Autophagy is a highly regulated catabolic pathway that is potently induced by stressors including starvation and infection. An essential component of the autophagy pathway is an ATG16L1-containing E3-like enzyme, which is responsible for lipidating LC3B and driving autophagosome formation. ATG16L1 polymorphisms have been linked to the development of Crohn’s disease (CD) and phosphorylation of CD-associated ATG16L1 (caATG16L1) has been hypothesized to contribute to cleavage and autophagy dysfunction. Here we show that ULK1 kinase directly phosphorylates ATG16L1 in response to infection and starvation. Moreover, we show that ULK1-mediated phosphorylation drives the destabilization of caATG16L1 in response to stress. Additionally, we found that phosphorylated ATG16L1 was specifically localized to the site of internalized bacteria indicating a role for ATG16L1 in the promotion of anti-bacterial autophagy. Lastly, we show that stable cell lines harbouring a phospho-dead mutant of ATG16L1 have impaired xenophagy. In summary, our results show that ATG16L1 is a novel target of ULK1 kinase and that ULK1-signalling to ATG16L1 is a double-edged sword, enhancing function of the wildtype ATG16L1, but promoting degradation of caATG16L1.

ULK1 (UNC-51 like kinase 1) is a serine/threonine protein kinase that has been shown to play a crucial role in autophagy, a process of self digestion implicated in maintaining cellular homeostasis and in mediating type II programmed cell death. However, the exact mechanism by which ULK1 controls autophagy remains elusive, mostly because none of the known ULK1 targets have been directly linked to autophagy. To address this issue, I have employed a protein microarray screening approach to identify novel ULK1 substrates. I found five putative targets: MERTK (proto-oncogene tyrosine-protein kinase MER), B-RAF (v-raf murine sarcoma viral oncogene homologue B1), NOL4 (nucleolar protein 4), TBC1D22B (TBC1 domain family member 22B) and ACVRL1 (activin A receptor type II-like 1). My preliminary experiments have not confirmed that MERTK or B-RAF can be phosphorylated by ULK1 in vitro. However, further investigation will be required to firmly rule out MERTK and B-RAF as downstream targets of ULK1 and to test the ability of ULK1 to phosphorylate the other candidates. In addition, I have identified by in-gel kinase assay a ULK1 kinase at 34-kDa whose ability to phosphorylate the kinase domain of ULK1 was increased upon starvation. Using the genome information, I predicted this upstream kinase to be Pim1 (Proto-oncogene serine/threonine-protein kinase pim-1). I confirmed that Pim1 phosphorylated ULK1 in vitro at S147 and S224. Results of site directed mutagenesis suggest that phosphorylation at S224 correlates with increased ULK1 activity. This is consistent with observation that Pim1 is capable of activating ULK1 in vitro. Furthermore, I present preliminary data suggesting that Pim1 promotes autophagy in HeLa cells.

Macroautophagy, hereafter referred to as autophagy, is a catabolic process involving the degradation of cellular proteins and structures sequestered into a vesicle known as an autophagosome. The initiation of autophagy involves the conversion of a protein microtubule-associated proteins 1A/1B light chain 3B (LC3) from form I to form II allowing interaction with the formation of the autophagosome. Using an LC3-II/I ratio, relative initiation of autophagy can be estimated since higher relative amounts of LC3-II suggests a higher conversion rate of LC3-I to LC3-II, therefore suggesting autophagosomes are being formed at a higher rate. Autophagy’s selectivity, or its ability to degrade specific targets, is dependent on the degradation of ubiquitinated proteins and a protein adaptors, the latter forming a physical bridge between the ubiquitin-tagged cargo and LC3-II present on the forming autophagosome. Without these protein adaptors, autophagy has no selectivity and portions of the cytosol that happen to be near the autophagosome formation site are the only cellular components captured and degraded. Because the entire contents of the autophagosome are degraded following lysosome fusion, the selectivity can be assessed by determining the levels of protein adaptor and ubiquitinated proteins. Autophagy is constitutively active but is strongly stimulated under nutrient deprivation, such as fasting. Impairments of autophagy have been implicated in contractile and/or metabolic deficiencies in muscle diseases, obesity, diabetes, and aging; however, regulation of skeletal muscle autophagy is poorly understood at the molecular level. Here, we examined the role of the two partially homologous unc-51 like autophagy activating kinases 1 and 2 (ULK1 and ULK2) in modulating autophagy and myofiber atrophy during fasting via a microRNA-specific knockdown of these proteins in mouse skeletal muscle and using a non-specific microRNA in the