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.

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.

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.

Mutant p53 blocks SESN1/AMPK/PGC-1α/UCP2 axis increasing mitochondrial O· 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.

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.

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.

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.

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 B.

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.

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).

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

The chaperone role of the pyridoxal 5'-phosphate and its implications for rare diseases involving B6-dependent enzymes.

The biologically active form of the B6 vitamers is pyridoxal 5'-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process.

Gly161 mutations associated with Primary Hyperoxaluria Type I induce the cytosolic aggregation and the intracellular degradation of the apo-form of alanine:glyoxylate aminotransferase.

Primary Hyperoxaluria Type I (PH1) is a severe rare disorder of metabolism due to inherited mutations on liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme whose deficiency causes the deposition of calcium oxalate crystals in the kidneys and urinary tract. PH1 is an extremely heterogeneous disease and there are more than 150 disease-causing mutations currently known, most of which are missense mutations. Moreover, the molecular mechanisms by which missense mutations lead to AGT deficiency span from structural, functional to subcellular localization defects.

Interaction of human Dopa decarboxylase with L-Dopa: spectroscopic and kinetic studies as a function of pH.

Human 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 335 nm and at 504 nm when excited at 420 nm. Absorbance and fluorescence titrations of hDDC-bound coenzyme identify a single pK(spec) of ~7.2.

Crystal structure of the S187F variant of human liver alanine: glyoxylate [corrected] aminotransferase associated with primary hyperoxaluria type I and its functional implications.

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5'-phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.

S250F variant associated with aromatic amino acid decarboxylase deficiency: molecular defects and intracellular rescue by pyridoxine.

Dopa or aromatic amino acid decarboxylase (DDC, AADC) is a pyridoxal 5'-phosphate-dependent enzyme that catalyses the production of the neurotransmitters dopamine and serotonin. Among the so far identified mutations associated with AADC deficiency, an inherited rare neurometabolic disease, the S250F mutation is the most frequent one. Here, for the first time, the molecular basis of the deficit of the S250F variant was investigated both in vitro and in cellular systems.