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

Abstract

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.

A molecular journey on the pathogenesis of primary hyperoxaluria

“…Human AGT localizes in the peroxisomal matrix thanks to a C-terminal targeting sequence, and utilizes pyridoxal phosphate (PLP) as coenzyme [ 10 ]. The protein forms a tight homodimer through a prevalently hydrophobic interface [ 16 ], as well as an N-terminal arm wrapping over the neighboring subunit, which strongly influences proper folding [ 17 , 18 ]. The equilibrium constant of transamination is largely shifted toward glyoxylate-to-glycine conversion, a peculiar property related to the physiological role of the enzyme [ 10 ].…”

The physiological functions of human peroxisomes

“…This exemplifies the importance of timely diagnosis so that appropriate treatment which includes pyridoxine supplementation, can be initiated. The beneficial effect of pyridoxine on AGT has not been worked out in detail except for a few pathogenic variants but is probably the result of at least three different mechanisms (177) including: (a.) direct stimulation of the catalytic activity of AGT thanks to pyridoxal-5phosphate as obligatory co-factor in the enzyme reaction; (b.)…”

Role of misfolding in rare enzymatic deficits and use of pharmacological chaperones as therapeutic approach

“…PLP is bound at the interface between the two subunits of the AGT dimer. The chaperone effect is mediated by a variety of mechanisms including (i) the promotion of dimerization [167,168]; (ii) an im-proved efficiency of the folding process, with also includes the correct targeting [163,166]; (iii) an overall stabilization of AGT native structure against electrostatically-driven aggregation [169]. Based on the chaperone role of PLP, which allows to rationalize the effects of PN administration to PH1 patients, cellular studies on other B6 vitamers have been performed.…”