Biology and pathobiology of lysosomal/vacuolar system

Lyososme/vacuole is an organelle responsible for the degradation of both endocytosed extracellular proteins and endogenous cell proteins. Degradation of endocytosed proteins via lysosome is named heterophagy. Heterophagy contributes to nutrient intake, down regulation of plasma membrane receptors, and, in some specialized cells such as macrophage, degradation of large particles and invasive microorganisms. Degradation of endogenous cell proteins via lysosome is called autophagy. Autophagy profoundly contributes to cellular protein turnover by degrading bulky cell constituents non-selectively and collectively. Recent studies from many laboratories have revealed that autophagy significantly contributes to quality control of the cell by removing injured organelles and proteins produced by various environmental stresses such as reactive oxygen species, UV irradiation, chemical substances, etc. It has been also shown that autophagy is activated in apoptosis and that it plays pivotal roles in type II cell death. Thus, proper functioning of lysosomal/vacuolar system in heterophagy and autophagy is critical for cell survival.

The aims of our studies at the Department of Biochemistry are to characterize molecular machineries essential for autophagy and heterophagy, and understand regulatory mechanisms of lysosomal protein degradation.

1) Mechanism of mammalian autophagy
(Drs. Masaaki Komatsu, Isei Tanida, Takashi Ueno)

In autophagy, various cytosolic proteins and cell organelles such as mitochondria are sequestered into double-membraned autophagosomes. Autophagosomes then fuse with lysosomes to mature into autolysosomes, in which sequestered cytoplasmic components are degraded by lysosomal proteinases. There are 15 APG genes that are essential for the formation of autophagosomes. One unique and novel aspect of APG genes is that 6 APG gene products are directly involved in two independent but mutually crosstalking protein-conjugation pathways. In one conjugation pathway, Apg12p activated by Apg7p, an E1-like protein-activating enzyme, is transferred to Apg10p, an E2-like enzyme, and subsequently conjugated with Apg5p. In the other conjugation pathway, Apg8p activated by Apg7p, is transferred to Apg3p, another E2-like enzyme, and conjugated with a phospholipid. We are currently focusing our studies on the characterization of mammalian homologues of Apg3p and Apg10p. We are also investigating lipidation mechanism of three mammalian Apg8p homologues, GABARAP, MAP-LC3, and GATE-16.

2) Mechanisms of neuronal ceroid-lipofuscinoses
(Drs. Junji Ezaki, Isei Tanida):

Neuronal ceroid lipofuscinoses are severe neuronal inherited disorders of children. Etiologically, the diseases are classified into 8 subtypes (CLN1~CLN8). Mutations in responsible genes have been identified in CLN1, which encodes a protein thioesterase 1; CLN2, which encodes tripeptidylpeptidase 1 (TPP1); CLN3, CLN5, CLN6, and CLN8, which encodes transmembrane proteins whose functions have yet to be clarified. Protein thioesterase 1, TPP1, and CLN3-gene product are localized on lysosomes and specific accumulation of subunit c of mitochondrial F1F0-ATPase are found in CLN2, CLN3, CLN4, CLN5, and CLN6. Being interested in pathological connection between CLN2, CLN3, and CLN6, we are currently studying two subjects: biosynthesis and sorting process of TPP1 and Cln3p, and isolation and identification of lysosomal proteins interacting with TPP1 and Cln3p.

In addition to these standing programs, which are being performed by the department staff, an inter-institutional collaborative research program is also in progress. In cooperation with the Department of Anatomy and Cell Biology, Osaka University (conducted by Dr. Uchiyama), pathological features of cathepsin D-deficient mice have been investigated, in particular, in relation to the dysfunction of autophagy. We also collaborate with Drs. Keiji Tanaka and Tomoki Chiba at the Department of Molecular Oncology, the Tokyo Metropolitan Institute of Medical Science, to study autophagy dysfunction in Apg7p-deficient mice.