Mammalian PIK3C3/VPS34: the key to autophagic processing in liver and heart

PIK3C3/Vps34 is the class III PtdIns3K that is evolutionarily conserved from yeast to mammals. Its central role in mammalian autophagy has been suggested through the use of pharmacological inhibitors and the study of its binding partners. However, the precise role of PIK3C3 in mammals is not clear. Using mouse strains that allow tissue-specific deletion of PIK3C3, we have described an essential role of PIK3C3 in regulating autophagy, and liver and heart function.     

Binding of Avibirnavirus VP3 to the PIK3C3-PDPK1 complex inhibits autophagy by activating the AKT-MTOR pathway

ABSTRACT

Macroautophagy/autophagy is a host natural defense response. Viruses have developed various strategies to subvert autophagy during their life cycle. Recently, we revealed that autophagy was activated by binding of Avibirnavirus to cells. In the present study, we report the inhibition of autophagy initiated by PIK3C3/VPS34 via the PDPK1-dependent AKT-MTOR pathway. Autophagy detection revealed that viral protein VP3 triggered inhibition of autophagy at the early stage of Avibirnavirus replication. Subsequent interaction analysis showed that the CC1 domain of VP3 disassociated PIK3C3-BECN1 complex by direct interaction with BECN1 and blocked autophagosome formation, while the CC3 domain of VP3 disrupted PIK3C3-PDPK1 complex via directly binding to PIK3C3 and inhibited both formation and maturation of autophagosome. Furthermore, we found that PDPK1 activated AKT-MTOR pathway for suppressing autophagy via binding to AKT. Finally, we proved that CC3 domain was critical for role of VP3 in regulating replication of Avibirnavirus through autophagy. Taken together, our study identified that Avibirnavirus VP3 links PIK3C3-PDPK1 complex to AKT-MTOR pathway and inhibits autophagy, a critical step for controlling virus replication.     

Two-layer regulation of PAQR3 on ATG14-linked class III PtdIns3K activation upon glucose starvation

As a central node of the macroautophagy/autophagy process, the BECN1/Beclin1-PIK3C3/VPS34 complex participates in different steps of autophagy by interacting with multiple molecules. The ATG14-associated PIK3C3 complex is involved in autophagy initiation, whereas the UVRAG-associated complex mainly modulates autophagosome maturation and endosome fusion. However, the molecular mechanism that coordinates the sequential execution of the autophagy program remains unknown. We have recently discovered that a Golgi-resident protein, PAQR3, regulates autophagy initiation as it preferentially facilitates the formation of the ATG14-linked PIK3C3 complex instead of the UVRAG-associated complex. Upon glucose starvation, AMPK directly phosphorylates T32 of PAQR3, which is crucial for the activation of the ATG14-associated class III PtdIns3K. Furthermore, Paqr3-deleted mice have a deficiency in exercise-induced autophagy as well as behavioral disorders. Thus, this work not only uncovers the regulatory mechanism of PAQR3 on autophagy initiation, but also provides a potential candidate therapeutic target for neurodegenerative diseases.     

PIK3C3/VPS34, the class III PtdIns 3-kinase,plays indispensable roles in the podocyte

The mammalian homolog of yeast Vps34 (PIK3C3/VPS34) is implicated in the regulation of autophagy, and recent studies have suggested that autophagy is a key mechanism in maintaining the integrity of renal glomerular podocytes. To date, however, the role of PIK3C3 in podocytes has remained unknown. We generated a line of podocyte-specific Pik3c3-knockout (Pik3c3^pdKO/mVps34^pdKO) mice and demonstrated an indispensable role for PIK3C3 in the regulation of intracellular vesicle trafficking and processing to protect the normal cellular metabolism, structure and function of podocytes.     

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy

ABSTRACT

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.

Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2     

Primary cilium-dependent autophagy drafts PIK3C2A to generate PtdIns3P in response to shear stress

ABSTRACT

Primary cilium-dependent macroautophagy/autophagy is induced by the urinary flow in epithelial cells of the kidney proximal tubule. A major physiological outcome of this cascade is the control of cell size. Some components of the ATG machinery are recruited at the primary cilium to generate autophagic structures. Shear stress induced by the liquid flow promotes PtdIns3P synthesis at the primary cilium, and this lipid is required both for ciliogenesis and initiation of autophagy. We showed that PtdIns3P is generated by PIK3C2A, but not by PIK3C3/VPS34, during flow-associated primary cilium-dependent autophagy, in a ULK1-independent manner. Along the same line BECN1 (beclin 1), a partner of PIK3C3 in starvation-induced autophagy, is not recruited at the primary cilium under shear stress. Thus, kidney epithelial cells mobilize different PtdIns 3-kinases, i.e., PIK3C2A or PIK3C3, to produce PtdIns3P in order to initiate autophagy depending on the stimuli (shear stress or starvation).     

The NEDD4-USP13 axis facilitates autophagy via deubiquitinating PIK3C3

ABSTRACT

Macroautophagy/autophagy, an evolutionarily conserved eukaryotic bioprocess, plays an important role in the bulk degradation of intracellular macromolecules, organelles, and invading pathogens. PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) functions as a key protein in autophagy initiation and progression. The activity of PIK3C3 is tightly regulated by multiple post-translational modifications, including ubiquitination, however, the regulatory mechanisms underpinning the reversible deubiquitination of PIK3C3 remain poorly understood. Recently, we identified the E3 ubiquitin ligase NEDD4/NEDD4-1 as a positive regulator of autophagy through decreasing the K48-linked ubiquitination of PIK3C3 by recruiting USP13.     

Autophagy protects PC12 cells against deoxynivalenol toxicity via the Class III PI3K/beclin 1/Bcl-2 pathway

Deoxynivalenol (DON) is a major mycotoxin from the trichothecene family of mycotoxins produced by Fusarium fungi. It can cause a variety of adverse effects on human and farm animal health. Here, we determined the effect of DON on the Class III phosphatidylinositol 3-kinase (PIK3C3)/beclin 1/B cell lymphoma-2 (Bcl-2) pathway in PC12 cells and the relationship between autophagy and apoptosis. The effects of DON were evaluated based on the apoptosis ratio; the typical indicators of autophagy, including cellular morphology, acridine orange- and monodansylcadaverine-labeled vacuoles, green fluorescent protein–microtubule associated protein 1 light chain 3 (LC3) localization, and LC3 immunofluorescence; and the expression of key autophagy-related genes and proteins, that is, PIK3C3, beclin 1, Bcl-2, LC3, and p62. The relationship between autophagy and apoptosis was analyzed by western blot analysis and flow cytometry. DON-induced PC12 cell morphological changes and autophagy significantly. PIK3C3, beclin 1, and LC3 increased in tandem with the DON concentration used; Bcl-2 and p62 expression decreased as DON concentrations increased. Moreover, the PIK3C3/beclin 1/Bcl-2 signaling pathway played a role in DON-induced autophagy. Our findings suggest that DON can induce autophagy by activating the PIK3C3/beclin 1/Bcl-2 signaling pathway and that autophagy may play a positive role in reducing DON-induced apoptosis.     

Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy

Autophagy is a cellular defense response to stress conditions, such as nutrient starvation. The type III phosphatidylinositol (PtdIns) 3-kinase, whose catalytic subunit is PIK3C3/VPS34, plays a critical role in intracellular membrane trafficking and autophagy induction. PIK3C3 forms multiple complexes and the ATG14-containing PIK3C3 is specifically involved in autophagy induction. Mechanistic target of rapamycin (MTOR) complex 1, MTORC1, is a key cellular nutrient sensor and integrator to stimulate anabolism and inhibit catabolism. Inactivation of TORC1 by nutrient starvation plays a critical role in autophagy induction. In this report we demonstrated that MTORC1 inactivation is critical for the activation of the autophagy-specific (ATG14-containing) PIK3C3 kinase, whereas it has no effect on ATG14-free PIK3C3 complexes. MTORC1 inhibits the PtdIns 3-kinase activity of ATG14-containing PIK3C3 by phosphorylating ATG14, which is required for PIK3C3 inhibition by MTORC1 both in vitro and in vivo. Our data suggest a mechanistic link between amino acid starvation and autophagy induction via the direct activation of the autophagy-specific PIK3C3 kinase.     

