Lipid kinase PIK3C3 maintains healthy brown and white adipose tissues to prevent metabolic diseases.

Adequate mass and function of adipose tissues (ATs) play essential roles in preventing metabolic perturbations. The pathological reduction of ATs in lipodystrophy leads to an array of metabolic diseases. Understanding the underlying mechanisms may benefit the development of effective therapies. Several cellular processes, including autophagy and vesicle trafficking, function collectively to maintain AT homeostasis. Here, we investigated the impact of adipocyte-specific deletion of the lipid kinase phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) on AT homeostasis and systemic metabolism in mice. We report that PIK3C3 functions in all ATs and that its absence disturbs adipocyte autophagy and hinders adipocyte differentiation, survival, and function with differential effects on brown and white ATs. These abnormalities cause loss of white ATs, whitening followed by loss of brown ATs, and impaired "browning" of white ATs. Consequently, mice exhibit compromised thermogenic capacity and develop dyslipidemia, hepatic steatosis, insulin resistance, and type 2 diabetes. While these effects of PIK3C3 largely contrast previous findings with the autophagy-related (ATG) protein ATG7 in adipocytes, mice with a combined deficiency in both factors reveal a dominant role of the PIK3C3-deficient phenotype. We have also found that dietary lipid excess exacerbates AT pathologies caused by PIK3C3 deficiency. Surprisingly, glucose tolerance is spared in adipocyte-specific PIK3C3-deficient mice, a phenotype that is more evident during dietary lipid excess. These findings reveal a crucial yet complex role for PIK3C3 in ATs, with potential therapeutic implications.

Autophagy inhibitors.

Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives.

Development of in vitro PIK3C3/VPS34 complex protein assay for autophagy-specific inhibitor screening.

Autophagy is an important catabolic program to respond to a variety of cellular stresses by forming a double membrane vesicle, autophagosome. Autophagy plays key roles in various cellular functions. Accordingly, dysregulation of autophagy is closely associated with diseases such as diabetes, neurodegenerative diseases, cardiomyopathy, and cancer. In this sense, autophagy is emerging as an important therapeutic target for disease control. Among the autophagy machineries, PIK3C3/VPS34 complex functions as an autophagy-triggering kinase to recruit the subsequent autophagy protein machineries on the phagophore membrane. Accumulating evidence showing that inhibition of PIK3C3/VPS34 complex successfully inhibits autophagy makes the complex an attractive target for developing autophagy inhibitors. However, one concern about PIK3C3/VPS34 complex is that many different PIK3C3/VPS34 complexes have distinct cellular functions. In this study, we have developed an in vitro PIK3C3/VPS34 complex monitoring assay for autophagy inhibitor screening in a high-throughput assay format instead of targeting the catalytic activity of the PIK3C3/VPS34 complex, which shuts down all PIK3C3/VPS34 complexes. We performed in vitro reconstitution of an essential autophagy-promoting PIK3C3/VPS34 complex, Vps34-Beclin1-ATG14L complex, in a microwell plate (96-well format) and successfully monitored the complex formation in many different conditions. This PIK3C3/VPS34 complex protein assay would provide a reliable tool for the screening of autophagy-specific inhibitors.

PIK3C3/VPS34 keeps body fats healthy.

Adipose tissue, or body fat, plays a critical role in the maintenance of health and the development of metabolic diseases. The pathological expansion of adipose tissue during obesity and the pathological reduction of adipose tissue during lipodystrophy can lead to a similar array of metabolic diseases that include diabetes, but mechanisms remain to be fully defined. In our recent studies, we explored the contribution of the lipid kinase PIK3C3/VPS34 to adipose tissue health and metabolic disease. We found that adipocyte-specific PIK3C3/VPS34 deficiency causes defects in the differentiation, survival and functional properties of adipocytes, resulting in reduced adipose tissue mass, altered blood lipid levels, fatty liver disease, diabetes, and defective body temperature control. These abnormalities mirror those observed in patients with lipodystrophy. These findings identify adipocyte PIK3C3/VPS34 as a potential target for therapeutic intervention in metabolic diseases.

