Autophagy Adaptor Research Articles

Heavy metal scavenger metallothionein mitigates deep hypothermia induced myocardial contractile anomalies:Role of autophagy

Metallothionein is a cysteine‐rich, low molecular weight heavy metal scavenger with potent cardioprotective properties. Deep hypothermia, if sustained, increased the risk of cardiovascular morbidity and mortality although the underlying mechanism is unclear. This study was designed to examine the impact of cardiac overexpression of metallothionein on deep hypothermia (4°C)‐induced myocardial contractile dysfunction, if any, and the underlying mechanism involved with a special focus on autophagy. Sustained deep hypothermia (4°C for 3 weeks) increased left ventricular end systolic and diastolic diameter, suppressed fractional shortening and perfused heart contractility, the effect of which was attenuated or ablated by metallothionein. Levels of autophagy marker LC3B, Atg7 and Beclin‐1, the autophagy adaptor protein p62 were increased following hypothermia. Fluorescence‐immunohistochemistry also revealed autophagy in murine hearts. Furthermore, sustained hypothermia triggered overtly reduced phosphorylation of the autophagy inhibitory signaling molecules Akt and mTOR (without affecting that of p70s6 kinase). Protein expression of pan Akt, mTOR and p70sk was unchanged. These hypothermia‐induced changes in autophagy and autophagic signaling were mitigated by metallothionein. Collectively, these data suggested that the heavy metal scavenger may protect against hypothermia‐induced cardiac anomalies through attenuation of cardiac autophagy.This research is supported by Wyoming INBRE (P20RR016474).

Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion

Autophagy defends the mammalian cytosol against bacterial infection.1-3 Efficient pathogen engulfment is mediated by cargo-selecting autophagy adaptors that rely on unidentified pattern-recognition or danger receptors to label invading pathogens as autophagy cargo, typically by poly-ubiquitin coating.4-9 Here we show that galectin-8, a cytosolic lectin, is a danger receptor that restricts Salmonella proliferation. Galectin-8 monitors endo-lysosomal integrity and detects bacterial invasion by binding host glycans exposed on damaged Salmonella-containing vacuoles. By recruiting NDP52 galectin-8 activates anti-bacterial autophagy. Galectin-8-dependent recruitment of NDP52 to Salmonella-containing vesicles is transient and followed by ubiquitin-dependent NDP52 recruitment. Since galectin-8 also detects sterile damage to endosomes or lysosomes, as well as invasion by Listeria or Shigella, we suggest galectin-8 serves as a versatile receptor for vesicle-damaging pathogens. Our results illustrate how cells deploy the danger receptor galectin-8 to combat infection by monitoring endo-lysososomal integrity based on the specific lack of complex carbohydrates in the cytosol.

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.

Triage of oxidation-prone proteins by Sqstm1/p62 within the mitochondria

As the mitochondrion is vulnerable to oxidative stress, cells have evolved several strategies to maintain mitochondrial integrity, including mitochondrial protein quality control mechanisms and autophagic removal of damaged mitochondria. Involvement of an autophagy adaptor, Sqstm1/p62, in the latter process has been recently described. In the present study, we provide evidence that a portion of p62 directly localizes within the mitochondria and supports stable electron transport by forming heterogeneous protein complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of mitochondrial proteins co-purified with p62 revealed that p62 interacts with several oxidation-prone proteins, including a few components of the electron transport chain complexes, as well as multiple chaperone molecules and redox regulatory enzymes. Accordingly, p62-deficient mitochondria exhibited compromised electron transport, and the compromised function was partially restored by in vitro delivery of p62. These results suggest that p62 plays an additional role in maintaining mitochondrial integrity at the vicinity of target machineries through its function in relation to protein quality control.

TBK1 Mediates Crosstalk Between the Innate Immune Response and Autophagy

The autophagic pathway participates in many physiological and pathophysiological processes. Autophagy plays an important role, as part of the innate immune response, in the first line of defense against intruding pathogens. Recognition of pathogens by the autophagic machinery is mainly mediated by autophagic adaptors, proteins that simultaneously interact with specific cargos and components of the autophagic machinery. However, the exact mechanisms and signaling pathways regulating this step are largely unknown. TANK-binding kinase 1 (TBK1) has been implicated recently in the autophagic clearance of the bacterium Salmonella enterica. After its activation by the invading bacteria, TBK1 directly phosphorylated the autophagic adaptor optineurin (OPTN). This modification led to enhanced interaction of OPTN with the family of mammalian Atg8 proteins, which are ubiquitin-like and essential for autophagy. Such interaction allows the autophagic machinery to be recruited to the intracellular loci of the bacteria, resulting in elimination of the bacteria by lysosomes. This study provides an example by which the innate immune response directly regulates cargo recruitment into autophagosomes.

Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy.