Effects of neuronal PIK3C3/Vps34 deletion on autophagy and beyond

PIK3C3/Vps34 plays important roles in the endocytic and autophagic pathways, both of which are essential for maintaining neuronal integrity. However, it is unclear how inactivating PIK3C3 may affect neuronal endosomal versus autophagic processes in vivo. We generated a conditional null allele of the Pik3c3 gene in mouse, and specifically deleted it in postmitotic sensory neurons. Subsequent analyses reveal several interesting and surprising findings.Key words: PIK3C3/Vps34, ATG7, sensory neurons, neurodegeneration, autophagy, abnormal endosomePIK3C3 (commonly known as Vps34) is the class III phosphatidylinositol 3-kinase (PtdIns3K) that specifically catalyzes the formation of phosphatidylinositol-3-phosphate (PtdIns3P). It is the only PtdIns3K that is conserved from lower eukaryotes to mammals, and represents the most ancient form of PtdIns3Ks. Studies in invertebrate organisms as well as mammalian cell lines show that PIK3C3/Vps34 regulates multiple aspects of both the endocytic and the autophagic pathways. On one hand, PIK3C3 is important for the progression of early endosome to late endosome, and the biogenesis of multivesicular bodies. On the other hand, PIK3C3 is critical for the initiation of autophagosome formation. A chemical inhibitor of PIK3C3, 3-MA, has been commonly used as a specific inhibitor for autophagy. The distinct functions of PIK3C3 are thought to be carried out by at least two different PIK3C3 complexes. In yeast, complex I (Vps34, Vps15, Atg6 and Atg14) is involved in autophagy, whereas complex II (Vps34, Vps15, Atg6 and Vps38) functions in the vacuolar protein sorting process. In mammals, the homologue of complex I (PIK3C3, p150, Beclin 1 and Atg14L) activates autophagy, whereas the homologue of complex II (PIK3C3, p150, Beclin 1 and UVRAG/Vps38) regulates endocytic trafficking.To characterize the in vivo function of PIK3C3 in mammals, we generated a conditional allele of the Pik3c3 gene in mouse and specifically deleted it in postmitotic sensory neurons (Pik3c3-cKO mouse). We focused our analyses on sensory neurons because Pik3c3 is most abundantly expressed in these neurons. Detailed analyses of the sensory ganglia in the knockout mice reveal rapid but differential neurodegenerations of different types of sensory neurons within a few days after birth. Large-diameter myelinated mechanosensory and proprioceptive neurons undergo fast degeneration, whereas mutant small-diameter unmyelinated nociceptive neurons degenerate slower and survive longer.Interestingly, the large-diameter Pik3c3-deleted neurons rapidly accumulate ubiquitin-positive aggregates as well as numerous enlarged vesicles, which are likely abnormal endosomes. The accumulation of enlarged vesicles not only sequesters the cellular membrane source, but also could create trafficking jams that block the transport of prosurvival signals and/or material and organelles, and thus may underlie the rapid demise of large neurons. By contrast, the small-diameter Pik3c3-deleted neurons contain a limited number of vacuoles but gradually build up lysosome- like organelles. The marked increase of lysosomes seems to be more tolerable by neurons, but the mechanism underlying this phenotype is unclear. It could represent a protective and homeostatic response of neurons challenged with stress and insults to their endomembrane system. Alternatively, since sorting of many lysosomal proteins requires PtdIns3P, this phenotype may also result from a build-up of nonfunctional lysosomes as was the case in cathepsin B and L knockout mice. It is also unclear why two types of sensory neurons respond differently to a universal insult. One speculative explanation is that the large-diameter neurons are constantly activated under normal physiological conditions by touch and body movement and thus they contain more active endocytic and membrane trafficking processes; whereas small-diameter pain-sensing neurons are normally not activated and have less endocytic events. These differences might allow the two types of neurons to respond differently to PIK3C3 deletion.We further show that the fast and differential degeneration phenotypes in the Pik3c3-cKO mice are caused primarily by a disruption in the endosomal but not the autophagic pathway. This is validated by comparing the neuronal phenotypes of Pik3c3-cKO mice with those of Atg7-cKO mice, in which the autophagy-specific gene Atg7 is deleted using the same sensory neuron-specific cre driver. Disrupting autophagy leads to a slow degeneration of all types of sensory neurons over a period of several months, and formation of very large intracellular inclusion bodies in all sensory neurons. No increase of lysosomes or accumulation of enlarged vesicles is observed. The completely distinct phenotypes observed in Atg7-cKO versus Pik3c3-cKO mice suggest that inactivation of PIK3C3 primarily disrupts the endosomal pathway rather than inhibiting autophagy (at least in neurons). It calls into attention that care needs to be taken to interpret the results of using PIK3C3 inhibitors such as 3-MA as autophagy-specific inhibitors.The most surprising finding is the existence and activation of a noncanonical, PIK3C3-independent macroautophagy pathway in small-diameter Pik3c3-mutant neurons. Although PIK3C3 is traditionally viewed as indispensable for autophagy initiation, several recent studies suggest a possible PIK3C3-independent autophagy pathway in various cell lines and in Drosophila. We show that this noncanonical autophagy pathway can occur in sensory neurons in vivo using three different assays: crossing Pik3c3-cKO mice to the GFP-LC3 reporter line, western blot analyses of LC3 isoforms, and performing autophagy flux experiments. Interestingly, analyses of Pik3c3/Atg7 double-mutant neurons indicate that this alternative autophagosome initiation pathway still requires ATG7 and hence the conventional conjugation systems. Therefore, this non-canonical autophagy is distinct from the newly reported ATG5/ATG7-independent but PIK3C3-dependent autophagy. We speculate that activation of this PIK3C3-independent autophagy in small-diameter mutant neurons is part of the reason for their longer survival period.The molecular mechanism underlying the PIK3C3-independent autophagosome formation is unknown. It is possible that PtdIns3P can be generated at a low level on the membrane of pre-autophagosomes/phagophores by salvage pathways using other lipid kinases or phosphatases. Alternatively, other mechanisms may direct the formation of the crescent-shaped double membrane structures. For instance, asymmetric insertion into the membrane of proteins with amphipathic helices can induce membrane curvature; BAR domain-containing proteins can also detect and facilitate the formation of curved membrane structures. Thus, these types of proteins might potentially be recruited to nucleate the formation of pre-autophagosomes in the absence of PIK3C3. Finally, the role of this PIK3C3-independent autophagy under normal physiological conditions in vivo needs to be explored.     

