ABSTRACT
Investigating differentially expressed proteins in a milieu rich in cholesterol oxidation products, we found via mass spectrometry-based proteomics that surface levels of heat shock protein 60 (HSP60) were upregulated on monocytic cells in the presence of 27-hydroxycholesterol (27OHChol). The elevated levels of cytoplasmic membrane HSP60 were verified via Western blot analysis and visualized by confocal microscopy. Treatment with 27OHChol also resulted in increased levels of cellular HSP60 without altering its transcription. Cholesterol, however, did not affect cell-surface levels and cellular amount of HSP60. GSK 2033, an LXR antagonist, inhibited expression of live X receptor α, but not of HSP60, induced by 27OHChol. Treatment with 27OHChol also resulted in increased release of HSP60 from monocytic cells, but the release was significantly reduced by inhibitors of endoplasmic reticulum-Golgi protein trafficking, brefeldin A and monensin. Results of the current study indicate that 27OHChol upregulates not only cell-surface and cellular levels of HSP60 but also its release from monocytic cells, thereby contributing to activation of the immune system.
Subject(s)
Chaperonin 60/genetics , Hydroxycholesterols/pharmacokinetics , Mitochondrial Proteins/genetics , Monocytes/drug effects , Amino Acid Sequence , Animals , Brefeldin A/pharmacology , Cell Line , Cell Membrane/drug effects , Cell Membrane/immunology , Cell Membrane/metabolism , Chaperonin 60/agonists , Chaperonin 60/metabolism , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/metabolism , Gene Expression Regulation , Golgi Apparatus/drug effects , Golgi Apparatus/immunology , Golgi Apparatus/metabolism , Humans , Hydroxycholesterols/metabolism , Immunity, Cellular , Liver X Receptors/antagonists & inhibitors , Liver X Receptors/genetics , Liver X Receptors/metabolism , Mitochondrial Proteins/agonists , Mitochondrial Proteins/metabolism , Monensin/pharmacology , Monocytes/cytology , Monocytes/immunology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Sulfonamides/pharmacology , Transcription, GeneticABSTRACT
The specific cochaperonin, chloroplast chaperonin (Cpn)20, consisting of two tandem GroES-like domains, is present abundantly in plant and algal chloroplasts, in addition to Cpn10, which is similar in size to GroES. How Cpn20 oligomers, containing six or eight 10-kDa domains, cooperate with the heptameric ring of chaperonin at the same time as encountering symmetry mismatch is unclear. In the present study, we characterized the functional cooperation of cochaperonins, including two plastidic Cpn20 homo-oligomers from Arabidopsis (AtCpn20) and Chlamydomonas (CrCPN20), and one algal CrCPNs hetero-oligomer, consisting of three cochaperonins, CrCPN11, CrCPN20 and CrCPN23, with two chaperonins, Escherichia coli GroEL and Chlamydomonas CrCPN60. AtCpn20 and CrCPNs were functional for assisting both chaperonins in folding model substrates ribulose bisphosphate carboxylase oxygenase from Rhodospirillum rubrum (RrRubisco) in vitro and complementing GroES function in E. coli. CrCPN20 cooperated only with CrCPN60 (and not GroEL) to refold RrRubisco in vitro and showed differential complementation with the two chaperonins in E. coli. Cochaperonin concatamers, consisting of six to eight covalently linked 10-kDa domains, were functionally similar to their respective native forms. Our results indicate that symmetrical match between chaperonin and cochaperonin is not an absolute requisite for functional cooperation.
Subject(s)
Algal Proteins/metabolism , Arabidopsis Proteins/metabolism , Bacterial Proteins/metabolism , Chloroplasts/metabolism , Group I Chaperonins/metabolism , Models, Molecular , Ribulose-Bisphosphate Carboxylase/metabolism , Algal Proteins/agonists , Algal Proteins/chemistry , Algal Proteins/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/agonists , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Bacterial Proteins/agonists , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Chaperonin 10/agonists , Chaperonin 10/chemistry , Chaperonin 10/genetics , Chaperonin 10/metabolism , Chaperonin 60/agonists , Chaperonin 60/chemistry , Chaperonin 60/genetics , Chaperonin 60/metabolism , Chlamydomonas/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/agonists , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Group I Chaperonins/agonists , Group I Chaperonins/chemistry , Group I Chaperonins/genetics , Molecular Weight , Protein Multimerization , Protein Refolding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Rhodospirillum rubrum/enzymology , Rhodospirillum rubrum/metabolism , Ribulose-Bisphosphate Carboxylase/chemistry , Ribulose-Bisphosphate Carboxylase/geneticsABSTRACT
Previous studies have shown that blocking DLL4 signaling reduced tumor growth by disrupting productive angiogenesis. We developed selective anti-human and anti-mouse DLL4 antibodies to dissect the mechanisms involved by analyzing the contributions of selectively targeting DLL4 in the tumor or in the host vasculature and stroma in xenograft models derived from primary human tumors. We found that each antibody inhibited tumor growth and that the combination of the two antibodies was more effective than either alone. Treatment with anti-human DLL4 inhibited the expression of Notch target genes and reduced proliferation of tumor cells. Furthermore, we found that specifically inhibiting human DLL4 in the tumor, either alone or in combination with the chemotherapeutic agent irinotecan, reduced cancer stem cell frequency, as shown by flow cytometric and in vivo tumorigenicity studies.