contralateral muscle to allow comparisons of ULK effects within the same animal. Our results revealed that deficiency of ULK1 caused LC3-I to accumulate in fasted muscle without changes in Lc3b mRNA, indicating an impairment in the step of LC3-I conversion into LC3-II (an essential step in autophagy initiation). Similar trends were observed with other LC3-like proteins (GABL1 and GABL2) suggesting a specific role for ULK1 in regulating autophagy initiation. Deficiency of ULK2 did not affect LC3 or LC3-like proteins suggesting that ULK2 does not regulate autophagy initiation. However, it led to accumulation of ubiquitinated proteins, and the autophagy adaptors p62 and NBR-1, under both basal and fasting conditions. Since autophagy adaptors bind to and are degraded together with ubiquitinated proteins, these findings are consistent with impaired involvement of adaptors and consequent deficient cargo recognition by autophagy. Of note, deficient expression of either ULK1, ULK2, or both ULK1 & ULK2 did not attenuate myofiber atrophy during fasting. Altogether, these results uncover fundamental divergent roles for ULK1 and ULK2 in modulating autophagy and its selectivity in muscle. Current and future studies in our laboratory will further expand the molecular signature of autophagy activation and selectivity in muscle in order to identify novel targets for therapy in conditions associated with autophagy deficiency.

The bulk degradative process of macroautophagy requires the dynamic growth of autophagosomes, which carry cellular contents to the lysosome for recycling. Atg9A, a multi-pass transmembrane protein, is an apical regulator of autophagosome growth, yet its regulatory mechanism remains unclear. Our work suggests that hypoxia (low glucose and oxygen) triggers a rearrangement of the small adapter protein 14-3-3ζ interactome. Our data suggest that the localization of mammalian Atg9A to autophagosomes requires phosphorylation on the C terminus of Atg9A at S761, which creates a 14-3-3z docking site. Under basal conditions, this phosphorylation is maintained at a low level and is dependent on both ULK1 and AMPK. However, upon induction of hypoxic stress, activated AMPK bypasses the requirement for ULK1 and mediates S761 phosphorylation directly, resulting in an increase in 14-3-3z interactions, recruitment of Atg9A to LC3-positive autophagosomes, and enhanced autophagosome production. These observations suggested to us that long unstructured C-terminus of Atg9A may be a site of protein docking and regulation. We used BioID, along with conventional interactomics, to map the C- and N-terminal proximity-based interactions of Atg9A. We identified a network of Atg9A C-terminal interactions that include members of the ULK1 complex. Using gel filtration, we find that Atg9A co-immunoprecipitates with the ULK1 complex in high molecular weight fractions. Moreover, phosphorylation of the Atg9A C-terminus at S761 occurs within the ULK1 complex under nutrient-replete conditions, while hypoxia triggers a redistribution of phosphorylated Atg9A to low molecular weight fractions. Probing these relationships further, we find that Atg13, a component of the ULK1 complex, directly interacts with Atg9A and is required for Atg9A C-terminal phosphorylation. Furthermore, a non-phosphorylatable mutant of Atg9A (S761A) accumulates with Atg13 in high molecular weight complexes. Together, these data suggest that Atg13 recruits Atg9A to the ULK1 complex at the phagophore assemble site (PAS) and that S761 phosphorylation triggers Atg9A retrieval from the PAS

Obesity is a rapidly growing epidemic. It is associated with preventable chronic disease and vast healthcare cost in the United States (about 200 billion per year). Therefore, dissecting pathogenic mechanisms of obesity would provide effective strategies to prevent its development and reduce related cost. Obesity is characterized by excessive expansion of white adipose tissue (WAT). Autophagy, a cellular self-digestive process, is associated with WAT expansion and may be a promising target for combating obesity. Both hormone signaling (e.g., ERα) and energy sensing factors (e.g., Sirt1) control metabolism and prevent adiposity, and in which they have been shown to play collaborate roles. However, how autophagy is involved in ERα and Sirt1's inhibitory roles on adiposity is unknown. These questions have been addressed in my dissertation studies. To address this fundamental questions, I have established a method to monitor autophagy flux during adipocyte differentiation, which better reflected the dynamic process of autophagy. Compared with preadipocytes, autophagy flux activity was increased in mature adipocytes after differentiation. And then, my thesis project has addressed three main questions. Firstly, the gender difference in visceral fat distribution (Males have higher deposit of visceral fat than females) is controlled