PIK3C3/VPS34 helps school T cells in the thymus.

The development of a broad repertoire of T cells in the immune system requires interaction of T cell receptors expressed by immature T cells with peptide/major histocompatibility complexes (MHCs) displayed by specialized epithelial cells in the thymus, in a process called T cell positive selection. Thymic epithelial cells (TECs) display unique antigen processing machinery which shapes the collection of self-peptides that drive positive selection. In our recent studies, we explored the contribution of the lipid kinase PIK3C3/VPS34 to the generation of positively selecting peptides in TECs. We found that TEC-specific PIK3C3/VPS34 facilitates the positive selection of CD4 but not CD8 T lineage cells, in a mechanism independent of its role in canonical macroautophagy/autophagy. Instead, we propose that PIK3C3/VPS34 alters vesicle trafficking in TECs that modulates lysosomal protease activity which, in turn, controls the generation of MHC class II-presented peptides optimized for positive selection of CD4 T cells.

Dendritic cell PIK3C3/VPS34 controls the pathogenicity of CNS autoimmunity independently of LC3-associated phagocytosis.

PIK3C3/VPS34 is a key player in macroautophagy/autophagy and MAP1LC3/LC3-associated phagocytosis (LAP), which play critical roles in dendritic cell (DC) function. In this study, we assessed the contribution of PIK3C3 to DC function during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We found that Pik3c3-deficient DCs exhibit attenuated capacity to reactivate encephalitogenic T cells in the central nervous system, leading to reduced incidence and severity of EAE in DC-specific Pik3c3-deficient mice. Additionally, animals with a DC-specific deficiency in Rb1cc1/Fip200 but not Rubcn were protected against EAE, suggesting that the EAE phenotype of DC-specific Pik3c3-deficient mice is due to defective canonical autophagy rather than LAP. Collectively, our studies have revealed a critical role of PIK3C3 in DC function and the pathogenicity of these cells during EAE, with important implications for the development of immunotherapies for autoimmune diseases such as MS.Abbreviations: ATG: autophagy-related; CNS: central nervous system; DC: dendritic cell; DEG: differentially expressed gene; EAE: experimental autoimmune encephalomyelitis; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; MOG: myelin oligodendrocyte glycoprotein; MS: multiple sclerosis; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; ROS: reactive oxygen species.

Regulation of oxidative stress-induced autophagy by ATG9A ubiquitination.

High levels of reactive oxygen species (ROS) result in oxidative stress, which damages cells and leads to the development of many diseases. Macroautophagy/autophagy plays an important role in protecting cells from diverse stress stimuli including oxidative stress. However, the molecular mechanisms of autophagy activation in response to oxidative stress remain largely unclear. In this study, we showed that TRAF6 mediates oxidative stress-induced ATG9A ubiquitination at two C-terminal lysine residues (K581 and K838). ATG9A ubiquitination promotes its association with BECN1, BECN1-PIK3C3/VPS34-UVRAG complex assembly and PIK3C3/VPS34 activation, thereby activating autophagy and endocytic trafficking. We also identified TNFAIP3/A20 as a negative regulator of oxidative-induced autophagy by counteracting TRAF6-mediated ATG9A ubiquitination. Moreover, ATG9A depletion attenuates LPS-induced autophagy and causes aberrant TLR4 signaling and inflammatory responses. Our findings revealed a critical role of ATG9A ubiquitination in oxidative stress-induced autophagy, endocytic trafficking and innate immunity.

Unsaturation, curvature and charge: effects of membrane parameters on PIK3C3/VPS34-containing complexes.

Phosphatidylinositol-3-phosphate (PtdIns3P) is essential for generating autophagosomes and regulating endocytic trafficking. Recently, we have shown that the activities of human PIK3C3/VPS34-containing complexes I and II, which synthesize PtdIns3P, are greatly affected by three membrane physicochemical parameters: lipid unsaturation, membrane curvature, and negative charge. Both complexes are more active on membranes composed of unsaturated lipids than saturated lipids, and high membrane curvature can compensate for the negative effect of high lipid saturation. Negatively charged phosphatidylserine (PS) activates the complexes, as well as PIK3C3/VPS34 alone. The kinase activity of complex I depends critically on the ATG14 BATS domain, whereas complex II relies on the BECN1 BARA domain. Our findings highlight the importance of the membrane character as sensed by the unique membrane binding motifs/domain of the complexes for regulating PIK3C3/VPS34 activity.

