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      Mitochondrion-associated protein LRPPRC suppresses the initiation of basal levels of autophagy via enhancing Bcl-2 stability

      research-article
      * , , , , , * , * , , 1
      Biochemical Journal
      Portland Press Ltd.
      ATG5, autophagy, Beclin 1, class III phosphoinositide 3-kinase (PI3KCIII), leucine-rich pentatricopeptide repeat-containing (LRPPRC), microtubule-associated protein 1 small form (MAP1S), mitochondrion, p27, AMPK, AMP-activated protein kinase, eIF4E, eukaryotic initiation factor 4E, HEK, human embryonic kidney, HRP, horseradish peroxidase, LAMP, lysosome-associated membrane protein, LC3, light chain 3, LRPPRC, leucine-rich pentatricopeptide repeat-containing, LSFC, Leigh syndrome, French-Canadian type, MAP1, microtubule-associated protein 1, MAP1S, MAP1 small form, mTOR, mammalian target of rapamycin, PI3K, phosphoinositide 3-kinase, PI3KCIII, class III PI3K, Tom20, translocase of the mitochondrial outer membrane 20, Vps, vacuolar protein sorting

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          Abstract

          The mitochondrion-associated protein LRPPRC (leucine-rich pentatricopeptide repeat-containing) interacts with one of the microtubule-associated protein family members MAP1S (microtubule-associated protein 1 small form), originally named C19ORF5 (chromosome 19 open reading frame 5), to form a complex. MAP1S interacts with LC3 (light chain 3), the mammalian homologue of yeast autophagy marker ATG8 and one of the most important autophagy markers in mammalian cells, and helps the attachment of autophagosomes with microtubules for trafficking and recruitment of substrate mitochondria into autophagosomes for degradation. MAP1S activates autophagosomal biogenesis and degradation to remove misfolded/aggregated proteins and dysfunctional organelles such as mitochondria and suppress oxidative stress-induced genomic instability and tumorigenesis. Previously, various studies have attributed LRPPRC nucleic acid-associated functions. Instead, in the present study, we show that LRPPRC associates with mitochondria, interacts with Beclin 1 and Bcl-2 and forms a ternary complex to maintain the stability of Bcl-2. Suppression of LRPPRC leads to reduction in mitochondrial potential and reduction in Bcl-2. Lower levels of Bcl-2 lead to release of more Beclin 1 to form the Beclin 1–PI3KCIII (class III phosphoinositide 3-kinase) complex to activate autophagy and accelerate the turnover of dysfunctional mitochondria through the PI3K (phosphoinositide 3-kinase)/Akt/mTOR (mammalian target of rapamycin) pathway. The activation of autophagy induced by LRPPRC suppression occurs upstream of the ATG5–ATG12 conjugate-mediated conversion of LC3-I into LC3-II and has been confirmed in multiple mammalian cell lines with multiple autophagy markers including the size of GFP–LC3 punctate foci, the intensity of LC3-II and p62 protein and the size of the vacuolar structure. The activated autophagy enhances the removal of mitochondria through lysosomes. LRPPRC therefore acts to suppress the initiation of basal levels of autophagy to clean up dysfunctional mitochondria and other cellular debris during the normal cell cycle.

          Abstract

          Mitochondrial protein LRPPRC interacts with LC3-interactive microtubule-associated MAP1S and regulates autophagy. It interacts with Beclin 1 and Bcl-2 to form a ternary complex to maintain Bcl-2 stability. LRPPRC suppression enriches the Beclin 1-PI3KCIII complex to activate autophagy and mitophagy.

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          Most cited references45

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          Guidelines for the use and interpretation of assays for monitoring autophagy.

          In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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            LC3 conjugation system in mammalian autophagy

            Autophagy is the bulk degradation of proteins and organelles, a process essential for cellular maintenance, cell viability, differentiation and development in mammals. Autophagy has significant associations with neurodegenerative diseases, cardiomyopathies, cancer, programmed cell death, and bacterial and viral infections. During autophagy, a cup-shaped structure, the preautophagosome, engulfs cytosolic components, including organelles, and closes, forming an autophagosome, which subsequently fuses with a lysosome, leading to the proteolytic degradation of internal components of the autophagosome by lysosomal lytic enzymes. During the formation of mammalian autophagosomes, two ubiquitylation-like modifications are required, Atg12-conjugation and LC3-modification. LC3 is an autophagosomal ortholog of yeast Atg8. A lipidated form of LC3, LC3-II, has been shown to be an autophagosomal marker in mammals, and has been used to study autophagy in neurodegenerative and neuromuscular diseases, tumorigenesis, and bacterial and viral infections. The other Atg8 homologues, GABARAP and GATE-16, are also modified by the same mechanism. In non-starved rats, the tissue distribution of LC3-II differs from those of the lipidated forms of GABARAP and GATE-16, GABARAP-II and GATE-16-II, suggesting that there is a functional divergence among these three modified proteins. Delipidation of LC3-II and GABARAP-II is mediated by hAtg4B. We review the molecular mechanism of LC3-modification, the crosstalk between LC3-modification and mammalian Atg12-conjugation, and the cycle of LC3-lipidation and delipidation mediated by hAtg4B, as well as recent findings concerning the other two Atg8 homologues, GABARAP and GATE-16. We also highlight recent findings regarding the pathobiology of LC3-modification, including its role in microbial infection, cancer and neuromuscular diseases.
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              The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy.

              Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.
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                Author and article information

                Journal
                Biochem J
                Biochem. J
                bic
                BJ
                Biochemical Journal
                Portland Press Ltd.
                0264-6021
                1470-8728
                3 July 2013
                29 August 2013
                15 September 2013
                : 454
                : Pt 3
                : 447-457
                Affiliations
                *Medical College, Nanchang University, No. 461 Bayi Road, Nanchang, Jiangxi Province 330006, China
                †Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M Health Science Center, 2121 W. Holcombe Blvd, Houston, TX, 77030, U.S.A.
                Author notes
                1To whom correspondence should be addressed (email lliu@ 123456ibt.tamhsc.edu ).
                Article
                BJ20130306
                10.1042/BJ20130306
                3778712
                23822101
                489ac7e3-343a-42ad-a8ab-61a65af8e2bb
                © 2013 The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Licence (CC-BY)(http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 26 February 2013
                : 27 June 2013
                : 3 July 2013
                Page count
                Figures: 8, References: 59, Pages: 11
                Categories
                Research Article

                Biochemistry
                atg5,autophagy,beclin 1,class iii phosphoinositide 3-kinase (pi3kciii),leucine-rich pentatricopeptide repeat-containing (lrpprc),microtubule-associated protein 1 small form (map1s),mitochondrion,p27,ampk, amp-activated protein kinase,eif4e, eukaryotic initiation factor 4e,hek, human embryonic kidney,hrp, horseradish peroxidase,lamp, lysosome-associated membrane protein,lc3, light chain 3,lrpprc, leucine-rich pentatricopeptide repeat-containing,lsfc, leigh syndrome, french-canadian type,map1, microtubule-associated protein 1,map1s, map1 small form,mtor, mammalian target of rapamycin,pi3k, phosphoinositide 3-kinase,pi3kciii, class iii pi3k,tom20, translocase of the mitochondrial outer membrane 20,vps, vacuolar protein sorting

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