Adenosine monophosphate-activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified ULK1 and ULK2, mammalian orthologs of the yeast protein kinase Atg1, which is required for autophagy. Genetic analysis of AMPK or ULK1 in mammalian liver and Caenorhabditis elegans revealed a requirement for these kinases in autophagy. In mammals, loss of AMPK or ULK1 resulted in aberrant accumulation of the autophagy adaptor p62 and defective mitophagy. Reconstitution of ULK1-deficient cells with a mutant ULK1 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival during starvation. These findings uncover a conserved biochemical mechanism coupling nutrient status with autophagy and cell survival.

The NMR structure of the p62 PB1 domain, a key protein in autophagy and NF-κB signaling pathway

In eukaryotic cells, proteins are degraded via two main pathways; One is the ubiquitin-proteasome system that degrades short-lived proteins, and the other is autophagy that degrades long-lived proteins and damaged organelles (Noda et al. 2009). The p62, also called ZIP (PKC-finteracting protein) or sequestosome 1, plays a crucial role in these protein degradation pathways (Sumimoto et al. 2007). In autophagy, polyubiquitinated aggregated proteins and damaged organelles are enclosed by the isolation membrane, eventually enwrapped by the autophagosome. The autophagosome is fused with the vacuole/lysosome and its inner content is delivered and then degraded. The p62, initially identified as a protein that binds to the SH2 domain of the tyrosine kinase Lck, functions as a receptor protein for aberrant proteins. It contains a PB1 domain at its N terminus, followed by a ZZ-type zinc-finger motif, a LC3 interacting region, and a UBA domain at its C-terminus (Geetha and Wooten 2002). The p62 interacts with ubiquitin through the UBA domain, and self-assembles through the PB1 domain to form large protein aggregates (Bjorkoy et al. 2005). The p62 also binds the autophagy adaptor LC3 through the WXXL motif in the LC3 interacting region (Noda et al. 2008). Defects in autophagy cause accumulation of protein aggregates that contain ubiquitin and p62, leading to severe liver damage such as steatohepatitis and hepatocellular carcinomas, and neurodegenerative diseases such as Parkinson’s disease, Alzheimer disease, and Huntington’s disease (Zatloukal et al. 2002). A recent study indicated that the oligomerization through the PB1 domain was important not only in the assembly of the targets, but also in the interaction with LC3 (Bjorkoy et al. 2005). In the ubiqutin-proteasome system, p62 acts as a shuttling factor that transports ubiquitinated proteins to the proteasome, by interaction through the PB1 domain with the proteasome subunits, S5a and Rpt1 (Seibenhener et al. 2004; Geetha et al. 2008). The p62 also works as a key factor in cell signal transduction. In the NF-jB signaling pathway, p62 controls osteoclastogenesis, T-cell differentiation, and tumor progression, via the PB1–PB1 interaction with PKCf (Geetha and Wooten 2002). Further, p62 controls adipogenesis and obesity via the interaction with ERK (Moscat et al. 2006), and apoptosis via ubiquitinated Caspase 8 (Jin et al. 2009). The p62 PB1 domain plays a variety of physiological roles, both through the PB1–PB1 interaction, and through ‘‘non-canonical’’ PB1 mediated interactions with proteins lacking the PB1 domain, such as S5a, Rpt1, ERK, and LCK. Thus, p62 PB1 can be expected to have characteristic features not common to other PB1 domains whose structures have been solved. The PB1 domain is classified into three types, type I, type II, and type I/II (Hirano et al. 2004). Type I contains a motif of 28 amino acid residues with highly conserved acidic and hydrophobic residues named the OPCA motif. Type II contains a conserved lysine residue on the side opposite to the OPCA motif. Type I/II contains the OPCA motif and the conserved lysine residue, and thus can selfinteract in a front-to-back topology. The p62 PB1 contains both the OPCA motif and the conserved lysine residue and T. Saio Graduate School of Life Science, Hokkaido University, Sapporo 001-0021, Japan

Shigella Phagocytic Vacuolar Membrane Remnants Participate in the Cellular Response to Pathogen Invasion and Are Regulated by Autophagy

Intracellular pathogens like Shigella flexneri enter host cells by phagocytosis. Once inside, the pathogen breaks the vacuolar membrane for cytosolic access. The fate and function of the vacuolar membrane remnants are not clear. Examining Shigella-infected nonmyeloid cells, we observed that proteins associated with vacuolar membrane remnants are polyubiquinated, recruit the autophagy marker LC3 and adaptor p62, and are targeted to autophagic degradation. Further, inflammasome components and caspase-1 were localized to these membranes and correlated with dampened inflammatory response and necrotic cell death. In Atg4B mutant cells in which autophagosome maturation is blocked, polyubiquitinated proteins and P62 accumulated on membrane remnants, and as in autophagy-deficient Atg5(-/-) cells, the early inflammatory and cytokine response was exacerbated. Our results suggest that host membranes, after rupture by an invading cytoplasm-targeted bacterium, contribute to the cellular responses to infection by acting as a signaling node, with autophagy playing a central role in regulating these responses.