about their application to PH.

Mutant p53 blocks SESN1/AMPK/PGC-1α/UCP2 axis increasing mitochondrial O2-· production in cancer cells.

BACKGROUND: The TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 gain-of-function isoforms actively promote cancer malignancy. METHODS: A panel of wild-type and mutant p53 cancer cell lines of different tissues, including pancreas, breast, skin, and lung were used, as well as chronic lymphocytic leukemia (CLL) patients with different TP53 gene status. The effects of mutant p53 were evaluated by confocal microscopy, reactive oxygen species production assay, immunoblotting, and quantitative reverse transcription polymerase chain reaction after cellular transfection. RESULTS: We demonstrate that oncogenic mutant p53 isoforms are able to inhibit SESN1 expression and consequently the amount of SESN1/AMPK complex, resulting in the downregulation of the AMPK/PGC-1α/UCP2 axis and mitochondrial O2-· production. We also show a correlation between the decrease of reduced thiols with a poorer clinical outcome of CLL patients bearing mutant TP53 gene. The restoration of the mitochondrial uncoupling protein 2 (UCP2) expression, as well as the addition of the radical scavenger N-acetyl-L-cysteine, reversed the oncogenic effects of mutant p53 as cellular hyper-proliferation, antiapoptotic effect, and resistance to drugs. CONCLUSIONS: The inhibition of the SESN1/AMPK/PGC-1α/UCP2 axis contributes to the pro-oxidant and oncogenic effects of mutant p53, suggesting pro-oxidant drugs as a therapeutic approach for cancer patients bearing mutant TP53 gene.

A novel p.(Glu111Val) missense mutation in GUCA1A associated with cone-rod dystrophy leads to impaired calcium sensing and perturbed second messenger homeostasis in photoreceptors.

Guanylate Cyclase-Activating Protein 1 (GCAP1) regulates the enzymatic activity of the photoreceptor guanylate cyclases (GC), leading to inhibition or activation of the cyclic guanosine monophosphate (cGMP) synthesis depending on its Ca2+- or Mg2+-loaded state. By genetically screening a family of patients diagnosed with cone-rod dystrophy, we identified a novel missense mutation with autosomal dominant inheritance pattern (c.332A>T; p.(Glu111Val); E111V from now on) in the GUCA1A gene coding for GCAP1. We performed a thorough biochemical and biophysical investigation of wild type (WT) and E111V human GCAP1 by heterologous expression and purification of the recombinant proteins. The E111V substitution disrupts the coordination of the Ca2+ ion in the high-affinity site (EF-hand 3, EF3), thus significantly decreasing the ability of GCAP1 to sense Ca2+ (∼80-fold higher Kdapp compared to WT). Both WT and E111V GCAP1 form dimers independently on the presence of cations, but the E111V Mg2+-bound form is prone to severe aggregation over time. Molecular dynamics simulations suggest a significantly increased flexibility of both the EF3 and EF4 cation binding loops for the Ca2+-bound form of E111V GCAP1, in line with the decreased affinity for Ca2+. In contrast, a more rigid backbone conformation is observed in the Mg2+-bound state compared to the WT, which results in higher thermal stability. Functional assays confirm that E111V GCAP1 interacts with the target GC with a similar apparent affinity (EC50); however, the mutant shifts the GC inhibition out of the physiological [Ca2+] (IC50E111V ∼10 µM), thereby leading to the aberrant constitutive synthesis of cGMP under conditions of dark-adapted photoreceptors.

Evolutionary Divergent Suppressor Mutations in Conformational Diseases.

Neutral and adaptive mutations are key players in the evolutionary dynamics of proteins at molecular, cellular and organismal levels. Conversely, largely destabilizing mutations are rarely tolerated by evolution, although their occurrence in diverse human populations has important roles in the pathogenesis of conformational diseases. We have recently proposed that divergence at certain sites from the consensus (amino acid) state during mammalian evolution may have rendered some human proteins more vulnerable towards disease-associated mutations, primarily by decreasing their conformational stability. We herein extend and refine this hypothesis discussing results from phylogenetic and structural analyses, structure-based energy calculations and structure-function studies at molecular and cellular levels. As proof-of-principle, we focus on different mammalian orthologues of the NQO1 (NAD(P)H:quinone oxidoreductase 1) and AGT (alanine:glyoxylate aminotransferase) proteins. We discuss the different loss-of-function pathogenic mechanisms associated with diseases involving the two enzymes, including enzyme inactivation, accelerated degradation, intracellular mistargeting, and aggregation. Last, we take into account the potentially higher robustness of mammalian orthologues containing certain consensus amino acids as suppressors of human disease, and their relation with different intracellular post-translational modifications and protein quality control capacities, to be discussed as sources of phenotypic variability between human and mammalian models of disease and as tools for improving current therapeutic approaches.