DIRAS3 regulates the autophagosome initiation complex in dormant ovarian cancer cells

DIRAS3 is an imprinted tumor suppressor gene that is downregulated in 60% of human ovarian cancers. Re-expression of DIRAS3 at physiological levels inhibits proliferation, decreases motility, induces autophagy, and regulates tumor dormancy. Functional inhibition of autophagy with choroquine in dormant xenografts that express DIRAS3 significantly delays tumor regrowth after DIRAS3 levels are reduced, suggesting that autophagy sustains dormant ovarian cancer cells. This study documents a newly discovered role for DIRAS3 in forming the autophagosome initiation complex (AIC) that contains BECN1, PIK3C3, PIK3R4, ATG14, and DIRAS3. Participation of BECN1 in the AIC is inhibited by binding of BECN1 homodimers to BCL2. DIRAS3 binds BECN1, disrupting BECN1 homodimers and displacing BCL2. Binding of DIRAS3 to BECN1 increases the association of BECN1 with PIK3C3 and ATG14, facilitating AIC activation. Amino acid starvation of cells induces DIRAS3 expression, reduces BECN1-BCL2 interaction and promotes autophagy, whereas DIRAS3 depletion blocks amino acid starvation-induced autophagy. In primary ovarian cancers, punctate expression of DIRAS3, BECN1, and the autophagic biomarker MAP1LC3 are highly correlated (P < 0.0001), underlining the clinical relevance of these mechanistic studies. Punctate expression of DIRAS3 and MAP1LC3 was detected in only 21–23% of primary ovarian cancers but in 81–84% of tumor nodules found on the peritoneal surface at second-look operations following primary chemotherapy. This reflects a 4-fold increase (P < 0.0001) in autophagy between primary disease and post-treatment recurrence. We suggest that DIRAS3 not only regulates the AIC, but induces autophagy in dormant, nutrient-deprived ovarian cancer cells that remain after conventional chemotherapy, facilitating their survival.     

