Browsing by Subject "Heat-Shock Proteins"

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Open Access
Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells.
(Cancer Biol Ther, 2010-01) Misra, Uma K; Pizzo, Salvatore V
We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.
Open Access
RNA-Seq and ChIP-Seq reveal SQSTM1/p62 as a key mediator of JunB suppression of NF-κB-dependent inflammation.
(J Invest Dermatol, 2015-04) Zhang, Xiaoling; Jin, Jane Y; Wu, Joseph; Qin, Xiaoxia; Streilein, Robert; Hall, Russell P; Zhang, Jennifer Y
Mice with epidermal deletion of JunB transcription factor displayed a psoriasis-like inflammation. The relevance of these findings to humans and the mechanisms mediating JunB function are not fully understood. Here we demonstrate that impaired JunB function via gene silencing or overexpression of a dominant negative mutant increased human keratinocyte cell proliferation but decreased cell barrier function. RNA-seq revealed over 500 genes affected by JunB loss of function, which included the upregulation of an array of proinflammatory molecules relevant to psoriasis. Among these were tumor necrosis factor α (TNFα), CCL2, CXCL10, IL6R, and SQSTM1, an adaptor protein involved in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Chromatin immunoprecipitation (ChIP)-Seq and gene reporter analyses showed that JunB directly suppressed SQSTM1 by binding to a consensus AP-1 cis element located around 2 kb upstream of SQSTM1-transcription start site. Similar to JunB loss of function, SQSTM1-overexpression induced TNFα, CCL2, and CXCL10. Conversely, NF-κB inhibition genetically with a mutant IκBα or pharmacologically with pyrrolidine dithiocarbamate (PDTC) prevented cytokine, but not IL6R, induction by JunB deficiency. Taken together, our findings indicate that JunB controls epidermal growth, barrier formation, and proinflammatory responses through direct and indirect mechanisms, pinpointing SQSTM1 as a key mediator of JunB suppression of NF-κB-dependent inflammation.
Open Access
The 70-kDa heat shock cognate protein (Hsc73) gene is enhanced by ovarian hormones in the ventromedial hypothalamus.
(Proc Natl Acad Sci U S A, 1999-02-16) Krebs, CJ; Jarvis, ED; Pfaff, DW
Estrogen (E) and progesterone (P) orchestrate many cellular responses involved in female reproductive physiology, including reproductive behaviors. E- and P-binding neurons important for lordosis behavior have been located within the ventromedial hypothalamus (VMH), and several hormone-responsive genes have been observed there as well. In attempts to identify additional E- and P-responsive genes in the VMH that may contribute to sexual behaviors, we used the differential display mRNA screening technique. One of the genes identified encodes the 73-kDa heat shock cognate protein (Hsc73). Quantitative in situ hybridization analysis of brains from naturally cycling female rats revealed a significant increase in Hsc73 mRNA in the VMH and arcuate nucleus of animals during proestrus compared with those at diestrus-1. To confirm that these increases were steroid hormone dependent, we compared vehicle-treated ovariectomized females with ovariectomized females treated with estradiol benzoate and P. Northern analysis and in situ hybridizations showed that the Hsc73 gene is enhanced by E and P in the pituitary and subregions of the VMH. Incidentally, by examining the primary amino acid sequence of rat, human, and chicken progesterone receptors, we noticed that putative Hsc73 binding sites are conserved across species with similar sites existing in the androgen and glucocorticoid receptors. Together these findings suggest a possible mechanism through which E could influence the activities of progesterone, androgen, and glucocorticoid receptors, by enhancing the expression of Hsc73 in cells where these proteins colocalize.
Open Access
Thermodynamic analysis of a molecular chaperone binding to unfolded protein substrates.
(Biochemistry, 2010-02-16) Xu, Ying; Schmitt, Sebastian; Tang, Liangjie; Jakob, Ursula; Fitzgerald, Michael C
Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins to facilitate protein folding and prevent nonspecific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports about the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H-D exchange), an H-D exchange and MALDI-based technique, in studying the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates, including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding-unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX-derived K(d) values for Hsp33 complexes with four different substrates were all found to be within the range of 3-300 nM.