Apg7p/Cvt2p: A Novel Protein-activating Enzyme Essential for Autophagy

Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
Molecular Biology of the Cell (Impact Factor: 4.47). 06/1999; 10(5):1367-79. DOI: 10.1091/mbc.10.5.1367
Source: PubMed

ABSTRACT In the yeast Saccharomyces cerevisiae, the Apg12p-Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc-tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p-Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p-Apg5p conjugation.

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    • "The autophagosome then fuses with the lysosome for the degradation of the encapsulated materials [25]. Autophagy ATG7 is an essential protein required for the elongation phase of autophagosome formation [26], [27], [28]. Therefore in this study we employed a mouse with Atg7 knockout specific to T lymphocytes only, in order to determine the role of autophagy in regulating T lymphocyte apoptosis and immune responses in sepsis. "
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    ABSTRACT: Background Although the role of autophagy in sepsis has been characterized in several organs, its role in the adaptive immune system remains to be ascertained. This study aimed to investigate the role of autophagy in sepsis-induced T cell apoptosis and immunosuppression, using knockout mice with T cell specific deletion of autophagy essential gene Atg7. Methods and Results Sepsis was induced in a cecal ligation and puncture (CLP) model, with T-cell-specific Atg7-knockout mice compared to control mice. Autophagic vacuoles examined by electron microscopy were decreased in the spleen after CLP. Autophagy proteins LC3-II and ATG7, and autophagosomes and autolysosomes stained by Cyto-ID Green and acridine orange were decreased in CD4+ and CD8+ splenocytes at 18 h and 24 h after CLP. This decrease in autophagy was associated with increased apoptosis of CD4+ and CD8+ after CLP. Moreover, mice lacking Atg7 in T lymphocytes showed an increase in sepsis-induced mortality, T cell apoptosis and loss of CD4+ and CD8+ T cells, in comparison to control mice. This was accompanied by suppressed cytokine production of Th1/Th2/Th17 by CD4+ T cells, reduced phagocytosis in macrophages and decreased bacterial clearance in the spleen after sepsis. Conclusion These results indicated that sepsis led to down-regulation of autophagy in T lymphocytes, which may result in enhanced apoptosis induction and decreased survival in sepsis. Autophagy may therefore play a protective role against sepsis-induced T lymphocyte apoptosis and immunosuppression.
    PLoS ONE 07/2014; 9(7):e102066. DOI:10.1371/journal.pone.0102066 · 3.23 Impact Factor
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    • "In the first step, newly synthesized Atg8 family proteins are immediately cleaved at its C terminus by Atg4, resulting in the exposure of the C-terminal conserved glycine residue (Hemelaar et al., 2003; He et al., 2003; Tanida et al., 2004; Li et al., 2011). Atg7 forms a thioester intermediate with Atg8, followed by the conjugation of phosphatidylethanolamine by Atg3, which anchors the Atg8 family proteins to the autophagosome membrane (Schlumpberger et al., 1997; Tanida et al., 1999; Taherbhoy et al., 2011). In this process, Atg8 family proteins play a role not only in the elongation of the autophagosome membrane, but also in the recruitment of specific cytosolic materials to the autophagosome membrane, such as unfolded or aggregated proteins, mitochondria, and bacterial pathogens, via adaptor proteins that have the LIR motif. "
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    ABSTRACT: Autophagy is a bulk degradation pathway that removes cytosolic materials to maintain cellular homeostasis. The autophagy-related gene 13 (Atg13) and microtubule associate protein 1 light chain 3 (LC3) proteins are required for autophagosome formation. We demonstrate that each of the human LC3 isoforms (LC3A, LC3B, and LC3C) interacts with Atg13 via the LC3 interacting region (LIR) of Atg13. Using X-ray crystallography, we solved the macromolecular structures of LC3A and LC3C, along with the complex structures of the LC3 isoforms with the Atg13 LIR. Together, our structural and binding analyses reveal that the side-chain of Lys49 of LC3 acts as a gatekeeper to regulate binding of the LIR. We verified this observation by mutation of Lys49 in LC3A, which significantly reduces LC3A positive puncta formation in cultured cells. Our results suggest that specific affinity of the LC3 isoforms to the Atg13 LIR is required for proper autophagosome formation.
    Structure 11/2013; 22(1). DOI:10.1016/j.str.2013.09.023 · 5.62 Impact Factor
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    • "A number of specific ATGs constitute two ubiquitin-like conjugation systems, the ATG8 and ATG12 conjugation systems [10] [11]. Atg7p (an E1-like enzyme) activates Atg12p [12], which is later transferred to the E2-like enzyme Atg10p and subsequently conjugated to Atg5p. Similar to Atg12p, Atg8p is activated by Atg7p and transferred to the E2-like enzyme Atg3p [11]. "
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    ABSTRACT: Autophagy plays a crucial role in a wide array of physiological processes. To uncover the complex regulatory networks and mechanisms underlying basal autophagy, we performed a quantitative proteomics analysis of autophagy-deficient mouse embryonic fibroblast cells (MEFs) using iTRAQ labeling coupled with on-line 2D LC/MS/MS. We quantified a total of 1,234 proteins and identified 114 proteins that were significantly altered (90% confidence interval), including 48 up-regulated proteins and 66 down-regulated proteins. We determined that F-actin was disassembled in autophagy-deficient Atg7(-/-) MEFs. Treatment of the WT MEFs with cytochalasin D (CD), which induces F-actin depolymerization, significantly induced autophagosome formation. However, treatment with cytochalasin D also increased the protein level of p62 under starvation conditions, suggesting that depolymerization of F-actin impaired autophagosome maturation and that the intact F-actin network is required for basal and starvation-induced autophagy. Our results demonstrate a close relationship between F-actin and autophagy and provide the basis for further investigation of their interactions.
    Biochemical and Biophysical Research Communications 07/2013; 437(3). DOI:10.1016/j.bbrc.2013.06.111 · 2.30 Impact Factor
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