52 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
Primary 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
Enzymes and Liquid-Liquid Phase Separation: A New Era for the Regulation of Enzymatic Activity
Liquid-liquid phase separation (LLPS) is recognized as a mechanism for regulation of enzymatic activity. Biochemical mechanisms include concentrating reactants to enhance reaction rates or sequester enzymes and reactants from each other to reduce the reaction rate. On the other hand, LLPS might also regulate the diffusion of small molecules or important parameters for enzymatic activity (such as modulators, macromolecular crowding and changing the media physicochemical features) increasing or decreasing the reaction rate of the enzymes. Furthermore, the co-compartmentalization of specific enzymes can favour or speed up specific metabolic fluxes. Here, we discuss how LLPS contributed to generate a new era for enzyme regulation and the new possible subtle regulation mechanisms still unexplored.journal articl
Hybrid Metamaterial Optical Tweezers for Dielectric Particles and Biomolecules Discrimination
Although plasmonic nanotweezers can achieve manipulation on a scale smaller
than the diffraction limit, direct trapping of sub-10~nm bioparticles in an
aqueous environment without modifications remains challenging. Here, we
demonstrate enzyme delivery-trapping and dynamic manipulation at the
single-entity level by employing photothermal-assisted trapping via
metamaterial optical tweezers at low trapping laser intensities. By analyzing
the probability density function, we are able to distinguish dielectric
particles from biomolecules. For enzyme-related data, the presence of two peaks
in violin plots indicates conformational changes or interactions with the
environment. We also observe that increasing the laser intensity increases the
local temperature, resulting in enzyme aggregation. This study provides an
alternative approach for identifying single enzymes and dielectric particles by
combining plasmonic fields and photothermal heat, while paving the way for
label-free characterization of biomolecules at the single-particle level
Pyridoxal 5âČ-Phosphate-Dependent Enzymes at the Crossroads of HostâMicrobe Tryptophan Metabolism
The chemical processes taking place in humans intersects the myriad of metabolic pathways occurring in commensal microorganisms that colonize the body to generate a complex biochemical network that regulates multiple aspects of human life. The role of tryptophan (Trp) metabolism at the intersection between the host and microbes is increasingly being recognized, and multiple pathways of Trp utilization in either direction have been identified with the production of a wide range of bioactive products. It comes that a dysregulation of Trp metabolism in either the host or the microbes may unbalance the production of metabolites with potential pathological consequences. The ability to redirect the Trp flux to restore a homeostatic production of Trp metabolites may represent a valid therapeutic strategy for a variety of pathological conditions, but identifying metabolic checkpoints that could be exploited to manipulate the Trp metabolic network is still an unmet need. In this review, we put forward the hypothesis that pyridoxal 5\u27-phosphate (PLP)-dependent enzymes, which regulate multiple pathways of Trp metabolism in both the host and in microbes, might represent critical nodes and that modulating the levels of vitamin B6, from which PLP is derived, might represent a metabolic checkpoint to re-orienteer Trp flux for therapeutic purposes
Biochemical Characterization of Aspergillus fumigatus AroH, a Putative Aromatic Amino Acid Aminotransferase
The rise in the frequency of nosocomial infections is becoming a major problem for public health, in particular in immunocompromised patients. Aspergillus fumigatus is an opportunistic fungus normally present in the environment directly responsible for lethal invasive infections. Recent results suggest that the metabolic pathways related to amino acid metabolism can regulate the fungus-host interaction and that an important role is played by enzymes involved in the catabolism of L-tryptophan. In particular, in A. fumigatus L-tryptophan regulates Aro genes. Among them, AroH encodes a putative pyridoxal 5'-phosphate-dependent aminotransferase. Here we analyzed the biochemical features of recombinant purified AroH by spectroscopic and kinetic analyses corroborated by in silico studies. We found that the protein is dimeric and tightly binds the coenzyme forming a deprotonated internal aldimine in equilibrium with a protonated ketoenamine form. By setting up a new rapid assay method, we measured the kinetic parameters for the overall transamination of substrates and we demonstrated that AroH behaves as an aromatic amino acid aminotransferase, but also accepts L-kynurenine and α-aminoadipate as amino donors. Interestingly, computational approaches showed that the predicted overall fold and active site topology of the protein are similar to those of its yeast ortholog, albeit with some differences in the regions at the entrance of the active site, which could possibly influence substrate specificity. Should targeting fungal metabolic adaptation be of therapeutic value, the results of the present study may pave the way to the design of specific AroH modulators as potential novel agents at the host/fungus interface
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
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 50
-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 membranepermeable 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.Italian Ministry of University and Research (SIR project
RBSI148BK3 to B.C.)Oxalosis and Hyperoxaluria Foundation (OHF2016, to B.C.). A.L.PERDF/Spanish Ministry of Science, Innovation and Universitiesâ
State Research Agency (Grant RTI2018-096246-B-I00)ConsejerĂa de EconomĂa, Conocimiento,
Empresas y Universidad, Junta de AndalucĂa (Grant P18-RT-2413)
Opposite effect of polymorphic mutations on the electrostatic aggregation of human alanine:glyoxylate aminotransferase: implications for the pathogenesis of Primary Hyperoxaluria Type I
Protein 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
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 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
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