Dindo Mirco, Ambrosini Giulia, Oppici Elisa, Pey Angel L, O'Toole Peter J, Marrison Joanne L, Morrison Ian E G, Butturini Elena, Grottelli Silvia, Costantini Claudio, Cellini Barbara
Department of Medicine and Surgery, University of Perugia, 06132 Perugia, Italy.
Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, 37134 Verona, Italy.
J Pers Med. 2021 Apr 6;11(4):273. doi: 10.3390/jpm11040273.
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.
过氧化物酶体基质蛋白以完全折叠的状态转运到过氧化物酶体中，但多聚体蛋白是以单体还是寡聚体形式导入仍存在争议。在这里，我们使用丙氨酸：乙醛酸转氨酶（AGT），一种依赖磷酸吡哆醛（PLP）的同二聚体酶，其缺陷会导致I型原发性高草酸尿症（PH1），作为模型蛋白，并比较了天然二聚体和人工单体形式的细胞内行为和过氧化物酶体导入情况。单体化强烈降低AGT的细胞内稳定性，并增加其聚集/降解倾向。此外，单体部分保留在细胞质中。为了评估导入动力学的可能差异，我们对AGT进行了改造，使其能够结合一种膜通透性染料，并跟踪其细胞内运输过程，同时不干扰其生化特性。通过光漂白后的荧光恢复，我们测量了活细胞中的导入速率。二聚体和单体AGT显示出相似的导入速率，这表明寡聚状态本身并不影响导入动力学。然而，当二聚化受损时，单体容易发生错误折叠事件，从而阻止过氧化物酶体导入，这一发现对于预测导致PH1的二聚体不稳定突变的后果至关重要。用吡哆醇处理表达单体AGT的细胞可促进二聚化和折叠，从而证明了PLP的伴侣作用。我们的数据支持这样一种模型，即二聚化代表细胞质中错误折叠和正确靶向之间十字路口的一个潜在关键检查点，这可能是其他寡聚过氧化物酶体蛋白的一种普遍机制。