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.

Primary 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.

Folding Defects Leading to Primary Hyperoxaluria.

Protein 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.

Metastatic Breast Cancer Cells Enter Into Dormant State and Express Cancer Stem Cells Phenotype Under Chronic Hypoxia.

Tumor dormancy is a poorly understood stage in cancer progression characterized by mitotic cycle arrest in G0/G1 phase and low metabolism. The cells survive in a quiescent state and wait for appropriate environmental conditions to begin proliferation again giving rise to metastasis. Despite their key role in cancer development and metastasis, the knowledge about their biology and origin is still very limited due to the poorness of established in vitro models that faithfully recapitulated tumor dormancy. Using at least three cycles of 1% O2 hypoxia and reoxygenation, we establish and characterize the hypoxia-resistant human breast cancer cell line chMDA-MB-231 that can stably survive under 1% O2 condition by entering into dormant state characterized by arrest in G0/G1 phase and low metabolism. This dormant state is reversible since once replaced in normoxia the cells recover the proliferation rate in 2 weeks. We show that chronic hypoxia induces autophagy that may be the survival mechanism of chMDA-MB-231 cells. Furthermore, the data in this work demonstrate that cycling hypoxic/reoxygenation stress selects MDA-MB-231 population that presents the cancer stem-like phenotype characterized by CD24- /CD44+ /ESA+ expression and spheroid forming capacity. We believe that our study presents a promising approach to select dormant breast cancer cells with stem-like phenotype using the hypoxia/reoxygenation regimen that may represent an area with profound implications for therapeutic developments in oncology. J. Cell. Biochem. 118: 3237-3248, 2017. © 2017 Wiley Periodicals, Inc.

Use of polymer conjugates for the intraperoxisomal delivery of engineered human alanine:glyoxylate aminotransferase as a protein therapy for primary hyperoxaluria type I.

Alanine:glyoxylate aminotransferase (AGT) is a liver peroxisomal enzyme whose deficit causes the rare disorder Primary Hyperoxaluria Type I (PH1). We now describe the conjugation of poly(ethylene glycol)-co-poly(L-glutamic acid) (PEG-PGA) block-co-polymer to AGT via the formation of disulfide bonds between the polymer and solvent-exposed cysteine residues of the enzyme. PEG-PGA conjugation did not affect AGT structural/functional properties and allowed the enzyme to be internalized in a cellular model of PH1 and to restore glyoxylate-detoxification. The insertion of the C387S/K390S amino acid substitutions, known to favor interaction with the peroxisomal import machinery, reduced conjugation efficiency, but endowed conjugates with the ability to reach the peroxisomal compartment. These results, along with the finding that conjugates are hemocompatible, stable in plasma, and non-immunogenic, hold promise for the development of polypeptide-based AGT conjugates as a therapeutic option for PH1 patients and represent the base for applications to other diseases related to deficits in peroxisomal proteins.

A Novel Pathway for Metabolism of the Cardiovascular Risk Factor Homoarginine by alanine:glyoxylate aminotransferase 2.

Low plasma concentrations of L-homoarginine are associated with an increased risk of cardiovascular events, while homoarginine supplementation is protective in animal models of metabolic syndrome and stroke. Catabolism of homoarginine is still poorly understood. Based on the recent findings from a Genome Wide Association Study we hypothesized that homoarginine can be metabolized by alanine:glyoxylate aminotransferase 2 (AGXT2). We purified human AGXT2 from tissues of AGXT2 transgenic mice and demonstrated its ability to metabolize homoarginine to 6-guanidino-2-oxocaproic acid (GOCA). After incubation of HepG2 cells overexpressing AGXT2 with isotope-labeled homoarginine-d4 we were able to detect labeled GOCA in the medium. We injected wild type mice with labeled homoarginine and detected labeled GOCA in the plasma. We found that AGXT2 knockout (KO) mice have higher homoarginine and lower GOCA plasma levels as compared to wild type mice, while the reverse was true for AGXT2 transgenic (Tg) mice. In summary, we experimentally proved the presence of a new pathway of homoarginine catabolism - its transamination by AGXT2 with formation of GOCA and demonstrated that endogenous AGXT2 is required for maintenance of homoarginine levels in mice. Our findings may lead to development of novel therapeutic approaches for cardiovascular pathologies associated with homoarginine deficiency.

