Autophagy Regulates Cytosol Protein Homeostasis And Lipid Metabolism

Autophagy eliminates "trash" in the cells to provide the nutrition and energy for cellular renewal and homeostasis.The malfunction of aggrephagy and lipophagy is related to human diseases,but the molecular mechanisms underlining these processes are still not clear.In this thesis,we focused on these two kinds of selective autophagy.The results were presented in two parts.In the first study,we found that the reduction of Tom 40(translocase of outer membrane 40),a subunit of the translocase of the outer mitochondrial membrane complex,reduced the activity of proteasome and disrupted the autophagy pathway.In Tom40 mutant cells,Ub(ubiquitin)-positive protein aggregates were accumulated.Autophagy was induced but further blocked at the fusion stage,which led to abnormal accumulation of large protein aggregates engulfed by Atg8a-positive membranes.In the nerve system,reducing the expression of Tom40 resulted protein accumulation in neurons and neurodegeneration.Tom40 knockdown also increased the accumulation of Huntingtin polyglutamine(Htt polyQ)protein and enhanced its neuronal toxicity.We proposed that defects in mitochondrial protein import may link imbalanced proteostasis and mitochondrial defects in neurodegenerative disease.In the second study,we identified two mutants that accumulate lipid droplets in adult fly eyes.We mapped the molecullar lesions of the mutants to a novel essential fly gene CG5745,encoding a Tre2-Bub2-Cdc16(TBC)domain containing protein We named CG5745 as dTBC22 after its mammalian ortho log TBC1D22A and TBC1D22B.TBC domain containing proteins usually function as GTPase-activating proteins(GAPs)to facilitate Rab proteins hydrolyzing GTP.Overe:xpression of a dominant-negative form of dTBC22(dTBC22RQ)led to accumulation of lipid droplets in multiple tissues,suggesting that the GAP activity of dTBC22 is critical for its function in lipid metabolism.We found dTBC22 interacted with the Rab40 and functioned as a Rab40 GAP.The ove re xpression of active form of Rab40 results in lipid droplets accumulation.Rab40 knockout animals were heavier than controls and survived longer than wild-type animals upon starvation.The loss of Rab40 reduced the lipid consumption in fat body tissues upon starvation.We found Rab40 bind to lipase Brummer(Bmm).In the fed wild type animals,the protein level of Bmm was low.The loss of Rab40 increased the level of Bmm.However,the level of Bmm was comparable between the wild type controls and Rab40 mutants upon starvation.It suggested that Rab40 reduced Bmm level and starvation released this reduction effects.We found that Rab40 is enriched on LDs in fed animals and it translocated to Golgi apparatus upon starvation.Further study found that the loss of Rab40 abolished starvation induced accumulation of autophagy marker Atg8a and lysosome membrane protein LAMP around lipid droplets,suggesting Rab40 is required for starvation induced lipophagy.The reduction of LAMP and Atg8a also reduced starvation induced LD consumption.We propose following model:In the fed animals,Rab40 localizes on LDs to reduce Bmm level.Upon starvation,Rab40 re-localizes to Golgi and releases its inhibition on Bmm to facilitate lipolysis and lipophagy.dTBC22 functions as Rab40's GAP to catalyze Rab40's GTP-GDP binding cycle,which is required for Rab40 functions.The loss of dTBC22 or overexpression of Rab40 disrupts the balance of Rab40 cycle and dominant-negatively inhibits Rab40 activity.