Specific molecular interaction between the insulin receptor and a D product of MHC class I

The density of MHC class I was determined on a murine thymoma cell line (R1), an H-2 negative variant (R1E), and R1E-derived cell lines in which H-2 expression was restored by transfection of various MHC class I genes (Db, Kb, and truncated Db) and/or a beta-2-microglobulin gene (beta 2-m; B2). Appreciable MHC class I expression was found on R1 cells and on the variants in which MHC class I expression was restored by transfection of Db/beta 2-m or Kb/beta 2-m genes. Only approximately 20% difference was observed between the number of Db molecules and Kb molecules on the R1E/B2/Db and on R1E/B2/Kb, respectively. However, specific insulin binding was significantly different between these lines. By using a computer assisted curve fitting program, the insulin binding data for R1 and R1E/B2/Db cell lines best fitted a two-site model (K approximately 6 x 10(-9) M for high-affinity sites and a 2 to 3 x 10(-7) M for low-affinity sites), whereas all other lines only expressed one type of insulin binding site. These sites were unrelated to IGF-I and IGF-II receptors. Cross-linking of 125I-labeled insulin demonstrated specific binding of the ligand to a Mr approximately 130,000 dalton band in all lines. In the R1E/B2/Db cells, insulin also cross-linked to cell membrane molecules with Mr approximately 48,000 and approximately 60,000 Da, which were identified by immunoprecipitation to be the H chain of MHC class I and the heavy chain of MHC class I plus beta 2-m, respectively. It is concluded that the insulin receptors in the cell membrane interact specifically with D-products of MHC class I and that class I molecules of MHC may have a crucial role in insulin receptor expression. This may reflect a more general nonimmunologic role of MHC class I.     

Polarized transport of MHC class II molecules in Madin-Darby canine kidney cells is directed by a leucine-based signal in the cytoplasmic tail of the beta-chain.

MHC class II molecules are found on the basolateral plasma membrane domain of polarized epithelial cells, where they can present Ag to intraepithelial lymphocytes in the vascular space. We have analyzed the sorting information required for efficient intracellular localization and polarized distribution of MHC class II molecules in stably transfected Madin-Darby canine kidney cells. These cells were able to present influenza virus particles to HLA-DR1-restricted T cell clones. Wild-type MHC class II molecules were located on the basolateral plasma membrane domain, in basolateral early endosomes, and in late multivesicular endosomes, the latter also containing the MHC class II-associated invariant chain and an HLA-DM fusion protein. A phenylalanine-leucine residue within the cytoplasmic tail of the beta-chain was required for basolateral distribution, efficient internalization, and localization of the MHC class II molecules to basolateral early endosomes. However, distribution to apically located, late multivesicular endosomes did not depend on signals in the class II cytoplasmic tails as both wild-type class II molecules and mutant molecules lacking the phenylalanine-leucine motif were found in these compartments. Our results demonstrate that sorting information in the tails of class II dimers is an absolute requirement for their basolateral surface distribution and intracellular localization.     

Deubiquitinase inhibition by WP1130 leads to ULK1 aggregation and blockade of autophagy

Autophagy represents an intracellular degradation process which is involved in both regular cell homeostasis and disease settings. In recent years, the molecular machinery governing this process has been elucidated. The ULK1 kinase complex consisting of the serine/threonine protein kinase ULK1 and the adapter proteins ATG13, RB1CC1, and ATG101, is centrally involved in the regulation of autophagy initiation. This complex is in turn regulated by the activity of different nutrient- or energy-sensing kinases, including MTOR, AMPK, and AKT. However, next to phosphorylation processes it has been suggested that ubiquitination of ULK1 positively influences ULK1 function. Here we report that the inhibition of deubiquitinases by the compound WP1130 leads to increased ULK1 ubiquitination, the transfer of ULK1 to aggresomes, and the inhibition of ULK1 activity. Additionally, WP1130 can block the autophagic flux. Thus, treatment with WP1130 might represent an efficient tool to inhibit the autophagy-initiating ULK1 complex and autophagy.     

Expression of a ULK1/2 binding-deficient ATG13 variant can partially restore autophagic activity in ATG13-deficient cells

Autophagy describes an intracellular process responsible for the lysosome-dependent degradation of cytosolic components. The ULK1/2 complex comprising the kinase ULK1/2 and the accessory proteins ATG13, RB1CC1, and ATG101 has been identified as a central player in the autophagy network, and it represents the main entry point for autophagy-regulating kinases such as MTOR and AMPK. It is generally accepted that the ULK1 complex is constitutively assembled independent of nutrient supply. Here we report the characterization of the ATG13 region required for the binding of ULK1/2. This binding site is established by an extremely short peptide motif at the C terminus of ATG13. This motif is mandatory for the recruitment of ULK1 into the autophagy-initiating high-molecular mass complex. Expression of a ULK1/2 binding-deficient ATG13 variant in ATG13-deficient cells resulted in diminished but not completely abolished autophagic activity. Collectively, we propose that autophagy can be executed by mechanisms that are dependent or independent of the ULK1/2-ATG13 interaction.     

