Selective Autophagy Pathway Research Articles

PLEKHM1 Regulates Autophagosome-Lysosome Fusion through HOPS Complex and LC3/GABARAP Proteins

The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways.

Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition

In addition to key roles in embryonic neurogenesis and myelinogenesis, γ-aminobutyric acid (GABA) serves as the primary inhibitory mammalian neurotransmitter. In yeast, we have identified a new role for GABA that augments activity of the pivotal kinase, Tor1. GABA inhibits the selective autophagy pathways, mitophagy and pexophagy, through Sch9, the homolog of the mammalian kinase, S6K1, leading to oxidative stress, all of which can be mitigated by the Tor1 inhibitor, rapamycin. To confirm these processes in mammals, we examined the succinic semialdehyde dehydrogenase (SSADH)-deficient mouse model that accumulates supraphysiological GABA in the central nervous system and other tissues. Mutant mice displayed increased mitochondrial numbers in the brain and liver, expected with a defect in mitophagy, and morphologically abnormal mitochondria. Administration of rapamycin to these mice reduced mTOR activity, reduced the elevated mitochondrial numbers, and normalized aberrant antioxidant levels. These results confirm a novel role for GABA in cell signaling and highlight potential pathomechanisms and treatments in various human pathologies, including SSADH deficiency, as well as other diseases characterized by elevated levels of GABA.

Regulation of ER-phagy by a Ypt/Rab GTPase module

Accumulation of misfolded proteins on intracellular membranes has been implicated in neurodegenerative diseases. One cellular pathway that clears such aggregates is endoplasmic reticulum autophagy (ER-phagy), a selective autophagy pathway that delivers excess ER to the lysosome for degradation. Not much is known about the regulation of ER-phagy. The conserved Ypt/Rab GTPases regulate all membrane trafficking events in eukaryotic cells. We recently showed that a Ypt module, consisting of Ypt1 and autophagy-specific upstream activator and downstream effector, regulates the onset of selective autophagy in yeast. Here we show that this module acts at the ER. Autophagy-specific mutations in its components cause accumulation of excess membrane proteins on aberrant ER structures and induction of ER stress. This accumulation is due to a block in transport of these membranes to the lysosome, where they are normally cleared. These findings establish a role for an autophagy-specific Ypt1 module in the regulation of ER-phagy. Moreover, because Ypt1 is a known key regulator of ER-to-Golgi transport, these findings establish a second role for Ypt1 at the ER. We therefore propose that individual Ypt/Rabs, in the context of distinct modules, can coordinate alternative trafficking steps from one cellular compartment to different destinations.

LAMP-2C regulation of MHC class II antigen presentation (P5020)

Abstract Chaperone-mediated autophagy (CMA) is a selective autophagy pathway, which relies on the lysosome-associated membrane protein (LAMP)-2A to translocate proteins from the cytoplasm into lysosomes. Alternative splicing of the LAMP-2 gene generates 3 highly conserved isoforms LAMP-2A, LAMP-2B and LAMP-2C. LAMP-2A overexpression increases MHC class II (MHCII) presentation of the cytoplasmic autoantigen, GAD, demonstrating a role for CMA in antigen presentation. Overexpression of LAMP-2B did not alter cytoplasmic GAD presentation, however, remarkably ectopic expression of LAMP-2C in B cells inhibited T cell responses to cytoplasmic GAD. Studies were undertaken here to understand the role of LAMP-2C in MHCII antigen presentation. The presentation of several exogenous proteins was not affected by LAMP-2C expression in B cells. Similar results were observed for the presentation of endogenous self and viral membrane proteins. These results suggest that LAMP-2C may be a specific inhibitor of antigen presentation via autophagy. Ectopic expression of LAMP-2C also failed to perturb MHCII presentation of the MP1-LC3 antigen via macroautophagy. Thus LAMP-2C appears to inhibit the presentation of cytoplasmic proteins that are processed through CMA and may not affect other autophagy pathways. Future studies are underway to determine mechanistically how LAMP-2C controls antigen presentation.

Adaptive plasticity of autophagic proteins to denervation in aging skeletal muscle

Aging muscle exhibits a progressive decline in mass and strength, known as sarcopenia, and a decrease in the adaptive response to contractile activity. The molecular mechanisms mediating this reduced plasticity have yet to be elucidated. The purposes of this study were 1) to determine whether denervation-induced muscle disuse would increase the expression of autophagy genes and 2) to examine whether selective autophagy pathways (mitophagy) are altered in aged animals. Denervation reduced muscle mass in young and aged animals by 24 and 16%, respectively. Moreover, young animals showed a 50% decrease in mitochondrial content following denervation, an adaptation that was not matched by aged animals. Basal autophagy protein expression was higher in aged animals, whereas young animals exhibited a greater induction of autophagy proteins following denervation. Localization of LC3II, Parkin, and p62 was significantly increased in the mitochondrial fraction of young and aged animals following denervation. Moreover, the unfolded protein response marker CHOP and the mitochondrial dynamics protein Fis1 were increased by 17- and 2.5-fold, respectively, in aged animals. Lipofuscin granules within lysosomes were evident with aging and denervation. Thus reductions in the adaptive plasticity of aged muscle are associated with decreases in disuse-induced autophagy. These data indicate that the expression of autophagy proteins and their localization to mitochondria are not decreased in aged muscle; however, the induction of autophagy in response to disuse, along with downstream events such as lysosome function, is impaired. This may contribute to an accumulation of dysfunctional mitochondria in aged muscle.

