Autophagy is a degradative process that recycles long-lived and faulty cellular components. It is linked to many diseases and is required for normal development. ULK1, a mammalian serine/threonine protein kinase, plays a key role in the initial stages of autophagy, though the exact molecular mechanism is unknown. Here we report identification of a novel protein complex containing ULK1 and two additional protein factors, FIP200 and ATG13, all of which are essential for starvationinduced autophagy. Both FIP200 and ATG13 are critical for correct localization of ULK1 to the pre-autophagosome and stability of ULK1 protein. Additionally, we demonstrate by using both cellular experiments and a de novo in vitro reconstituted reaction that FIP200 and ATG13 can enhance ULK1 kinase activity individually but both are required for maximal stimulation. Further, we show that ATG13 and ULK1 are phosphorylated by the mTOR pathway in a nutrient starvation-regulated manner, indicating that the ULK1⅐ATG13⅐FIP200 complex acts as a node for integrating incoming autophagy signals into autophagosome biogenesis.Macroautophagy (herein referred to as autophagy) is a catabolic process whereby long-lived proteins and damaged organelles are shuttled to lysosomes for degradation. This process is conserved in all eukaryotes. Under normal growth conditions a housekeeping level of autophagy exists. Under stress, such as nutrient starvation, autophagy is strongly induced resulting in the engulfment of cytosolic components and organelles in specialized double-membrane structures termed autophagosomes. Following fusion of the outer autophagosomal membrane with lysosomes, the inner membrane and its cytoplasmic cargo are degraded and recycled (1-3). Recent work has implicated autophagy in many disease pathologies, including cancer, neurodegeneration, as well as in eliminating intracellular pathogens (4 -8).The morphology of autophagy was first described in mammalian cells over 50 years ago (9). However, it is only recently through yeast genetic screens, that multiple autophagy-related (ATG) genes have been identified (10 -12). The yeast ATG proteins have been classified into four major groups: the Atg1 protein kinase complex, the Vps34 phosphatidylinositol 3-phosphate kinase complex, the Atg8/Atg12 conjugation systems, and the Atg9 recycling complex (13). Even though many ATG genes are now known, most of which have functional homologs in mammalian cells (14, 15), the molecular mechanism by which they sense the initial triggers and subsequently dictate autophagy-specific intracellular membrane events is far from understood.In yeast, one of the earliest autophagy-specific events is believed to involve the Atg1 protein kinase complex. Atg1 is a serine/threonine protein kinase and a key autophagy-regulator (16). Atg1 is complexed to at least two other proteins during autophagy, Atg13 and Atg17, both of which are required for normal Atg1 function and autophagosome generation (17-19). Classical signaling pathways such as the cAMP-dependent kinase (PKA) pat...

The tumor suppressor PTEN, a critical regulator for multiple cellular processes, is mutated or deleted frequently in various human cancers. Subtle reductions in PTEN expression levels have profound impacts on carcinogenesis. Here we show that PTEN level is regulated by ubiquitin-mediated proteasomal degradation, and purified its ubiquitin ligase as HECT-domain protein NEDD4-1. In cells NEDD4-1 negatively regulates PTEN stability by catalyzing PTEN polyubiquitination. Consistent with the tumor-suppressive role of PTEN, overexpression of NEDD4-1 potentiated cellular transformation. Strikingly, in a mouse cancer model and multiple human cancer samples where the genetic background of PTEN was normal but its protein levels were low, NEDD4-1 was highly expressed, suggesting that aberrant upregulation of NEDD4-1 can posttranslationally suppress PTEN in cancers. Elimination of NEDD4-1 expression inhibited xenotransplanted tumor growth in a PTEN-dependent manner. Therefore, NEDD4-1 is a potential proto-oncogene that negatively regulates PTEN via ubiquitination, a paradigm analogous to that of Mdm2 and p53.

