The MAP4 Projection Domain Accelerates Hypoxia-Induced Mitophagy Disruption through LIR Motif in Cardiomyocytes

Previously, we and other investigators have demonstrated that phosphorylated microtubule-associated protein 4 (p-MAP4) impacts myocardial hypertrophy and ischemic heart failure. However, the detailed mechanism behind this remains under elucidated. Published studies have suggested that impaired mitophagy contributes to hypoxia-induced myocardial damage, hence the involvement of p-MAP4 in mitophagy in cardiomyocytes was investigated. The results herein revealed that there was increased degradation of mitochondria, accumulated mitophagosomes and disrupted autophagic flux in both neonatal and adult ones of MAP4-knockin (KI) mice. This indicated that p-MAP4 persistently degraded mitochondria through activating mitophagy. Next, Tom70 was found as the importer of p-MAP4 in the context of mitochondrial translocation. And, the LC3-interacting region (LIR) motif (47–50aa) caused p-MAP4-induced mitochondrial engulfment, and the ubiquitin-interacting motif (UIM) domain determined the characteristics of p-MAP4-induced mitophagosomes, which were structure and membrane potential-independent. Moreover, p-MAP4 enhanced hypoxia-induced mitophagic flux impairment, and p-MAP4 LIR (47–50aa) mutation decreased hypoxia-induced autophagy both in MAP4 knockout and wildtype cardiomyocytes. Overall, this study identified that p-MAP4 as a novel mediator and cargo receptor in mitophagy, and that the degradation of the MAP4 PJ domain as a promising therapeutic target for improving the cardiac function of hypoxia-related heart failure or cardiac remodelling.

NudCL2 is an autophagy receptor that mediates selective autophagic degradation of CP110 at mother centrioles to promote ciliogenesis

Primary cilia extending from mother centrioles are essential for vertebrate development and homeostasis maintenance. Centriolar coiled-coil protein 110 (CP110) has been reported to suppress ciliogenesis initiation by capping the distal ends of mother centrioles. However, the mechanism underlying the specific degradation of mother centriole-capping CP110 to promote cilia initiation remains unknown. Here, we find that autophagy is crucial for CP110 degradation at mother centrioles after serum starvation in MEF cells. We further identify NudC-like protein 2 (NudCL2) as a novel selective autophagy receptor at mother centrioles, which contains an LC3-interacting region (LIR) motif mediating the association of CP110 and the autophagosome marker LC3. Knockout of NudCL2 induces defects in the removal of CP110 from mother centrioles and ciliogenesis, which are rescued by wild-type NudCL2 but not its LIR motif mutant. Knockdown of CP110 significantly attenuates ciliogenesis defects in NudCL2-deficient cells. In addition, NudCL2 morphants exhibit ciliation-related phenotypes in zebrafish, which are reversed by wild-type NudCL2, but not its LIR motif mutant. Importantly, CP110 depletion significantly reverses these ciliary phenotypes in NudCL2 morphants. Taken together, our data suggest that NudCL2 functions as an autophagy receptor mediating the selective degradation of mother centriole-capping CP110 to promote ciliogenesis, which is indispensable for embryo development in vertebrates.

ULK1 Promotes Mitophagy via Phosphorylation and Stabilization of BNIP3.

UNC51-like kinase-1 (ULK1) is the catalytic component of the autophagy pre-initiation complex that stimulates autophagy via phosphorylation of ATG14, BECLN1 and other autophagy proteins. ULK1 has also been shown to specifically promote mitophagy but the mechanistic basis of how has remained unclear. Here we show that ULK1 phosphorylates the BNIP3 mitochondrial cargo receptor on a critical serine residue (S17) adjacent to its amino terminal LIR motif. ULK1 similarly phosphorylates BNIP3L on S35. Phosphorylation of BNIP3 on S17 by ULK1 promotes interaction with LC3 and mitophagy. ULK1 interaction also promotes BNIP3 protein stability by limiting its turnover at the proteasome. The ability of ULK1 to regulate BNIP3 protein stability depends on an intact “BH3” domain and deletion of its “BH3” domain reduces BNIP3 turnover and increases BNIP3 protein levels independent of ULK1. In summary ULK1 promotes mitophagy by both stabilization of BNIP3 protein and via phosphorylation of S17 to stimulate interaction with LC3.

