Cryptic genetic variations of alanine:glyoxylate aminotransferase shape its fitness and dynamics

Genetic variations expand the conformational landscape of proteins and may underlie cryptic functions able to influence protein adaptability under unfavorable conditions. Cryptic functions usually associate with regions of increased frustration or even intrinsically disordered, whose role as drivers of innovation is increasingly being recognized. Hence, the balance between protein stability and controlled disorder translates in the dichotomy between conservation and innovability, and should be regarded as the more comprehensive measure of protein fitness. In this context, understanding how genetic variations affect protein fitness is not trivial, since cryptic functions behind frustrated regions are not easily detectable. Herein, we used as model the pyridoxal 5-phosphate (PLP)-dependent enzyme alanine:glyoxylate aminotransferase (AGT), which is present as a common major allelic form (AGT-Ma) and a minor polymorphic form (AGT-Mi) expressed in 20% of Caucasian population and considered a lower limit of AGT fitness. By solving the structure of AGT-Mi, we could show that three distinct regions, that are structured in AGT-Ma, are disordered in AGT-Mi. Molecular dynamics shows that AGT-Mi samples more flexible conformations than AGT-Ma, supporting a plasticity effect propagated to all the structure. In-depth biochemical characterisation of mutants from a small library of variants encompassing the three regions reproduces the fitness window between AGT-Ma and AGT-Mi. Cellular studies highlight the consequences of this plasticity at functional level and suggest the existence of cryptic functions likely related to protein-protein interactions. These results establish that naturally-occurring genetic variations tip the balance between protein stability and frustration to encode cryptic functions that expand the potential innovability of the protein.

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.

Cycloserine enantiomers are reversible inhibitors of human alanine:glyoxylate aminotransferase: implications for Primary Hyperoxaluria type 1

Peroxisomal alanine:glyoxylate aminotransferase (AGT) is responsible for glyoxylate detoxification in human liver and utilizes pyridoxal 5′-phosphate (PLP) as coenzyme. The deficit of AGT leads to Primary Hyperoxaluria Type I (PH1), a rare disease characterized by calcium oxalate stones deposition in the urinary tract as a consequence of glyoxylate accumulation. Most missense mutations cause AGT misfolding, as in the case of the G41R, which induces aggregation and proteolytic degradation. We have investigated the interaction of wild-type AGT and the pathogenic G41R variant with d-cycloserine (DCS, commercialized as Seromycin), a natural product used as a second-line treatment of multidrug-resistant tuberculosis, and its synthetic enantiomer l-cycloserine (LCS). In contrast with evidences previously reported on other PLP-enzymes, both ligands are AGT reversible inhibitors showing inhibition constants in the micromolar range. While LCS undergoes half-transamination generating a ketimine intermediate and behaves as a classical competitive inhibitor, DCS displays a time-dependent binding mainly generating an oxime intermediate. Using a mammalian cellular model, we found that DCS, but not LCS, is able to promote the correct folding of the G41R variant, as revealed by its increased specific activity and expression as a soluble protein. This effect also translates into an increased glyoxylate detoxification ability of cells expressing the variant upon treatment with DCS. Overall, our findings establish that DCS could play a role as pharmacological chaperone, thus suggesting a new line of intervention against PH1 based on a drug repositioning approach. To a widest extent, this strategy could be applied to other disease-causing mutations leading to AGT misfolding.