Autophagy is a cellular catabolic process critical for cell viability and homeostasis. Inhibition of mammalian target of rapamycin (mTOR) complex-1 (mTORC1) activates autophagy. A puzzling observation is that amino acid starvation triggers more rapid autophagy than pharmacological inhibition of mTORC1, although they both block mTORC1 activity with similar kinetics. Here we find that in addition to mTORC1 inactivation, starvation also causes an increase in phosphatase activity toward ULK1, an mTORC1 substrate whose dephosphorylation is required for autophagy induction. We identify the starvation-stimulated phosphatase for ULK1 as the PP2A-B55α complex. Treatment of cells with starvation but not mTORC1 inhibitors triggers dissociation of PP2A from its inhibitor Alpha4. Furthermore, pancreatic ductal adenocarcinoma cells (PDACs), whose growth depends on high basal autophagy, possess stronger basal phosphatase activity toward ULK1 and require ULK1 for sustained anchorage-independent growth. Taken together, concurrent mTORC1 inactivation and PP2A-B55α stimulation fuel ULK1-dependent autophagy.

Analogs of vitamin E (tocols) are under development as radioprophylactic agents because of their high efficacy and lack of toxicity. Gamma-tocotrienol (GT3) is of particular interest because, in addition to being an antioxidant, it also inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase and accumulates to greater extent in endothelial cells than other tocols. We addressed in vivo whether HMG-CoA reductase inhibition contributes to the radioprotection conferred by GT3. Groups of mice were treated with vehicle, mevalonate (the product of the reaction catalyzed by HMG-CoA reductase), GT3 alone or GT3 in combination with mevalonate. Lethality and standard parameters of injury to the hematopoietic, intestinal and vascular/ endothelial systems were assessed after exposure to total-body irradiation. GT3 improved postirradiation survival and decreased radiation-induced vascular oxidative stress, an effect that was reversible by mevalonate. GT3 also enhanced hematopoietic recovery, reduced intestinal radiation injury, and accelerated the recovery of soluble markers of endothelial function. These parameters were not reversed by mevalonate co-administration. Our data confirm GT3's radioprophylactic properties against hematopoietic injury and, for the first time, demonstrate benefits in terms of protection against gastrointestinal and vascular injury. The radioprotective efficacy of GT3 against vascular injury is related to its properties as an HMG-CoA reductase inhibitor.

Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.

The heterostructures have the HER and OER in water-splitting taking place separately on the two components with the STH energy conversion efficiencies up to 10.5%. The Se-vacancy makes the two reactions occur spontaneously in pure water.

Real-time quantitative polymerase chain reaction (RT-qPCR), a reliable technique for quantifying gene expression, requires stable reference genes to normalize its data. Salicornia europaea, a stem succulent halophyte with remarkable salt resistance and high capacity for ion accumulation, has not been investigated with regards to the selection of appropriate reference genes for RT-qPCR. In this study, the expression of 11 candidate reference genes, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), Actin, α-Tub (α-tubulin), β-Tub (β-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), UBQ (Polyubiquitin), CYP (Cyclophilin), TIP41 (TIP41-like protein), CAC (Clathrin adaptor complexes), and DNAJ (DnaJ-like protein), was analyzed in S. europaea samples, which were classified into groups according to various abiotic stresses (NaCl, nitrogen, drought, cold and heat), tissues and ages. Three commonly used software programs (geNorm, NormFinder and BestKeeper) were applied to evaluate the stability of gene expression, and comprehensive ranks of stability were generated by aggregate analysis. The results show that the relatively stable genes for each group are the following: (1) CAC and UBC for whole samples; (2) CAC and UBC for NaCl stress; (3) Actin and α-Tub for nitrogen treatment; (4) Actin and GAPDH for drought stress; (5) α-Tub and UBC for cold stress; (6) TIP41 and DNAJ for heat stress; (7) UBC and UBQ for different tissues; and (8) UBC and Actin for various developmental stages. These genes were validated by comparing transcriptome profiles. Using two stable reference genes was recommended in the normalization of RT-qPCR data. This study identifies optimal reference genes for RT-qPCR in S. europaea, which will benefit gene expression analysis under these conditions.

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