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Title: Molecular mechanisms underlying selective clearance of protein aggregates and mitochondria by autophagy
Authors: Tan, Sijie
Keywords: DRNTU::Science::Biological sciences::Cytology
Issue Date: 2017
Source: Tan, S. (2017). Molecular mechanisms underlying selective clearance of protein aggregates and mitochondria by autophagy. Doctoral thesis, Nanyang Technological University, Singapore.
Abstract: Autophagy is an important quality control mechanism that degrades unwanted or damaged cellular components in the lysosomes to maintain cellular homeostasis. Autophagy was once thought as a non-selective catabolic process. However, it is now recognize that the cells can upregulate selective forms of autophagy to target specific cellular cargo for lysosomal degradation. Selective autophagy plays a major role in cellular stress resistance. Selective autophagic removal of pathogenic protein aggregates (aggrephagy) and damaged mitochondria (mitophagy) are crucial aspects of cellular physiology. Impairment in these processes is a common denominator underlying aging and neurodegeneration. Hence, this study aims to understand the mechanisms underlying efficient cargo recognition and handling in the selective removal of protein inclusions and dysfunctional mitochondria, and to derive ways to augment both processes to protect against cellular toxicity. The first part of this study looked at post-translational regulation of aggrephagy. Synphilin-1 (Sph1), a common component of protein inclusions in α-synucleopathies, has been shown to mediate different types of aggrephagy. Sph1 mediates constitutive turnover of small cytosolic aggregates (Agg) basally (basal aggrephagy), and facilitates formation and autophagic disposal of large perinuclear protein inclusion known as the aggresome (Agm). Sph1 is both ubiquitinated and phosphorylated. K63-linked polyubiquitination (K63U) of Sph1 promotes the stress-induced aggrephagy, but has no influence on the basal aggrephagy. The effect of phosphorylation on Sph1 aggrephagy activity remains unexplored; hence, the role of phosphorylation in influencing Sph1 basal and inducible aggrephagy was examined. In-silico and mass spectrometry analysis revealed Sph1 is highly phosphorylated, and chemical inhibition as well as genetic knockdown (KD) of PKC, CKII and GSK3β isoforms perturbed basal and inducible aggrephagy. These observations highlight phosphorylation as a molecular determinant for Sph1-mediated basal and inducible aggrephagy. Furthermore, inhibition of GSK3β reduced the recruitment and activation of E3 ligase Parkin to Sph1, as well as the levels of K63U linkages on Sph1 under proteasomal stress. This finding demonstrates the crosstalk between phosphorylation and ubiquitination, mediated by an interplay between GSK3β and Parkin, underlies the mechanism for stress-induced aggrephagy. The next part of this study focused on the functional food, pomegranate, in the regulation of selective autophagy. The beneficial effects of pomegranate and its constituents to alleviate protein amyloid loads and enhance mitochondrial functions in-vivo were partially overlapped with autophagy induction. Hence, using a pomegranate extract (PE), the potential of pomegranate and its constituents to maintain proteostasis and mitochondrial health via upregulation of selective autophagy (aggrephagy and mitophagy) were examined. Our study demonstrates PE as a regulator of autophagy-lysosomal fitness. Furthermore, activation of transcription factor EB (TFEB) via the lysosomal-Ca2+ signaling underscores the mechanism for PE-induced autophagy. These findings highlight pomegranate as a novel activator of TFEB to upregulate autophagy. Further examination of the effects of PE-induced autophagy under proteotoxicity revealed that PE did not mediate the turnover of oxidized proteins or protein aggregates under proteasomal stress; instead, PE-induced autophagy enhanced the mitochondrial competence basally to facilitate mitophagy upon onset of mitochondrial stress. PE induced the formation of characteristic “donut-shaped” mitochondria, and promoted the recruitment of autophagosomes to the mitochondria under basal condition. Upon mitochondrial stress, PE preserved the mitochondrial health, and protected the mitochondria against reactive oxygen species (ROS)-induced redox toxicity by promoting PINK1-Parkin mediated mitophagy. KD of TFEB abrogated mitophagy induction and the protective effects of PE on the mitochondria. Hence, our study uncovered the mechanism underlying the beneficial effects of pomegranate and its constituents on mitochondrial health. PE activates TFEB to prime the mitochondria for clearance under basal condition, and promote selective autophagic clearance of damaged mitochondria under mitochondrial stress. Collectively, our study uncovered novel autophagy mechanisms to facilitate the selective removal of protein aggregates and damaged mitochondria under physiological and pathological conditions. These understandings provide new avenues for enhancing aggrephagy and mitophagy to counteract proteotoxicity and mitochondrial dysfunction observed in aging and diseased states to promote health and longevity.
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