Ubiquitination Is a Novel Post-Translational Modification of VMP1 in Autophagy of Human Tumor Cells.

Autophagy is a tightly regulated catabolic process involved in the degradation and recycling of proteins and organelles. Ubiquitination plays an important role in the regulation of autophagy. Vacuole Membrane Protein 1 (VMP1) is an essential autophagy protein. The expression of VMP1 in pancreatic cancer stem cells carrying the activated Kirsten rat sarcoma viral oncogene homolog (KRAS) triggers autophagy and enables therapy resistance. Using biochemical and cellular approaches, we identified ubiquitination as a post-translational modification of VMP1 from the initial steps in autophagosome biogenesis. VMP1 remains ubiquitinated as part of the autophagosome membrane throughout autophagic flux until autolysosome formation. However, VMP1 is not degraded by autophagy, nor by the ubiquitin-proteasomal system. Mass spectrometry and immunoprecipitation showed that the cell division cycle protein cdt2 (Cdt2), the substrate recognition subunit of the E3 ligase complex associated with cancer, cullin-RING ubiquitin ligase complex 4 (CRL4), is a novel interactor of VMP1 and is involved in VMP1 ubiquitination. VMP1 ubiquitination decreases under the CRL inhibitor MLN4924 and increases with Cdt2 overexpression. Moreover, VMP1 recruitment and autophagosome formation is significantly affected by CRL inhibition. Our results indicate that ubiquitination is a novel post-translational modification of VMP1 during autophagy in human tumor cells. VMP1 ubiquitination may be of clinical relevance in tumor-cell-therapy resistance.

Impaired HPA axis function in diabetes involves adrenal apoptosis and phagocytosis.

PURPOSE: The aim of the present study was to analyze the involvement of oxidative stress and inflammation in the modulation of glucocorticoid production in the adrenal cortex of diabetic rats. METHODS: Male Wistar rats were treated with or without streptozotocin (STZ, an insulinopenic model of diabetes) and either α-lipoic (90 mg/kg ip.), α-tocopherol (200 mg/kg po.) or with STZ and supplemented with insulin (STZ + INS: 2.5U/day) for 4 weeks. Oxidative/nitrosative stress parameters and antioxidant enzymes were determined in adrenocortical tissues. Apoptosis and macrophage activation were evaluated by immunohistochemistry (TUNEL and ED1+). Basal and ACTH-stimulated corticosterone production were assessed by RIA and plasma ACTH levels were determined by an immunometric assay. RESULTS: Diabetic rats showed a diminished response to exogenous ACTH stimulation along with higher basal corticosterone and lower plasma ACTH levels. In the adrenal cortex we determined an increase in the levels of lipoperoxides, S-nitrosothiols, nitric oxide synthase activity and nitro-tyrosine modified proteins while catalase activity and heme oxygenase-1 expression levels were also elevated. Antioxidant treatments were effective in the prevention of these effects, and in the increase in the number of apoptotic and phagocytic (ED1+) cells detected in diabetic rats. No changes were observed in the STZ + INS group. CONCLUSIONS: Generation of oxidative/nitrosative stress in the adrenal cortex of diabetic rats leads to the induction of apoptosis and the activation of adrenocortical macrophages and is associated with an elevated basal corticosteronemia and the loss of the functional capacity of the gland.