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.
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.
Liver X receptor (LXR)α is a nuclear receptor that responds to oxysterols and cholesterol overload by stimulating cholesterol efflux, transport, conversion to bile acids, and excretion. LXRα binds to and is regulated by synthetic (T-0901317, GW3695) and endogenous (oxysterols) ligands. LXRα activity is also modulated by FAs, but the ligand binding specificity of FA and acyl-CoA derivatives for LXRα remains unknown. We investigated whether LXRα binds FA or FA acyl-CoA with affinities that mimic in vivo concentrations, examined the effect of FA chain length and the degree of unsaturation on binding, and investigated whether FAs regulate LXRα activation. Saturated medium-chain FA (MCFA) displayed binding affinities in the low nanomolar concentration range, while long-chain fatty acyl-CoA did not bind or bound weakly to LXRα. Circular dichroic spectra and computational docking experiments confirmed that MCFA bound to the LXRα ligand binding pocket similar to the known synthetic agonist of LXRα (T0901317), but with limited change to the conformation of the receptor. Transactivation assays showed that MCFA activated LXRα, whereas long-chain FA caused no effect. Our results suggest that LXRα functions as a receptor for saturated FA or acyl-CoA of C10 and C12 in length.
Early detection of lung cancer (LC) significantly increases the likelihood of successful treatment and improves LC survival rates. Currently, screening (mainly low-dose CT scans) is recommended for individuals at high risk. However, the recent increase in the number of LC cases unrelated to the well-known risk factors, and the high false-positive rate of low-dose CT, indicate a need to develop new, non-invasive methods for LC detection. Therefore, we evaluated the use of differential scanning calorimetry (DSC) for LC patients’ diagnosis and predicted survival. Additionally, by applying mass spectrometry, we investigated whether changes in O- and N-glycosylation of plasma proteins could be an underlying mechanism responsible for observed differences in DSC curves of LC and control subjects. Our results indicate selected DSC curve features could be useful for differentiation of LC patients from controls with some capable of distinction between subtypes and stages of LC. DSC curve features also correlate with LC patients’ overall/progression free survival. Moreover, the development of classification models combining patients’ DSC curves with selected plasma protein glycosylation levels that changed in the presence of LC could improve the sensitivity and specificity of the detection of LC. With further optimization and development of the classification method, DSC could provide an accurate, non-invasive, radiation-free strategy for LC screening and diagnosis.
Invertase catalyzes the hydrolysis of sucrose to glucose and fructose. Invertase from Saccharomyces cerevisiae has been used in studies of the enzyme catalytic mechanism for more than a century. Isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) were used to determine the kinetics and physical characteristics of invertase, respectively. Two subspecies of invertase with unique melting points of ∼57 and ∼65 °C are identified using DSC. The rate of invertase-catalyzed sucrose hydrolysis was measured with ITC. ITC has the distinct advantage of directly measuring the rate of reaction, which makes it easy to determine when the enzyme exhibits maximal activity. ITC revealed a previously unknown significant delay after the injections of sucrose before invertase reaches maximal activity. The delay in maximum activity was decreased by a statistically significant (p > 0.05) 85% by an increase in invertase concentration from 1 to 47 μg/mL. Additionally, increasing the temperature of the reaction mixture from 25 to 55 °C resulted in a 66% decrease in the delay to reach maximum activity. However, increasing the sucrose concentration had no effect of the delay in maximum activity. These data are consistent with a change in the conformational or oligomeric state preceding the enzyme reaching full catalytic activity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.