bafilomycin-a and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Epidermal growth factor receptor (EGFR) is highly expressed in head and neck squamous cell carcinoma (HNSCC) and correlates with poor prognosis. EGFR has been demonstrated to be associated with cancer stem cell traits in HNSCC. However, the underlying molecular mechanism is far from elucidated. Here, SOX2, one of the most important stem cell markers, was identified as a binding partner and substrate of EGFR. EGFR signaling inhibition decreases SOX2 expression by promoting its autophagic degradation. Mechanistically, EGFR activation induces SOX2 phosphorylation at the Y277 site and reduces its ubiquitination, which inhibits its association with p62 and subsequent autophagic degradation. Gefitinib, an EGFR tyrosine kinase inhibitor, shows in vitro and in vivo protective effects against oral cancer cells that can be reversed through autophagy inhibition. Our study suggests that EGFR plays an important role in the development of cancer stem cells by stabilizing SOX2. Targeting EGFR in combination with conventional chemotherapy might be a promising strategy for the treatment of HNSCC through elimination of cancer stem cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Autophagy; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Gefitinib; Humans; Leupeptins; Macrolides; Male; Mice; Mouth Neoplasms; Mutagenesis; Neoplastic Stem Cells; Protein Kinase Inhibitors; Protein Stability; Proteolysis; SOXB1 Transcription Factors; Squamous Cell Carcinoma of Head and Neck; Xenograft Model Antitumor Assays

Lithium chloride (LiCl) is a widely used drug for the treatment of bipolar disorders, but as a side effect, 40% of the patients develop diabetes insipidus. LiCl affects the activity of the glycogen synthase kinase 3 (GSK3), and mice deficient for GSK3β showed a reduction in the urine concentration capability. The cellular and molecular mechanisms are not fully understood. We used primary cultured inner medullary collecting duct cells to analyze the underlying mechanisms. LiCl and the inhibitor of GSK3 (SB216763) induced a decrease in the aquaporin-2 (Aqp2) protein level. LiCl induced downregulation of Aqp2 mRNA expression while SB216763 had no effect and TWS119 led to increase in expression. The inhibition of the lysosomal activity with bafilomycin or chloroquine prevented both LiCl- and SB216763-mediated downregulation of Aqp2 protein expression. Bafilomycin and chloroquine induced the accumulation of Aqp2 in lysosomal structures, which was prevented in cells treated with dibutyryl cyclic adenosine monophosphate (dbcAMP), which led to phosphorylation and membrane localization of Aqp2. Downregulation of Aqp2 was also evident when LiCl was applied together with dbcAMP, and dbcAMP prevented the SB216763-induced downregulation. We showed that LiCl and SB216763 induce downregulation of Aqp2 via different mechanisms. While LiCl also affected the mRNA level, SB216763 induced lysosmal degradation. Specific GSK3β inhibition had an opposite effect, indicating a more complex regulatory mechanism.

Topics: Animals; Aquaporin 2; Aquaporin 3; Aquaporin 4; Cells, Cultured; Chloroquine; Cyclic AMP Response Element-Binding Protein; Down-Regulation; Female; Glycogen Synthase Kinase 3; Indoles; Kidney Tubules, Collecting; Leupeptins; Lithium Chloride; Macrolides; Maleimides; Protein Kinase Inhibitors; Proteolysis; Pyrimidines; Pyrroles; Rats, Wistar; RNA, Messenger

Store-operated calcium entry (SOCE) channels composed of Stim and Orai proteins play a critical role in diverse biological processes. Upon endoplasmic reticulum (ER)-mediated calcium (Ca(2+)) depletion, Stim proteins oligomerize with Orai to initiate Ca(2+) influx across the plasma membrane. The ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of ubiquilin 1 are involved in the degradation of presenilin and polyglutamine proteins. Through screening of Orai1 interaction partner(s) that might have an effect on SOCE, ubiquilin 1 was identified as a target of Orai1. However, the UBL and UBA domains of ubiquilin 1 were dispensable for this interaction. Additionally, ubiquilin 1 and Orai1 colocalized in the cytosolic compartment. Ubiquilin 1 increased the ubiquitination of Orai1, resulting in the formation of a high-molecular-weight form. MG132, a proteasome inhibitor, failed to block the degradation of Orai1, whereas bafilomycin A, a lysosome inhibitor, prevented Orai1 degradation. Confocal microscopy studies demonstrated that a fraction of Orai1 colocalized with ubiquilin 1 and the autophagosomal marker LC3. Because Orai1 is a constituent of SOCE, we determined the effect of ubiquilin 1 on Orai1-mediated Ca(2+) influx. As we expected, intracellular Ca(2+) mobilization, a process normally potentiated by Orai1, was downregulated by ubiquilin 1. Taken together, these findings suggest that ubiquilin 1 downregulates intracellular Ca(2+) mobilization and its downstream signaling by promoting the ubiquitination and lysosomal degradation of Orai1.

