iLIR@viral : a web resource for LIR motif-containing proteins in viruses

Autophagy has been shown to mediate the lysosomal degradation of pathogenic bacteria and viruses (xenophagy), and to contribute to the activation of innate and adaptative immune responses. Autophagy can serve as an antiviral defense mechanism but also as a proviral process during infection. ATG8-family proteins play a central role in the autophagy process due to their ability to interact with components of the autophagy machinery as well as selective autophagy receptors and adaptor proteins. Such interactions are usually mediated through LC3-interacting region (LIR) motifs. So far, only one viral protein has been experimentally shown to have a functional LIR motif, leaving open a vast field for investigation. Here, we have developed the iLIR@viral database (http://ilir.uk/virus/) as a freely accessible web resource listing all the putative canonical LIR motifs identified in viral proteins. Additionally, we used a curated text-mining analysis of the literature to identify novel putative LIR motif-containing protein (LICRPs) in viruses. We anticipate that iLIR@viral will assist with elucidating the full complement of LIRCPs in viruses

iLIR : a web resource for prediction of Atg8-family interacting proteins

Macroautophagy was initially considered to be a nonselective process for bulk breakdown of cytosolic material. However, recent evidence points toward a selective mode of autophagy mediated by the so-called selective autophagy receptors (SARs). SARs act by recognizing and sorting diverse cargo substrates (e.g., proteins, organelles, pathogens) to the autophagic machinery. Known SARs are characterized by a short linear sequence motif (LIR-, LRS-, or AIM-motif) responsible for the interaction between SARs and proteins of the Atg8 family. Interestingly, many LIR-containing proteins (LIRCPs) are also involved in autophagosome formation and maturation and a few of them in regulating signaling pathways. Despite recent research efforts to experimentally identify LIRCPs, only a few dozen of this class of—often unrelated—proteins have been characterized so far using tedious cell biological, biochemical, and crystallographic approaches. The availability of an ever-increasing number of complete eukaryotic genomes provides a grand challenge for characterizing novel LIRCPs throughout the eukaryotes. Along these lines, we developed iLIR, a freely available web resource, which provides in silico tools for assisting the identification of novel LIRCPs. Given an amino acid sequence as input, iLIR searches for instances of short sequences compliant with a refined sensitive regular expression pattern of the extended LIR motif (xLIR-motif) and retrieves characterized protein domains from the SMART database for the query. Additionally, iLIR scores xLIRs against a custom position-specific scoring matrix (PSSM) and identifies potentially disordered subsequences with protein interaction potential overlapping with detected xLIR-motifs. Here we demonstrate that proteins satisfying these criteria make good LIRCP candidates for further experimental verification. Domain architecture is displayed in an informative graphic, and detailed results are also available in tabular form. We anticipate that iLIR will assist with elucidating the full complement of LIRCPs in eukaryotes

GMAP is an Atg8a-interacting protein that regulates Golgi turnover in Drosophila

Selective autophagy receptors and adapters contain short linear motifs called LIR motifs (LC3-interacting re- gion), which are required for the interaction with the Atg8-family proteins. LIR motifs bind to the hydrophobic pockets of the LIR motif docking site (LDS) of the respective Atg8-family proteins. The physiological signif- icance of LDS docking sites has not been clarified in vivo. Here, we show that Atg8a-LDS mutant Drosophila flies accumulate autophagy substrates and have reduced lifespan. Using quantitative proteomics to identify the proteins that accumulate in Atg8a-LDS mutants, we identify the cis-Golgi protein GMAP (Golgi microtu- bule-associated protein) as a LIR motif-containing protein that interacts with Atg8a. GMAP LIR mutant flies exhibit accumulation of Golgi markers and elongated Golgi morphology. Our data suggest that GMAP medi- ates the turnover of Golgi by selective autophagy to regulate its morphology and size via its LIR motif-medi- ated interaction with Atg8a

GMAP is an Atg8a-interacting protein that regulates Golgi turnover In Drosophila

Selective autophagy receptors and adapters contain short linear motifs called LIR motifs (LC3-interacting region), which are required for the interaction with the Atg8-family proteins. LIR motifs bind to the hydrophobic pockets of the LIR motif docking site (LDS) of the respective Atg8-family proteins. The physiological significance of LDS docking sites has not been clarified in vivo. Here, we show that Atg8a-LDS mutant Drosophila flies accumulate autophagy substrates and have reduced lifespan. Using quantitative proteomics to identify the proteins that accumulate in Atg8a-LDS mutants, we identify the cis-Golgi protein GMAP (Golgi microtubule-associated protein) as a LIR motif-containing protein that interacts with Atg8a. GMAP LIR mutant flies exhibit accumulation of Golgi markers and elongated Golgi morphology. Our data suggest that GMAP mediates the turnover of Golgi by selective autophagy to regulate its morphology and size via its LIR motif-mediated interaction with Atg8a