Caenorhabditis elegans AGXT-1 is a mitochondrial and temperature-adapted ortholog of peroxisomal human AGT1: New insights into between-species divergence in glyoxylate metabolism.

In humans, glyoxylate is an intermediary product of metabolism, whose concentration is finely balanced. Mutations in peroxisomal alanine:glyoxylate aminotransferase (hAGT1) cause primary hyperoxaluria type 1 (PH1), which results in glyoxylate accumulation that is converted to toxic oxalate. In contrast, glyoxylate is used by the nematode Caenorhabditis elegans through a glyoxylate cycle to by-pass the decarboxylation steps of the tricarboxylic acid cycle and thus contributing to energy production and gluconeogenesis from stored lipids. To investigate the differences in glyoxylate metabolism between humans and C. elegans and to determine whether the nematode might be a suitable model for PH1, we have characterized here the predicted nematode ortholog of hAGT1 (AGXT-1) and compared its molecular properties with those of the human enzyme. Both enzymes form active PLP-dependent dimers with high specificity towards alanine and glyoxylate, and display similar three-dimensional structures. Interestingly, AGXT-1 shows 5-fold higher activity towards the alanine/glyoxylate pair than hAGT1. Thermal and chemical stability of AGXT-1 is lower than that of hAGT1, suggesting temperature-adaptation of the nematode enzyme linked to the lower optimal growth temperature of C. elegans. Remarkably, in vivo experiments demonstrate the mitochondrial localization of AGXT-1 in contrast to the peroxisomal compartmentalization of hAGT1. Our results support the view that the different glyoxylate metabolism in the nematode is associated with the divergent molecular properties and subcellular localization of the alanine:glyoxylate aminotransferase activity.

Mutant p53 proteins counteract autophagic mechanism sensitizing cancer cells to mTOR inhibition.

Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy.

Natural and Unnatural Compounds Rescue Folding Defects of Human Alanine: Glyoxylate Aminotransferase Leading to Primary Hyperoxaluria Type I.

The 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 progress 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.

Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine.

Liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) enzyme, exists as two polymorphic forms, the major (AGT-Ma) and the minor (AGT-Mi) haplotype. Deficit of AGT causes Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive rare disease. Although ~one-third of the 79 disease-causing missense mutations segregates on AGT-Mi, only few of them are well characterized. Here for the first time the molecular and cellular defects of G47R-Mi are reported. When expressed in Escherichia coli, the recombinant purified G47R-Mi variant exhibits only a 2.5-fold reduction of its kcat, and its apo form displays a remarkably decreased PLP binding affinity, increased dimer-monomer equilibrium dissociation constant value, susceptibility to thermal denaturation and to N-terminal region proteolytic cleavage, and aggregation propensity. When stably expressed in a mammalian cell line, we found ~95% of the intact form of the variant in the insoluble fraction, and proteolyzed (within the N-terminal region) and aggregated forms both in the soluble and insoluble fractions. Moreover, the intact and nicked forms have a peroxisomal and a mitochondrial localization, respectively. Unlike what already seen for G41R-Mi, exposure of G47R-Mi expressing cells to pyridoxine (PN) remarkably increases the expression level and the specific activity in a dose-dependent manner, reroutes all the protein to peroxisomes, and rescues its functionality. Although the mechanism of the different effect of PN on the variants G47R-Mi and G41R-Mi remains elusive, the chaperoning activity of PN may be of value in the therapy of patients bearing the G47R mutation.

Pyridoxamine and pyridoxal are more effective than pyridoxine in rescuing folding-defective variants of human alanine:glyoxylate aminotransferase causing primary hyperoxaluria type I.

Vitamin B6 in the form of pyridoxine (PN) is one of the most widespread pharmacological therapies for inherited diseases involving pyridoxal phosphate (PLP)-dependent enzymes, including primary hyperoxaluria type I (PH1). PH1 is caused by a deficiency of liver-peroxisomal alanine: glyoxylate aminotransferase (AGT), which allows glyoxylate oxidation to oxalate leading to the deposition of insoluble calcium oxalate in the kidney. Only a minority of PH1 patients, mostly bearing the F152I and G170R mutations, respond to PN, the only pharmacological treatment currently available. Moreover, excessive doses of PN reduce the specific activity of AGT in a PH1 cellular model. Nevertheless, the possible effect(s) of other B6 vitamers has not been investigated previously. Here, we compared the ability of PN in rescuing the effects of the F152I and G170R mutations with that of pyridoxamine (PM) and PL. We found that supplementation with PN raises the intracellular concentration of PN phosphate (PNP), which competes with PLP for apoenzyme binding leading to the formation of an inactive AGT-PNP complex. In contrast, PNP does not accumulate in the cell upon PM or PL supplementation, but higher levels of PLP and PM phosphate (PMP), the two active forms of the AGT coenzyme, are found. This leads to an increased ability of PM and PL to rescue the effects of the F152I and G170R mutations compared with PN. A similar effect was also observed for other folding-defective AGT variants. Thus, PM and PL should be investigated as matter of importance as therapeutics for PH1 patients bearing folding mutations.