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.     

A fine-tuning mechanism underlying self-control for autophagy: deSUMOylation of BECN1 by SENP3

ABSTRACT

The roles of SUMOylation and the related enzymes in autophagic regulation are unclear. Based on our previous studies that identified the SUMO2/3-specific peptidase SENP3 as an oxidative stress-responsive molecule, we investigated the correlation between SUMOylation and macroautophagy/autophagy. We found that Senp3^± mice showed increased autophagy in the liver under basal and fasting conditions, compared to Senp3^+/+ mice. We constructed a liver-specific senp3 knockout mouse; these Senp3-deficient liver tissues showed increased autophagy as well. Autophagic flux was accelerated in hepatic and other cell lines following knockdown of SENP3, both before and after the cells underwent starvation in the form of the serum and amino acid deprivation. We demonstrated that BECN1/beclin 1, the core molecule of the BECN1-PIK3C3 complex, could be SUMO3-conjugated by PIAS3 predominantly at K380 and deSUMOylated by SENP3. The basal SUMOylation of BECN1 was increased upon cellular starvation, which enhanced autophagosome formation by facilitating BECN1 interaction with other complex components UVRAG, PIK3C3 and ATG14, thus promoting PIK3C3 activity. In contrast, SENP3 deSUMOylated BECN1, which impaired BECN1-PIK3C3 complex formation or stability to suppress the PIK3C3 activity. DeSUMOylation of BECN1 restrained autophagy induction under basal conditions and especially upon starvation when SENP3 had accumulated in response to the increased generation of reactive oxygen species. Thus, while reversible SUMOylation regulated the degree of autophagy, SENP3 provided an intrinsic overflow valve for fine-tuning autophagy induction.     

Selective autophagy inhibition through disruption of the PIK3C3-containing complex I

ABSTRACT

The PIK3C3/VPS34-containing phosphatidylinositol 3-kinase (PtdIns3K) initiation complex (complex I) is necessary for macroautophagy/autophagy initiation and is comprised of PIK3R4/VPS15-PIK3C3/VPS34-BECN1-ATG14, while the endosomal trafficking complex (complex II) is necessary for vesicle trafficking and is comprised of PIK3R4/VPS15-PIK3C3/VPS34-BECN1-UVRAG. This composition difference was exploited to identify novel and specific autophagy inhibitors that disrupted the BECN1-ATG14 protein-protein interaction, without affecting vesicle trafficking. A cellular NanoBRET assay was implemented to identify these inhibitors, and one compound was able to successfully disrupt the BECN1-ATG14 interaction and inhibit autophagy, with limited impact on vesicle trafficking. These results reveal the first protein-protein interaction inhibitor targeting the autophagy initiation machinery and demonstrate the viability of targeting protein-protein interactions for the discovery of autophagy-specific modulators.     

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.     

NRBF2 is involved in the autophagic degradation process of APP-CTFs in Alzheimer disease models

Alzheimer disease (AD) is the most common neurodegenerative disease characterized by the deposition of amyloid plaque in the brain. The autophagy-associated PIK3C3-containing phosphatidylinositol 3-kinase (PtdIns3K) complex has been shown to interfere with APP metabolism and amyloid beta peptide (Aβ) homeostasis via poorly understood mechanisms. Here we report that NRBF2 (nuclear receptor binding factor 2), a key component and regulator of the PtdIns3K, is involved in APP-CTFs homeostasis in AD cell models. We found that NRBF2 interacts with APP in vivo and its expression levels are reduced in hippocampus of 5XFAD AD mice; we further demonstrated that NRBF2 overexpression promotes degradation of APP C-terminal fragments (APP-CTFs), and reduces Aβ_1–40 and Aβ_1-42 levels in human mutant APP-overexpressing cells. Conversely, APP-CTFs, Aβ_1–40 and Aβ_1-42 levels were increased in Nrbf2 knockdown or nrbf2 knockout cells. Furthermore, NRBF2 positively regulates autophagy in neuronal cells and NRBF2-mediated reduction of APP-CTFs levels is autophagy dependent. Importantly, nrbf2 knockout attenuates the recruitment of APP and APP-CTFs into phagophores and the sorting of APP and APP-CTFs into endosomal intralumenal vesicles, which is accompanied by the accumulation of the APP and APP-CTFs into RAB5-positive early endosomes. Collectively, our results reveal the potential connection between NRBF2 and the AD-associated protein APP by showing that NRBF2 plays an important role in regulating degradation of APP-CTFs through modulating autophagy.     