HUWE1 E3 ligase promotes PINK1/PARKINindependent mitophagy by regulating AMBRA1 activation via IKKa

The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells

FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326–380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the “Claw” for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation

The selectivity and specificity of autophagy in drosophila

Autophagy is a process of cellular self-degradation and is a major pathway for elimination of cytoplasmic material by the lysosomes. Autophagy is responsible for the degradation of damaged organelles and protein aggregates and therefore plays a significant role in cellular homeostasis. Despite the initial belief that autophagy is a nonselective bulk process, there is growing evidence during the last years that sequestration and degradation of cellular material by autophagy can be accomplished in a selective and specific manner. Given the role of autophagy and selective autophagy in several disease related processes such as tumorigenesis, neurodegeneration and infections, it is very important to dissect the molecular mechanisms of selective autophagy, in the context of the system and the organism. An excellent genetically tractable model organism to study autophagy is Drosophila, which appears to have a highly conserved autophagic machinery compared with mammals. However, the mechanisms of selective autophagy in Drosophila have been largely unexplored. The aim of this review is to summarize recent discoveries about the selectivity of autophagy in Drosophila

Hepatic cytochromes P450: structural degrons and barcodes, posttranslational modifications and cellular adapters in the ERAD-endgame.

The endoplasmic reticulum (ER)-anchored hepatic cytochromes P450 (P450s) are enzymes that metabolize endo- and xenobiotics i.e. drugs, carcinogens, toxins, natural and chemical products. These agents modulate liver P450 content through increased synthesis or reduction via inactivation and/or proteolytic degradation, resulting in clinically significant drug-drug interactions. P450 proteolytic degradation occurs via ER-associated degradation (ERAD) involving either of two distinct routes: Ubiquitin (Ub)-dependent 26S proteasomal degradation (ERAD/UPD) or autophagic lysosomal degradation (ERAD/ALD). CYP3A4, the major human liver/intestinal P450, and the fast-turnover CYP2E1 species are degraded via ERAD/UPD entailing multisite protein phosphorylation and subsequent ubiquitination by gp78 and CHIP E3 Ub-ligases. We are gaining insight into the nature of the structural determinants involved in CYP3A4 and CYP2E1 molecular recognition in ERAD/UPD [i.e. K48-linked polyUb chains and linear and/or "conformational" phosphodegrons consisting either of consecutive sequences on surface loops and/or disordered regions, or structurally-assembled surface clusters of negatively charged acidic (Asp/Glu) and phosphorylated (Ser/Thr) residues, within or vicinal to which, Lys-residues are targeted for ubiquitination]. Structural inspection of select human liver P450s reveals that such linear or conformational phosphodegrons may indeed be a common P450-ERAD/UPD feature. By contrast, although many P450s such as the slow-turnover CYP2E1 species and rat liver CYP2B1 and CYP2C11 are degraded via ERAD/ALD, little is known about the mechanism of their ALD-targeting. On the basis of our current knowledge of ALD-substrate targeting, we propose a tripartite conjunction of K63-linked Ub-chains, P450 structural "LIR" motifs and selective cellular "cargo receptors" as plausible P450-ALD determinants

Signal transducer and activator of transcription-1 localizes to the mitochondria and modulates mitophagy

The signal transducer and activator of transcription (STAT) proteins are latent transcription factors that have been shown to be involved in cell proliferation, development, apoptosis, and autophagy. STAT proteins undergo activation by phosphorylation at tyrosine 701 and serine 727 where they translocate to the nucleus to regulate gene expression. STAT1 has been shown to be involved in promoting apoptotic cell death in response to cardiac ischemia/reperfusion and has recently been shown by our laboratory to be involved in negatively regulating autophagy. These processes are thought to promote cell death and restrict cell survival leading to the generation of an infarct. Here we present data that shows STAT1 localizes to the mitochondria and co-immunoprecipitates with LC3. Furthermore, electron microscopy studies also reveal mitochondria from ex vivo I/R treated hearts of STAT1KO mice contained within a double membrane autophagosome indicating that STAT1 may be involved in negatively regulating mitophagy. This is the first description of STAT1 being localized to the mitochondria and also having a role in mitophagy