Effect of meal and propranolol on whole body and splanchnic oxygen consumption in patients with cirrhosis

Our aim was to measure whole body energy expenditure after a mixed liquid meal, with and without simultaneous propranolol infusion, in patients with cirrhosis. We also wanted to investigate the effect of propranolol on substrate fluxes and oxygen uptake in the tissues drained by the hepatic vein and azygos vein in the postprandial period in these patients. Whole-body oxygen uptake, hepatic blood flow, hepatic venous pressure gradient and net-hepatic fluxes of oxygen, lactate, glucose, glycerol, and free fatty acids (FFA) were measured in 12 patients with alcoholic cirrhosis before and for 2 h after ingestion of a mixed liquid meal (700 kcal). Half of the patients (n = 6) were randomized to a treatment group receiving intravenous infusion of propranolol in combination with the meal. The meal-induced energy expenditure was significantly lower in patients given propranolol [15.0 +/- 18.9 vs. 67.0 +/- 26.1 kJ/120 min (means +/- SD), P < 0.01]. Meal-induced whole body oxygen uptake was lower in patients receiving propranolol (19.2 +/- 38 vs. 135.7 +/- 61 mmol/120 min, P < 0.01), and the meal-induced increase in splanchnic oxygen uptake was nonexistent when propranolol was administered in combination (-13.2 +/- 34.8 vs. 110.4 +/- 34.8 mmol/120 min, P = 0.04). Postprandially, the propranolol group had a tendency toward a reduced splanchnic glucose output, and the FFA uptake was significantly reduced. Propranolol reduces meal-induced whole body oxygen uptake and energy expenditure as well as splanchnic oxygen uptake. The splanchnic reduction in oxygen consumption can explain almost the entire reduction in whole body oxygen consumption.     

The fluorescent cholesterol analog dehydroergosterol induces liquid-ordered domains in model membranes

The fluorescent sterol dehydroergosterol (DHE) is often used as a marker for cholesterol in cellular studies. We show by vesicle fluctuation analysis that DHE has a lower ability than cholesterol to stiffen lipid bilayers suggesting less efficient packing with phospholipid acyl chains. Despite this difference, we found by fluorescence and atomic force microscopy, that DHE induces liquid-ordered/-disordered coexistent domains in giant unilamellar vesicles (GUVs) and supported bilayers made of dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) and DHE or cholesterol. DHE-induced phases have a height difference of 0.9-1 nm similar as known for cholesterol-containing domains. DHE not only promotes formation of liquid-liquid immiscibility but also shows strong partition preference for the liquid-ordered phase further supporting its suitability as cholesterol probe.     

Coordination of membrane events during autophagy by multiple class III PI3-kinase complexes

In response to DNA damage, cells activate a phosphorylation-based signaling cascade known as the DNA damage response (DDR). One of the main outcomes of DDR activation is inhibition of cyclin-dependent kinase (Cdk) activity to restrain cell cycle progression until lesions are healed. Recent studies have revealed a reverse connection by which Cdk activity modulates processing of DNA break ends and DDR activation. However, the specific contribution of individual Cdks to this process remains poorly understood. To address this issue, we have examined the DDR in murine cells carrying a defined set of Cdks. Our results reveal that genome maintenance programs of postreplicative cells, including DDR, are regulated by the overall level of Cdk activity and not by specific Cdks.     

Proteomics reveals lowering oxygen alters cytoskeletal and endoplasmatic stress proteins in human endothelial cells

A proteomic approach was applied to explore the signalling pathways elicited by lowering O₂ in endothelial cells. Endothelial cells isolated from native umbilical cords were subjected to 21, 5, or 1% O₂ for 24 h. 2‐D PAGE was performed and candidate proteins were identified using LC‐MS/MS. Lowering of O₂ from 21 to 5% induced upregulation of cofilin‐1, cyclophilin A, tubulin and tubulin fragments, a fragment of glucose‐regulated protein 78 (Grp78) and calmodulin. The upregulation of Grp78 suggested that ER stress proteins were altered and indeed Grp94 and caspase 12 expression were increased in cells exposed to 5% O₂. The presence of ER stress is also supported by findings of blunted caffeine‐evoked ER calcium release in cells exposed to 5 and 1% O₂. Exposure to 1% O₂ caused increases in cofilin‐1, cyclophilin A, and caspase 12 as well as a decrease of β‐actin, but it did not alter the expression of calmodulin, tubulin, Grp78, and Grp94. Incubation with CoCl₂, a stabilizer of the hypoxia‐inducible factor, increased the expression of several of the proteins. The present investigations reveal that lowering O₂, probably in part through hypoxia‐inducible factor, alter the expression of a series of proteins mainly involved in cytoskeletal changes (e.g. cofilin‐1, tubulin, and β‐actin) and in ER stress/apoptosis (e.g. Grp78/94, caspase 12, and cyclophilin A).