SAMM50 acts with p62 in piecemeal basal- and OXPHOS-induced mitophagy of SAM and MICOS components

Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.

Decoding three distinct states of the Syntaxin17 SNARE motif in mediating autophagosome–lysosome fusion

Syntaxin17, a key autophagosomalN-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR–GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17–SNAP29–VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome–lysosome fusion process.

A Conserved LIR Motif in Connexins Mediates Ubiquitin-Independent Binding to LC3/GABARAP Proteins

Gap junctions (GJ) are specialized cell-cell contacts formed by connexins (Cxs), which provide direct communication between adjacent cells. Cx43 ubiquitination has been suggested to induce the internalization of GJs, as well as the recruitment of the autophagy receptor p62 to mediate binding to LC3B and degradation by macroautophagy. In this report, we describe a functional LC3 interacting region (LIR), present in the amino terminal of most Cx protein family members, which can mediate the autophagy degradation of Cx43 without the need of ubiquitin. Mutation of the LIR motif on Cx37, Cx43, Cx46 and Cx50 impairs interaction with LC3B and GABARAP without compromising protein ubiquitination. Through in vitro protein-protein interaction assays, we demonstrate that this LIR motif is required for the binding of Cx43 to LC3B and GABARAP. Overall, our findings describe an alternative mechanism whereby Cxs interact with LC3/GABARAP proteins, envisioning a new model for the autophagy degradation of connexins.

Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites

The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.

LIR Motif-Containing Hyperdisulfide β-Ginkgotide is Cytoprotective, Adaptogenic, and Scaffold-Ready

Grafting a bioactive peptide onto a disulfide-rich scaffold is a promising approach to improve its structure and metabolic stability. The ginkgo plant-derived β-ginkgotide β-gB1 is a highly unusual molecule: Small, hyperdisulfide, and found only in selected ancient plants. It also contains a conserved 16-amino-acid core with three interlocking disulfides, as well as a six-amino-acid inter-cysteine loop 2 suitable for grafting peptide epitopes. However, very little is known about this recently-discovered family of molecules. Here, we report the biophysical and functional characterizations of the β-ginkgotide β-gB1 from G. biloba. A circular dichroism spectroscopy analysis at 90 °C and proteolytic treatments of β-gB1 supported that it is hyperstable. Data mining revealed that the β-gB1 loop 2 contains the canonical LC3 interacting region (LIR) motif crucial for selective autophagy. Cell-based assays and pull-down experiments showed that β-gB1 is an adaptogen, able to maintain cellular homeostasis through induced autophagosomes formation and to protect cells by targeting intracellular proteins from stress-mediated damage against hypoxia and the hypoxia-reoxygenation of induced cell death. This is the first report of an LIR-containing peptide natural product. Together, our results suggest that the plant-derived β-ginkgotide is cytoprotective, capable of targeting intracellular proteins, and holds promise as a hyperdisulfide scaffold for engineering peptidyl therapeutics with enhanced structural and metabolic stability.

Atg8a interacts with transcription factor Sequoia to control the expression of autophagy genes in Drosophila

SUMMARYAutophagy is a fundamental, evolutionarily conserved, process in which cytoplasmic material is degraded through the lysosomal pathway [1–7]. One of the most important and well-studied autophagy-related proteins is LC3 [Microtubule-associated protein 1 light chain 3, (called Atg8 in yeast and Drosophila)], which participates in autophagosome formation and autophagy cargo selection in the cytoplasm, and is one of the most widely utilized markers of autophagy [8, 9]. Despite growing evidence that LC3 is enriched in the nucleus, little is known about the mechanisms involved in targeting LC3 to the nucleus and the nuclear components it interacts with [10–13]. Here we show that Drosophila Atg8a protein, homologous to mammalian LC3 and yeast Atg8, interacts with the transcription factor Sequoia in a LIR-motif dependent manner. We show that Sequoia depletion induces autophagy in nutrient rich conditions through enhanced expression of autophagy genes. We also show that Atg8a interacts with YL-1, a component of a nuclear acetyltransferase complex, and is acetylated at position K46. Additionally, we show that Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation in order to activate autophagy. Our results suggest a mechanism of regulation of expression of autophagy genes by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1 and Sir2.