Role of class III phosphatidylinositol 3-kinase during programmed nuclear death of Tetrahymena thermophila

Programmed nuclear death (PND) in the ciliate protozoan Tetrahymena thermophila is a novel type of autophagy that occurs during conjugation, in which only the parental somatic macronucleus is destined to die and is then eliminated from the progeny cytoplasm. Other coexisting nuclei, however, such as new micro- and macronuclei are unaffected. PND starts with condensation in the nucleus followed by apoptotic DNA fragmentation, lysosomal acidification, and final resorption. Because of the peculiarity in the process and the absence of some ATG genes in this organism, the mechanism of PND has remained unclear. In this study, we focus on the role of class III phosphatidylinositol 3-kinase (PtdIns3K, corresponding to yeast Vps34) in order to identify central regulators of PND. We identified the sole Tetrahymena thermophila ortholog (TtVPS34) to yeast Vps34 and human PIK3C3 (the catalytic subunit of PtdIns3K), through phylogenetic analysis, and generated the gene knockdown mutant for functional analysis. Loss of TtVPS34 activity prevents autophagosome formation on the parental macronucleus, and this nucleus escapes from the lysosomal pathway. In turn, DNA fragmentation and final resorption of the nucleus are drastically impaired. These phenotypes are similar to the situation in the ATG8Δ mutants of Tetrahymena, implying an inextricable link between TtVPS34 and TtATG8s in controlling PND as well as general macroautophagy. On the other hand, TtVPS34 does not appear responsible for the nuclear condensation and does not affect the progeny nuclear development. These results demonstrate that TtVPS34 is critically involved in the nuclear degradation events of PND in autophagosome formation rather than with an involvement in commitment to the death program.     

DIRAS3 regulates the autophagosome initiation complex in dormant ovarian cancer cells

DIRAS3 is an imprinted tumor suppressor gene that is downregulated in 60% of human ovarian cancers. Re-expression of DIRAS3 at physiological levels inhibits proliferation, decreases motility, induces autophagy, and regulates tumor dormancy. Functional inhibition of autophagy with choroquine in dormant xenografts that express DIRAS3 significantly delays tumor regrowth after DIRAS3 levels are reduced, suggesting that autophagy sustains dormant ovarian cancer cells. This study documents a newly discovered role for DIRAS3 in forming the autophagosome initiation complex (AIC) that contains BECN1, PIK3C3, PIK3R4, ATG14, and DIRAS3. Participation of BECN1 in the AIC is inhibited by binding of BECN1 homodimers to BCL2. DIRAS3 binds BECN1, disrupting BECN1 homodimers and displacing BCL2. Binding of DIRAS3 to BECN1 increases the association of BECN1 with PIK3C3 and ATG14, facilitating AIC activation. Amino acid starvation of cells induces DIRAS3 expression, reduces BECN1-BCL2 interaction and promotes autophagy, whereas DIRAS3 depletion blocks amino acid starvation-induced autophagy. In primary ovarian cancers, punctate expression of DIRAS3, BECN1, and the autophagic biomarker MAP1LC3 are highly correlated (P < 0.0001), underlining the clinical relevance of these mechanistic studies. Punctate expression of DIRAS3 and MAP1LC3 was detected in only 21–23% of primary ovarian cancers but in 81–84% of tumor nodules found on the peritoneal surface at second-look operations following primary chemotherapy. This reflects a 4-fold increase (P < 0.0001) in autophagy between primary disease and post-treatment recurrence. We suggest that DIRAS3 not only regulates the AIC, but induces autophagy in dormant, nutrient-deprived ovarian cancer cells that remain after conventional chemotherapy, facilitating their survival.     

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.