Autophagy-related protein PIK3C3/VPS34 controls T cell metabolism and function.

The PIK3C3/VPS34 subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is a key early player in macroautophagy/autophagy. In this study, we assessed the contribution of PIK3C3 to T cell metabolism and function. We found that Pik3c3-deficient T cells exhibited impaired cellular metabolism, and Pik3c3-deficient CD4+ T cells failed to differentiate into T helper 1 cells. These alterations were associated with reduced levels of active mitochondria upon T cell activation. In addition, conditional Pik3c3-deficient animals failed to mount autoreactive T cell responses and were resistant to experimental autoimmune encephalomyelitis (EAE). Interestingly, the deletion of Pik3c3 had little effect on the capacity of animals to clear tumor metastases. Collectively, our studies have revealed a critical role of PIK3C3 in T cell metabolism and the pathogenicity of these cells during EAE. Our findings also have important implications for the development of immunotherapies to treat multiple sclerosis and other inflammatory diseases by targeting PIK3C3.Abbreviations: CNS: central nervous system; DC: dendritic cell; DEG: differentially expressed gene; EAE: experimental autoimmune encephalomyelitis; ECAR: extracellular acidification rate; iNKT: invariant natural killer T; LAP: LC3-associated phagocytosis; LLC: Lewis lung carcinoma; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MDSC: myeloid-derived suppressor cell; MOG: myelin oligodendrocyte glycoprotein; NK: natural killer; OCR: oxygen consumption rate; PI: propidium iodide; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; RNA-seq: RNA-sequencing; TCR: T cell receptor; TMRE: tetramethylrhodamine ethyl ester perchlorate.

Autophagy: An Essential Degradation Program for Cellular Homeostasis and Life.

Autophagy is a lysosome-dependent cellular degradation program that responds to a variety of environmental and cellular stresses. It is an evolutionarily well-conserved and essential pathway to maintain cellular homeostasis, therefore, dysfunction of autophagy is closely associated with a wide spectrum of human pathophysiological conditions including cancers and neurodegenerative diseases. The discovery and characterization of the kingdom of autophagy proteins have uncovered the molecular basis of the autophagy process. In addition, recent advances on the various post-translational modifications of autophagy proteins have shed light on the multiple layers of autophagy regulatory mechanisms, and provide novel therapeutic targets for the treatment of the diseases.

Membrane Trafficking in Autophagy.

Macroautophagy is an intracellular pathway used for targeting of cellular components to the lysosome for their degradation and involves sequestration of cytoplasmic material into autophagosomes formed from a double membrane structure called the phagophore. The nucleation and elongation of the phagophore is tightly regulated by several autophagy-related (ATG) proteins, but also involves vesicular trafficking from different subcellular compartments to the forming autophagosome. Such trafficking must be tightly regulated by various intra- and extracellular signals to respond to different cellular stressors and metabolic states, as well as the nature of the cargo to become degraded. We are only starting to understand the interconnections between different membrane trafficking pathways and macroautophagy. This review will focus on the membrane trafficking machinery found to be involved in delivery of membrane, lipids, and proteins to the forming autophagosome and in the subsequent autophagosome fusion with endolysosomal membranes. The role of RAB proteins and their regulators, as well as coat proteins, vesicle tethers, and SNARE proteins in autophagosome biogenesis and maturation will be discussed.

In Vitro Characterization of VPS34 Lipid Kinase Inhibition by Small Molecules.

