Macroautophagy is an intracellular, vesicle-mediated mechanism for the sequestration and ultimate lysosomal degradation of cytoplasmic proteins, organelles and macromolecules. The macroautophagy process and many of the autophagy-specific (Atg) proteins are remarkably well conserved in higher eukaryotes. In yeast, the Atg1 kinase complex includes Atg1, Atg13, Atg17, and at least four other interacting proteins, some of which are phosphorylated in a TOR-dependent manner, placing the Atg1 signaling complex downstream of a major nutrient-sensing pathway. Atg1 orthologs, including mammalian unc-51-like kinase 1 (ULK1), have been identified in higher eukaryotes and have been functionally linked to autophagy. This suggests that other components of the Atg1 complex exist in higher eukaryotes. Recently, a putative human Atg13 ortholog, FLJ20698, was identified by gapped-BLAST analysis. We show here that FLJ20698 (Atg13) is a ULK1-interacting phosphoprotein that is essential for macroautophagy. Furthermore, we identify a novel, human Atg13-interacting protein, FLJ11773, which we have termed Atg101. Atg101 is essential for autophagy and interacts with ULK1 in an Atg13-dependent manner. Additionally, we present evidence that intracellular localization of the ULK1 complex is regulated by nutrient conditions. Finally, we demonstrate that Atg101 stabilizes the expression of Atg13 in the cell, suggesting that Atg101 contributes to Atg13 function by protecting Atg13 from proteasomal degradation. Therefore, the identification of the novel protein, Atg101, and the validation of Atg13 and Atg101 as ULK1-interacting proteins, suggests an Atg1 complex is involved in the induction of macroautophagy in mammalian cells.

nuclear mechanistic links between sugar and fatty acid regulation remain elusive. Recent evidence suggests that peroxisome proliferator-activated receptors (PPARs), ligand-activated transcription factors belonging to the nuclear hormone receptor superfamily, play a central role in energy homeostasis by initiating transcription of multiple genes involved in fatty acid oxidation and glucose metabolism. In liver, PPAR ␣ induces transcription of genes involved in long-chain fatty acid (LCFA) uptake and transport (e.g., liver fatty acid binding protein [L-FABP]), fatty acid degradation by ␤ -oxidation, and lipoprotein metabolism ( 4, 5 ). Thus, activation of PPAR ␣ induces transcription of a number of lipid metabolic proteins whose abnormal regulation may contribute to diabetes and obesity.Although it is known that exogenous LCFAs activate PPAR ␣ and that certain PPAR ␣ -targeting drugs (fi brates) used in cardiovascular and diabetes therapy enhance glucose uptake, increase fatty acid metabolism, and improve insulin sensitivity ( 6, 7 ), the identity of endogenous, highaffi nity PPAR ␣ ligands has proven more elusive. Only recently was it shown that PPAR ␣ exhibits high affi nity for unsaturated (but not saturated) LCFA ( 8, 9 ) and all examined CoA thioesters of LCFA (LCFA-CoA) ( 9, 10 ). Upon binding these lipids, PPAR ␣ undergoes a conformational change and increased activation, consistent with LCFA and LCFA-CoA being endogenous ligands. The latter is especially likely as nuclear concentrations of LCFA and LCFA-CoA are in the range of PPAR ␣ affi nity for these ligands ( 11,12 ). New fi ndings show that glucose is also an Elevated serum fatty acids and sugars are signifi cant cardiovascular risk factors in diabetes, obesity, and metabolic syndrome ( 1-4 ). While these nutrients regulate transcription of multiple genes involved in their own metabolism,

Background: Differential Scanning Calorimetry (DSC) is a technique traditionally used to study thermally induced macromolecular transitions, and it has recently been proposed as a novel approach for diagnosis and monitoring of several diseases. We report a pilot study applying Thermal Liquid Biopsy (TLB, DSC thermograms of plasma samples) as a new clinical approach for diagnostic assessment of melanoma patients. Methods: Multiparametric analysis of DSC thermograms of patient plasma samples collected during treatment and surveillance (63 samples from 10 patients) were compared with clinical and diagnostic imaging assessment to determine the utility of thermograms for diagnostic assessment in melanoma. Nine of the ten patients were stage 2 or 3 melanoma subjects receiving adjuvant therapy after surgical resection of their melanomas. The other patient had unresectable stage 4 melanoma and was treated with immunotherapy. Two reference groups were used: (A) 36 healthy subjects and (B) 13 samples from 8 melanoma patients who had completed successful surgical management of their disease and were determined by continued clinical assessment to have no evidence of disease. Results: Plasma thermogram analysis applied to melanoma patients generally agrees with clinical evaluation determined by physical assessment or diagnostic imaging (~80% agreement). No false negatives were obtained from DSC thermograms. Importantly, this methodology was able to detect changes in disease status before it was identified clinically. Conclusions: Thermal Liquid Biopsy could be used in combination with current clinical assessment for the earlier detection of melanoma recurrence and metastasis. General Significance: TLB offers advantages over current diagnostic techniques (PET/CT imaging), limited in frequency by radiation burden and expense, in providing a minimally-invasive, low-risk, low-cost clinical test for more frequent personalized patient monitoring to assess recurrence and facilitate clinical decision-making.

No abstract

Diluted (1%) plasma induces migration of malignant cell lines much more strongly than potent pro-metastatic factors. To characterize the factor(s) present in diluted plasma responsible for this phenomenon we performed i) heat inactivation, ii) dialysis, iii) proteinase K treatment, and iv) molecular size filtration studies. We found that this remarkable pro-migratory activity of diluted normal plasma is associated with a ~50–100-kD protein that interacts with GαI protein-coupled receptors and activates p42/44 MAPK and AKT signaling in target cells. Since this pro-migratory activity of 1% plasma decreases at higher plasma concentrations (> 20%), but is retained in serum, we hypothesized that fibrinogen may be involved as a chaperone of the protein(s). To identify the pro-migratory protein(s) present in diluted plasma and fibrinogen-depleted serum, we performed gel filtration and hydrophobic interaction chromatography followed by mass spectrometry analysis. We identified several putative protein candidates that were further tested in in vitro experiments. We found that this pro-migratory factor chaperoned by fibrinogen is vitronectin, which activates uPAR, and that this effect can be inhibited by fibrinogen. These results provide a novel mechanism for the metastasis of cancer cells to lymphatics and body cavities, in which the concentration of fibrinogen is low, and thus suggests that free vitronectin stimulates migration of tumor cells.