by an estradiol (E2)-autophagy axis. In C57BL/6J and wild type control mice, a higher visceral fat mass was detected in the males than in the females, which was associated with lower expression of estrogen receptor  (ER) and more active autophagy in males vs. females. ER knockout normalized this difference. Mechanistically, E2-ER- mTOR-ULK1-autophagy signaling contributed to the gender difference in visceral fat distribution. Secondly, in vitro and in vivo studies demonstrated that Sirt1 suppressed autophagy and reduced adipogenesis and adiposity via inducing mTOR-ULK1 signaling. Specific activation and overexpression of Sirt1 induced mTOR-ULK1 signaling to suppress autophagy and adipogenesis. And knockdown of Sirt1 exhibited opposite effects. The first and second studies revealed that ER and Sirt1 acted on mTOR-ULK1 signaling pathway, underlying the importance of their interaction in inhibiting autophagy and adipogenesis. As such, the third study was conducted and it unraveled that ER acted as upstream of Sirt1, possibly through its direct binding to Sirt1 promoter. Specifically, E2 signaling suppressed autophagy and adipogenesis. But when Sirt1 was knockdown, the effects of E2 on autophagy and adipogenesis were abolished. Taken together, my dissertation project underscores the importance for future research to consider gender difference and how E2-ER-autophagy axis contributes to this difference in other metabolic diseases. Also, the unraveled interaction between ERα and Sirt1 might lead to new therapeutic approach to adiposity and metabolic dysfunction in post-menopausal women or individuals with abnormal estrogen secretion. For example, dietary intervention or exercise challenge to activate Sirt1 may partially compensate estrogen deficiency. / Doctor of Philosophy / Obesity is a rapidly growing epidemic, which is associated with chronic disease and vast healthcare cost in the United States. Understanding the pathogenic mechanism of obesity is of critical importance. Recent studies have implicated autophagy, a cellular self-digestive process, in WAT development and expansion. It was also shown that hormone (e.g., via estrogen receptor ERα) and energy (e.g., via Sirt1) signaling control metabolism and adiposity. However, it is unclear whether and how autophagy interacts with ERα and Sirt1 in the regulation of adiposity. My dissertation project unraveled the mechanism of how hormone signaling (e.g., ERα) and energy sensing factors (e.g., Sirt1) interacted with autophagy to control adipogenesis and adiposity. My thesis project has addressed three main questions. Firstly, the gender difference in visceral fat distribution (Males have higher deposit of visceral fat than females) is controlled by an estradiol (E2)-autophagy axis, ER knockout normalized this difference. Mechanistically, E2- ER- mTOR-ULK1-autophagy signaling contributed to the gender difference in visceral fat distribution. Secondly, in vitro and in vivo studies demonstrated that Sirt1 induced mTOR-ULK1 signaling, suppressed autophagy and reduced adipogenesis and adiposity (ER similar effects). As such, the third study was conducted and it unraveled that ER acted as upstream of Sirt1, possibly through its direct binding to Sirt1 promoter. Taken together, my dissertation study has explored how hormone signaling (ER) and energy signaling (Sirt1) interact with autophagy to control adipogenesis and adiposity individually and collaboratively, which may provide new therapeutical approach to control obesity.

Autophagy, as the name suggests, is a cellular process of self-eating in which cytoplasmic debris is engulfed by a double membrane vesicle dubbed the autophagosome and is ultimately degraded and recycled by proteases in the lysosome. The process is initiated by a group of core ATG proteins, including a multi-pass transmembrane protein called ATG9A. Although ATG9A has been shown to be essential for both stress induced and basal autophagy, its mechanism and interaction network remain largely illusive. Our current study employs BioID proteomics to identify a network of interactors, including regulators of membrane fusion and vesicle trafficking, such as TRAPP, EARP, GARP, exocyst, AP-1 and AP-4 complexes, as well as members of the ULK1 autophagy kinase complex. Further investigations confirm that two components of the ULK1 complex, ATG13 and ATG101, directly interact with ATG9A. Using CRISPR, we show that deletion of ATG13 or ATG101 disrupts ATG9A trafficking and causes an accumulation of ATG9A at p62/SQSTM1-positive ubiquitin clusters. Lentivirus reconstitution and split-mVenus approaches using an ULK1 binding deficient mutant of ATG13 reveal that ATG9A interacts with ATG13 and ATG101 in an ULK1-independent manner. Together, these data reveal ATG9A interactions in vesicle trafficking and autophagy pathways, including a role for an ULK1- independent ATG13 complex in regulating ATG9A.