VPS34 is a class III phosphoinositide 3-kinase that acts on vesicle trafficking. This kinase has recently attracted significant attention because of the function it plays in the machinery involved in the early steps of autophagy. Moreover, because significant progress had been made in the optimization of specific kinase inhibitors, its potential to be targeted by catalytic inhibitors has been investigated by different groups. The aim of this review is to present the key in vitro assays necessary for characterizing inhibitors of the catalytic activity of VPS34. The review covers catalytic (IC50 on purified recombinant protein) and binding assays (KD, ka, kd on purified recombinant protein), and a cell-based assay (IC50 in GFP-FYVE expressing cell line). The methodology for crystallization of VPS34 protein is also presented as it can provide guidance for the design by medicinal chemistry of small molecular mass kinase inhibitor.

Novel pharmacological modulators of autophagy: an updated patent review (2012-2015).

INTRODUCTION: Autophagy is a lysosome-dependent degradation pathway that maintains cellular homeostasis in response to a variety of cellular stresses. Accumulating reports based on animal models have indicated the importance of this catabolic program in many human pathophysiological conditions, including diabetes, neurodegenerative diseases, aging, and cancers. Therefore, autophagy has been highlighted as a novel therapeutic target with a wide range of beneficial effects on human diseases. Here, we review the recent advances of our knowledge toward autophagy, as well as the efforts for developing autophagy modulators. Areas covered: The relevant patents (published at 2012-2015) and the research literature claiming the pharmacological modulation of autophagy are reviewed. Also, their molecular mechanisms and potential therapeutic utilities are discussed. Expert opinion: Considering the molecular machinery involved in autophagy induction, the targeting of autophagy-specific protein is very important to design the therapeutic interventions for specifically treating a variety of autophagy-associated disorders. Many patents and the research literature described in this review have shown promising applications of the relevant autophagy modulators for cancer or neurodegeneration treatments, a few of which are already being considered for clinical evaluation. However, most patents have claimed the modulators of autophagy with little information regarding their mechanisms of action. To design highly potent therapeutics, further work, such as developing compounds that specifically target the autophagy-specific machinery, are required.

SAR405, a PIK3C3/Vps34 inhibitor that prevents autophagy and synergizes with MTOR inhibition in tumor cells.

Autophagy plays an important role in cancer and it has been suggested that it functions not only as a tumor suppressor pathway to prevent tumor initiation, but also as a prosurvival pathway that helps tumor cells endure metabolic stress and resist death triggered by chemotherapeutic agents. We recently described the discovery of inhibitors of PIK3C3/Vps34 (phosphatidylinositol 3-kinase, catalytic subunit type 3), the lipid kinase component of the class III phosphatidylinositol 3-kinase (PtdIns3K). This PtdIns3K isoform has attracted significant attention in recent years because of its role in autophagy. Following chemical optimization we identified SAR405, a low molecular mass kinase inhibitor of PIK3C3, highly potent and selective with regard to other lipid and protein kinases. We demonstrated that inhibiting the catalytic activity of PIK3C3 disrupts vesicle trafficking from late endosomes to lysosomes. SAR405 treatment also inhibits autophagy induced either by starvation or by MTOR (mechanistic target of rapamycin) inhibition. Finally our results show that combining SAR405 with everolimus, the FDA-approved MTOR inhibitor, results in a significant synergy on the reduction of cell proliferation using renal tumor cells. This result indicates a potential therapeutic application for PIK3C3 inhibitors in cancer.

V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation.

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H(+)-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.

Divergent roles of BECN1 in LC3 lipidation and autophagosomal function.

BECN1/Beclin 1 is regarded as a critical component in the class III phosphatidylinositol 3-kinase (PtdIns3K) complex to trigger autophagy in mammalian cells. Despite its significant role in a number of cellular and physiological processes, the exact function of BECN1 in autophagy remains controversial. Here we created a BECN1 knockout human cell line using the TALEN technique. Surprisingly, the complete loss of BECN1 had little effect on LC3 (MAP1LC3B/LC3B) lipidation, and LC3B puncta resembling autophagosomes by fluorescence microscopy were still evident albeit significantly smaller than those in the wild-type cells. Electron microscopy (EM) analysis revealed that BECN1 deficiency led to malformed autophagosome-like structures containing multiple layers of membranes under amino acid starvation. We further confirmed that the PtdIns3K complex activity and autophagy flux were disrupted in BECN1(-/-) cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.