The Chaperoning Activity of Amino-oxyacetic Acid on Folding-Defective Variants of Human Alanine:Glyoxylate Aminotransferase Causing Primary Hyperoxaluria Type I.

The rare disease Primary Hyperoxaluria Type I (PH1) results from the deficit of liver peroxisomal alanine:glyoxylate aminotransferase (AGT), as a consequence of inherited mutations on the AGXT gene frequently leading to protein misfolding. Pharmacological chaperone (PC) therapy is a newly developed approach for misfolding diseases based on the use of small molecule ligands able to promote the correct folding of a mutant enzyme. In this report, we describe the interaction of amino-oxyacetic acid (AOA) with the recombinant purified form of two polymorphic species of AGT, AGT-Ma and AGT-Mi, and with three pathogenic variants bearing previously identified folding defects: G41R-Ma, G170R-Mi, and I244T-Mi. We found that for all these enzyme AOA (i) forms an oxime at the active site, (ii) behaves as a slow, tight-binding inhibitor with KI values in the nanomolar range, and (iii) increases the thermal stability. Furthermore, experiments performed in mammalian cells revealed that AOA acts as a PC by partly preventing the intracellular aggregation of G41R-Ma and by promoting the correct peroxisomal import of G170R-Mi and I244T-Mi. Based on these data, we carried out a small-scale screening campaign. We identified four AOA analogues acting as AGT inhibitors, even if only one was found to act as a PC. The possible relationship between the structure and the PC activity of these compounds is discussed. Altogether, these results provide the proof-of-principle for the feasibility of a therapy with PCs for PH1-causing variants bearing folding defects and provide the scaffold for the identification of more specific ligands.

Liver peroxisomal alanine:glyoxylate aminotransferase and the effects of mutations associated with Primary Hyperoxaluria Type I: An overview.

Liver peroxisomal alanine:glyoxylate aminotransferase (AGT) (EC 2.6.1.44) catalyses the conversion of l-alanine and glyoxylate to pyruvate and glycine, a reaction that allows glyoxylate detoxification. Inherited mutations on the AGXT gene encoding AGT lead to Primary Hyperoxaluria Type I (PH1), a rare disorder characterized by the deposition of calcium oxalate crystals primarily in the urinary tract. Here we describe the results obtained on the biochemical features of AGT as well as on the molecular and cellular effects of polymorphic and pathogenic mutations. A complex scenario on the molecular pathogenesis of PH1 emerges in which the co-inheritance of polymorphic changes and the condition of homozygosis or compound heterozygosis are two important factors that determine the enzymatic phenotype of PH1 patients. All the reported data represent relevant steps toward the understanding of genotype/phenotype correlations, the prediction of the response of the patients to the available therapies, and the development of new therapeutic approaches. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

S81L and G170R mutations causing Primary Hyperoxaluria type I in homozygosis and heterozygosis: an example of positive interallelic complementation.

Primary Hyperoxaluria type I (PH1) is a rare disease due to the deficit of peroxisomal alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal-5'-phosphate (PLP) enzyme present in humans as major (Ma) and minor (Mi) allele. PH1-causing mutations are mostly missense identified in both homozygous and compound heterozygous patients. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, whereas the molecular aspects of the genotype-enzymatic-clinical phenotype relationship in compound heterozygous patients are completely unknown. Here, for the first time, we elucidate the enzymatic phenotype linked to the S81L mutation on AGT-Ma, relative to a PLP-binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functionality, is present in the second allele. By using a bicistronic eukaryotic expression vector, we demonstrate that (i) S81L-Ma is mainly in its apo-form and has a significant peroxisomal localization and (ii) S81L and G170R monomers interact giving rise to the G170R-Mi/S81L-Ma holo-form, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the heterodimer and the homodimeric counterparts in their purified recombinant form, (i) highlight the molecular basis of the pathogenicity of S81L-Ma and (ii) provide evidence for a positive interallelic complementation between the S81L and G170R monomers. Our study represents a valid approach to investigate the molecular pathogenesis of PH1 in compound heterozygous patients.