L'arthrose est la maladie musculo-squelettique la plus commune dans le monde. Elle est l'une des principales causes de douleur et d’incapacité chez les adultes, et elle représente un fardeau considérable sur le système de soins de santé. L'arthrose est une maladie de l’articulation entière, impliquant non seulement le cartilage articulaire, mais aussi la synoviale, les ligaments et l’os sous-chondral. L’arthrose est caractérisée par la dégénérescence progressive du cartilage articulaire, la formation d’ostéophytes, le remodelage de l'os sous-chondral, la détérioration des tendons et des ligaments et l'inflammation de la membrane synoviale. Les traitements actuels aident seulement à soulager les symptômes précoces de la maladie, c’est pour cette raison que l'arthrose est caractérisée par une progression presque inévitable vers la phase terminale de la maladie. La pathogénie exacte de l'arthrose est encore inconnue, mais on sait que l'événement clé est la dégradation du cartilage articulaire. Le cartilage articulaire est composé uniquement des chondrocytes; les cellules responsables de la synthèse de la matrice extracellulaire et du maintien de l'homéostasie du cartilage articulaire. Les chondrocytes maintiennent la matrice du cartilage en remplaçant les macromolécules dégradées et en répondant aux lésions du cartilage et aux dégénérescences focales en augmentant l'activité de synthèse locale. Les chondrocytes ont un taux faible de renouvellement, c’est pour cette raison qu’ils utilisent des mécanismes endogènes tels que l'autophagie (un processus de survie cellulaire et d’adaptation) pour enlever les organelles et les macromolécules endommagés et pour maintenir l'homéostasie du cartilage articulaire. i L'autophagie est une voie de dégradation lysosomale qui est essentielle pour la survie, la différenciation, le développement et l’homéostasie. Elle régule la maturation et favorise la survie des chondrocytes matures sous le stress et des conditions hypoxiques. Des études effectuées par nous et d'autres ont montré qu’un dérèglement de l’autophagie est associé à une diminution de la chondroprotection, à l'augmentation de la mort cellulaire et à la dégénérescence du cartilage articulaire. Carames et al ont montré que l'autophagie est constitutivement exprimée dans le cartilage articulaire humain normal. Toutefois, l'expression des inducteurs principaux de l'autophagie est réduite dans le vieux cartilage. Nos études précédentes ont également identifié des principaux gènes de l’autophagie qui sont exprimés à des niveaux plus faibles dans le cartilage humain atteint de l'arthrose. Les mêmes résultats ont été montrés dans le cartilage articulaire provenant des modèles de l’arthrose expérimentaux chez la souris et le chien. Plus précisément, nous avons remarqué que l'expression d’Unc-51 like kinase-1 (ULK1) est faible dans cartilage humain atteint de l'arthrose et des modèles expérimentaux de l’arthrose. ULK1 est la sérine / thréonine protéine kinase et elle est l’inducteur principal de l’autophagie. La perte de l’expression de ULK1 se traduit par un niveau d’autophagie faible. Etant donné qu’une signalisation adéquate de l'autophagie est nécessaire pour maintenir la chondroprotection ainsi que l'homéostasie du cartilage articulaire, nous avons proposé l’hypothèse suivante : une expression adéquate de ULK1 est requise pour l’induction de l’autophagie dans le cartilage articulaire et une perte de cette expression se traduira par une diminution de la chondroprotection, et une augmentation de la mort des chondrocytes ce qui conduit à la dégénérescence du cartilage articulaire. Le rôle exact de ULK1 dans la pathogénie de l'arthrose est inconnue, j’ai alors créé pour la première fois, des souris KO ULK1spécifiquement dans le cartilage en utilisant la technologie Cre-Lox et j’ai ensuite soumis ces souris à la déstabilisation du ménisque médial (DMM), un modèle de l'arthrose de la souris pour élucider le rôle spécifique in vivo de ULK1 dans pathogenèse de l'arthrose. Mes résultats montrent que ULK1 est essentielle pour le maintien de l'homéostasie du cartilage articulaire. Plus précisément, je montre que la perte de ULK1 dans le cartilage articulaire a causé un phénotype de l’arthrose accéléré, associé à la dégénérescence accélérée du cartilage, l’augmentation de la mort cellulaire des chondrocytes, et l’augmentation de l'expression des facteurs cataboliques. En utilisant des chondrocytes