Investigation of Physiological Meaning and Molecular Machinery of Autophagy in Mammalian.

在两年的研究期间，我们研究了几种与酵母自噬必需基因产物相关的哺乳动物蛋白质，作为参与哺乳动物自噬的候选分子。获得的结果如下：1)LC3：大鼠LC3与自噬必需的酵母Apg8p同源。利用抗Lc3和Lc3的抗体，我们发现大约一半的Lc3分子在合成后的2-3小时内被加工成2-3 kDa的小分子，小的Lc3特异地定位于参与自噬和自噬的细胞器中，而大的Lc3分布在细胞质上。在诱导自噬和一些药物治疗的启动条件下，小的Lc3增加了小的Lc3。这些数据表明，小的Lc3在哺乳动物的自噬中可能起到一定的作用。2)SKd1：小鼠SKd1是与自噬相关的酵母Csclp高度同源的AAA型ATPase。我们产生了一个突变体，与显性的负突变体Csclp同源。在培养的动物细胞中过表达突变的SKD1导致异常的早期内小体的形成和转铁蛋白受体从早期内小体到质膜的循环被抑制。从这些结果，我们认为SKD1调节早期内小体的形态和运输功能，然后这种内体功能可能需要进行自噬。3)mAPG5：我们发现酵母Apg5p与Apgl2p共价连接，并且自噬需要这种连接。此外，我们还证明了它们的人类同源基因hAPG5和hAPG12形成了共价结合。为了在细胞、组织和个体水平阐明自噬的生理意义，我们建立了Apg5p同源基因缺失的小鼠MAPG5.我们已经克隆了mAPG5的基因组DNA，并将在ES细胞中干扰mAPG5基因。

We have investigated several mammalian proteins related to products of yeast genes essential for autophagy as candidates of molecules involved in mammalian autophagy, during the two-years research period.Obtained results are as follows.1) LC3 : The rat LC3 is homologous to yeast Apg8p essential for autophagy. Using an antibody against LC3 and LC3 cDNA, we showed that about half of LC3 molecules were processed to 2-3 kDa-smaller form during 2-3 hours after synthesis and that the smaller form LC3 was specifically localized in the membrane organella involved in autophagy, autophagosomes, whereas the larger form LC3 was distributed over the cytoplasm.Starvation condition which induces autophagy and some drug treatment increase the smaller form LC3.These data suggest that the smaller form LC3 may play some role in mammalian autophagy.2) SKD1 : The mouse SKD1 is an AAA-type ATPase highly homologous to the yeast Csclp implicated in autophagy.We generated a mutant SKD1 analogous to the dominant negative mutant Csclp. Overexpression of the mutant SKD1 in cultured animal cells caused formation of aberrant early endosomes and inhibition of recycling of transferrin receptor from early endosomes to the plasma membrane.From the results, we suggest that SKD1 regulates morphology and transport function of early endosomes, and then that such endosomal function may be required to proceed autophagy.3) mAPG5 : We found that yeast Apg5p was conjugated covalently to Apgl2p and the conjugation was required for autophagy.Furthermore we also showed that their human homologue, hAPG5 and hAPG12, formed the covalent conjugation.To elucidate physiological meanings of autophagy at level of cell, tissue, and individual, we are generating mice deficient for the homologue of Apg5p, mAPG5.We already cloned genomic DNA of mAPG5 and will disrupt mAPG5 gene in ES cells.