Mo1779 Interleukin-33 Induces Distinct Gastric Epithelial Alterations and Plays an Important Role in the Pathogenesis of Murine Gastritis

active in CD Paneth cells. TEM high magnification observation revealed that autophagy activation was associated with a significant decrease in the number of secretory granules in CD patients. The granules appeared to be wrapped around autophagolysosomal vacuoles and abnormally degraded. Conclusion: This work describes of a novel selective autophagy pathway, called crinophagy that targets specifically secretory granules of CD Paneth cells. This process consists of the self-digestion of secretory granules by the autophago-lysosomal pathway. Crinophagy may explain the decrease of CD secretory granule observed in our study as well as the disorganization of the secretory granules and the exocytosis defect previously reported in CD.

Yeast methylotrophy and autophagy in a methanol-oscillating environment on growing Arabidopsis thaliana leaves.

The yeast Candida boidinii capable of growth on methanol proliferates and survives on the leaves of Arabidopsis thaliana. The local methanol concentration at the phyllosphere of growing A. thaliana exhibited daily periodicity, and yeast cells responded by altering both the expression of methanol-inducible genes and peroxisome proliferation. Even under these dynamically changing environmental conditions, yeast cells proliferated 3 to 4 times in 11 days. Among the C1-metabolic enzymes, enzymes in the methanol assimilation pathway, but not formaldehyde dissimilation or anti-oxidizing enzymes, were necessary for yeast proliferation at the phyllosphere. Furthermore, both peroxisome assembly and pexophagy, a selective autophagy pathway that degrades peroxisomes, were necessary for phyllospheric proliferation. Thus, the present study sheds light on the life cycle and physiology of yeast in the natural environment at both the molecular and cellular levels.

S513 A COMBINATION OF MULTIPLE AUTONOMIC PARAMETERS, RATHER THAN EACH PARAMETER ALONE, YIELDS SIGNIFICANT DIFFERENTIATION BETWEEN HEAT PAIN INTENSITIES

Damaged mitochondria are detrimental to cellular homeostasis. One mechanism for removal of damaged mitochondria involves the PINK1-PARKIN pathway, which poly-ubiquitylates damaged mitochondria to promote mitophagy. We report that assembly of ubiquitin chains on mitochondria triggers autophagy adaptor recruitment concomitantly with activation of the TBK1 kinase, which physically associates with OPTN, NDP52, and SQSTM1. TBK1 activation in HeLa cells requires OPTN and NDP52 and OPTN ubiquitin chain binding. In addition to the known role of S177 phosphorylation in OPTN on ATG8 recruitment, TBK1-dependent phosphorylation on S473 and S513 promotes ubiquitin chain binding in vitro as well as TBK1 activation, OPTN mitochondrial retention, and efficient mitophagy in vivo. These data reveal a self-reinforcing positive feedback mechanism that coordinates TBK1-dependent autophagy adaptor phosphorylation with the assembly of ubiquitin chains on mitochondria to facilitate efficient mitophagy, and mechanistically links genes mutated in Parkinson’s disease and amyotrophic lateral sclerosis in a common selective autophagy pathway.

Zymophagy, a Novel Selective Autophagy Pathway Mediated by VMP1-USP9x-p62, Prevents Pancreatic Cell Death*♦

Autophagy has recently elicited significant attention as a mechanism that either protects or promotes cell death, although different autophagy pathways, and the cellular context in which they occur, remain to be elucidated. We report a thorough cellular and biochemical characterization of a novel selective autophagy that works as a protective cell response. This new selective autophagy is activated in pancreatic acinar cells during pancreatitis-induced vesicular transport alteration to sequester and degrade potentially deleterious activated zymogen granules. We have coined the term "zymophagy" to refer to this process. The autophagy-related protein VMP1, the ubiquitin-protease USP9x, and the ubiquitin-binding protein p62 mediate zymophagy. Moreover, VMP1 interacts with USP9x, indicating that there is a close cooperation between the autophagy pathway and the ubiquitin recognition machinery required for selective autophagosome formation. Zymophagy is activated by experimental pancreatitis in genetically engineered mice and cultured pancreatic acinar cells and by acute pancreatitis in humans. Furthermore, zymophagy has pathophysiological relevance by controlling pancreatitis-induced intracellular zymogen activation and helping to prevent cell death. Together, these data reveal a novel selective form of autophagy mediated by the VMP1-USP9x-p62 pathway, as a cellular protective response.

BAG3 mediates chaperone‐based aggresome‐targeting and selective autophagy of misfolded proteins

Increasing evidence indicates the existence of selective autophagy pathways, but the manner in which substrates are recognized and targeted to the autophagy system is poorly understood. One strategy is transport of a particular substrate to the aggresome, a perinuclear compartment with high autophagic activity. In this paper, we identify a new cellular pathway that uses the specificity of heat-shock protein 70 (Hsp70) to misfolded proteins as the basis for aggresome-targeting and autophagic degradation. This pathway is regulated by the stress-induced co-chaperone Bcl-2-associated athanogene 3 (BAG3), which interacts with the microtubule-motor dynein and selectively directs Hsp70 substrates to the motor and thereby to the aggresome. Notably, aggresome-targeting by BAG3 is distinct from previously described mechanisms, as it does not depend on substrate ubiquitination.