provenant des patients atteints de l’arthrose et qui ont été transfectées avec le plasmide d'expression ULK1, je montre qu’ULK1 est capable de réduire l’expression de la protéine mTOR (principal régulateur négatif de l’autophagie) et de diminuer l’expression des facteurs cataboliques comme MMP-13 et ADAMTS-5 et COX-2. Mes résultats jusqu'à présent indiquent que ULK1 est une cible thérapeutique potentielle pour maintenir l'homéostasie du cartilage articulaire. / Osteoarthritis (OA) is the most common musculoskeletal disease worldwide. It is one of the leading causes of pain and disability among adults, and represents a considerable burden on the healthcare system. OA is a disease of the entire joint, involving not only the articular cartilage but also the synovium, ligaments and subchondral bone. It is characterized by the progressive degeneration of the articular cartilage, osteophyte formation, remodelling of the subchondral bone, deterioration of tendons and ligaments and various degrees of inflammation of the synovium. While current therapies and management strategies can help alleviate symptoms early in the disease process, OA is characterized by almost inevitable progression towards end-stage disease. The exact pathogenesis of OA is largely unknown but the key event in OA is the degradation of the articular cartilage. The articular cartilage is only composed of chondrocytes; cells responsible for the synthesis of the extracellular matrix (ECM) and maintenance of articular cartilage homeostasis. Chondrocytes maintain the articular cartilage matrix by replacing degraded macromolecules and respond to focal cartilage injury or degeneration by increasing local synthesis activity. Since chondrocytes exhibit low levels of turnover, they rely on endogenous mechanisms such as autophagy (a cell survival and adaptation process) to remove damaged organelles and macromolecules in order to maintain articular cartilage homeostasis. Autophagy is a lysosomal degradation pathway that is essential for survival, differentiation, development and homeostasis. It regulates maturation and promotes survival of terminally differentiated chondrocytes under stress and hypoxic conditions. Studies by us and others have shown that compromised autophagy is associated with decreased chondroprotection, increased cell death and articular cartilage degeneration. Carames et al showed that autophagy is constitutively expressed in normal human articular cartilage. However, expression of key autophagy inducers is reduced in ageing cartilage. Our previous studies have also identified a panel of key autophagy genes that are expressed in low levels in human OA cartilage as well as in the articular cartilage from mouse and dog models of experimental OA. Specifically, we identified that expression of unc-51 like kinase-1 (ULK1) is suppressed in human OA cartilage and experimental OA models. ULK1 is a serine/threonine protein kinase and is the most upstream autophagy inducer. Loss of ULK1 results in disruption of autophagy induction. Since adequate autophagy signaling is required for maintaining chondroprotection as well as articular cartilage homeostasis, we hypothesized that ULK1 is required for autophagy induction in the articular cartilage and loss of it will result in decreased chondroprotection and enhanced chondrocyte death leading to the degeneration of articular cartilage. Since the exact role of ULK1 in pathogenesis of OA is unknown, I created for the first time, an inducible cartilage- specific ULK1 knockout (KO) mice using Cre-Lox technology and subjected these mice to the destabilization of the medial meniscus (DMM) mouse OA model to specifically elucidate the specific in vivo role of ULK1 in OA pathogenesis. My results show that ULK1 is essential for maintaining articular cartilage homeostasis. Specifically I show that loss of ULK1 in the articular cartilage results in an accelerated OA phenotype; which is associated with accelerated cartilage degeneration, enhanced chondrocyte cell death, increased expression of catabolic MMP-13. Using human OA chondrocytes transfected with ULK1 expression plasmid I show that ULK1 is able to reduce the expression of mTOR (major negative regulator of autophagy) and decrease the expression of OA catabolic factors including MMP-13, ADAMTS-5 and COX-2. My results so far suggest that ULK-1 is a potential therapeutic target to maintain articular cartilage homeostasis.