Topics: Adaptor Proteins, Signal Transducing; Autophagy-Related Proteins; Blotting, Western; Calcium; Calcium Channels; Calcium Signaling; Carrier Proteins; Cell Cycle Proteins; Cysteine Proteinase Inhibitors; Cytosol; Enzyme Inhibitors; HEK293 Cells; HeLa Cells; Humans; Immunoprecipitation; Leupeptins; Lysosomes; Macrolides; Membrane Proteins; Neoplasm Proteins; ORAI1 Protein; Phagosomes; Plasmids; Proteasome Endopeptidase Complex; Proton-Translocating ATPases; Signal Transduction; Stromal Interaction Molecule 1; Two-Hybrid System Techniques; Ubiquitination

PINK1 and Parkin mutations cause recessive Parkinson's disease (PD). In Drosophila and SH-SY5Y cells, Parkin is recruited by PINK1 to damaged mitochondria, where it ubiquitinates Mitofusins and consequently promotes mitochondrial fission and mitophagy.Here, we investigated the impact of mutations in endogenous PINK1 and Parkin on the ubiquitination of mitochondrial fusion and fission factors and the mitochondrial network structure. Treating control fibroblasts with mitochondrial membrane potential (Δψ) inhibitors or H(2)O(2) resulted in ubiquitination of Mfn1/2 but not of OPA1 or Fis1. Ubiquitination of Mitofusins through the PINK1/Parkin pathway was observed within 1 h of treatment. Upon combined inhibition of Δψ and the ubiquitin proteasome system (UPS), no ubiquitination of Mitofusins was detected. Regarding morphological changes, we observed a trend towards increased mitochondrial branching in PD patient cells upon mitochondrial stress.For the first time in PD patient-derived cells, we demonstrate that mutations in PINK1 and Parkin impair ubiquitination of Mitofusins. In the presence of UPS inhibitors, ubiquitinated Mitofusin is deubiquitinated by the UPS but not degraded, suggesting that the UPS is involved in Mitofusin degradation.

Topics: Carbonyl Cyanide m-Chlorophenyl Hydrazone; Fibroblasts; GTP Phosphohydrolases; Humans; Hydrogen Peroxide; Leupeptins; Macrolides; Membrane Proteins; Membrane Transport Proteins; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Proteins; Models, Biological; Mutation; Oligopeptides; Proteasome Endopeptidase Complex; Protein Kinases; Protein Transport; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination; Valinomycin

Type I collagen is a major component of the extracellular matrix, and mutations in the collagen gene cause several matrix-associated diseases. These mutant procollagens are misfolded and often aggregated in the endoplasmic reticulum (ER). Although the misfolded procollagens are potentially toxic to the cell, little is known about how they are eliminated from the ER. Here, we show that procollagen that can initially trimerize but then aggregates in the ER are eliminated by an autophagy-lysosome pathway, but not by the ER-associated degradation (ERAD) pathway. Inhibition of autophagy by specific inhibitors or RNAi-mediated knockdown of an autophagy-related gene significantly stimulated accumulation of aggregated procollagen trimers in the ER, and activation of autophagy with rapamycin resulted in reduced amount of aggregates. In contrast, a mutant procollagen which has a compromised ability to form trimers was degraded by ERAD. Moreover, we found that autophagy plays an essential role in protecting cells against the toxicity of the ERAD-inefficient procollagen aggregates. The autophagic elimination of aggregated procollagen occurs independently of the ERAD system. These results indicate that autophagy is a final cell protection strategy deployed against ER-accumulated cytotoxic aggregates that are not able to be removed by ERAD.

Topics: Animals; Autophagy; Blotting, Western; Cell Line; Collagen Type I; Endoplasmic Reticulum; Enzyme Inhibitors; Fibroblasts; Fluorescence Recovery After Photobleaching; Green Fluorescent Proteins; HSP47 Heat-Shock Proteins; Immunoprecipitation; Leupeptins; Lysosomes; Macrolides; Mice; Mice, Knockout; Microscopy, Fluorescence; Microscopy, Immunoelectron; Mutation; Protein Folding; Recombinant Fusion Proteins; RNA Interference; Transfection

BPV-4 E5 inhibits transcription of the bovine MHC class I heavy chain (HC) gene, increases degradation of HC and downregulates surface expression of MHC class I by retaining the complex in the Golgi apparatus (GA). Here we report that transcription inhibition can be alleviated by interferon treatment and the degradation of HC can be reversed by treatment with inhibitors of proteasomes and lysosomes. However, the inhibition of transport of MHC class I to the cell surface is irreversible. We show that E5 is capable of physically interacting with HC. Together with the inhibition of the vacuolar ATPase (due to the interaction between E5 and 16k subunit c), the interaction between E5 and HC is likely to be responsible for retention of MHC class I in the GA. C-terminus deletion mutants of E5 are incapable of either downregulating surface MHC class I or interacting with HC, establishing that the C-terminus domain of E5 is important in the inhibition of MHC class I.

Topics: Animals; Antiviral Agents; Bovine papillomavirus 1; Bovine papillomavirus 4; Cattle; Cell Transformation, Viral; Cysteine Proteinase Inhibitors; Down-Regulation; Enzyme Inhibitors; Fetus; Golgi Apparatus; Histocompatibility Antigens Class I; Immunoprecipitation; Interferon-beta; Interferon-gamma; Leupeptins; Macrolides; Oncogene Proteins, Viral; Protein Biosynthesis; Protein-Tyrosine Kinases; Sequence Deletion; Transcription, Genetic; Vacuolar Proton-Translocating ATPases