Anti-HSP90 alpha Antibody [2G5.G3] (A305031)

The adenosine A2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90a and HSP70-1A) that interact with and retain partially folded A2A receptor prior to ER exit. Complex formation between the A2A receptor and HSP90a (but not HSP90ß) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.

Inhibition of B-cell receptor (BCR) signaling pathways in chronic lymphocytic leukemia (CLL) provides significant clinical benefit to patients, mainly by blocking adhesion of CLL cells in the lymph node microenvironment. The currently applied inhibitors ibrutinib and idelalisib have limited capacity however to induce cell death as monotherapy and are unlikely to eradicate the disease. Acquired resistance to therapy in CLL is often caused by mutations in the response network being targeted, both for DNA damage or BCR signaling pathways. Thus, drugs with dual targeting capacity could offer improved therapeutic value. Here, the potency of CC-115, a novel inhibitor of mammalian target of rapamycin kinase (TORK) and DNA-dependent protein kinase (DNA-PK), was evaluated in primary CLL cells in vitro and in CLL patients. Combined TORK and DNA-PK inhibition in vitro resulted in caspase-dependent cell killing irrespective of p53, ATM, NOTCH1, or SF3B1 status. Proliferation induced by CD40(+) interleukin-21 stimulation was completely blocked by CC-115, and CD40-mediated resistance to fludarabine and venetoclax could be reverted by CC-115. BCR-mediated signaling was inhibited by CC-115 and also in CLL samples obtained from patients with acquired resistance to idelalisib treatment. Clinical efficacy of CC-115 was demonstrated in 8 patients with relapsed/refractory CLL/small lymphocytic lymphoma harboring ATM deletions/mutations; all but 1 patient had a decrease in lymphadenopathy, resulting in 1 IWCLL partial response (PR) and 3 PRs with lymphocytosis. In conclusion, these preclinical results, along with early promising clinical activity, suggest that CC-115 may be developed further for treatment of CLL. The trial was registered at www.clinicaltrials.gov as #NCT01353625.

MAP17 is a small nonglycosylated membrane protein that is overexpressed in a high percentage of carcinomas. High levels of MAP17 enhance the tumorigenic properties of tumor cells by increasing oxidative stress, which is dependent on Na(+)-coupled cotransport. Here, we show that MAP17 is associated with proteins involved in protein degradation and that proteasome inhibition induces autophagy. To analyze whether MAP17 could also alter this process, we used the proteasome inhibitor bortezomib (Velcade, PS-341), which is approved for the treatment of multiple myeloma and mantle cell lymphoma, although it has a high rate of resistance emergence and poor efficacy in solid tumors. We provide evidence that bortezomib induces a cytoprotective effect by activating autophagy and NFκB nuclear translocation, responses that are repressed in the presence of high levels of MAP17 both in vitro and in vivo. Indeed, patients with multiple myeloma treated with bortezomib showed higher response rates and a longer time to progression associated with increased levels of MAP17 expression. The MAP17-induced sensitivity to bortezomib is dependent on the oxidative status of the cells and the activity of Na(+)-coupled transporters because treatment with antioxidants or the inhibitor furosemide restores the cytoprotective activity induced by bortezomib. Therefore, bortezomib induces a prosurvival response through cytoprotective autophagy and NFκB nuclear translocation, which is repressed by high levels of MAP17. We propose that the levels of MAP17 could be used as a prognostic marker to predict the response to bortezomib in hematologic malignancies and in other tissues that are not commonly responsive to the drug.

Airway smooth muscle cell (ASMC) migration is an important mechanism postulated to play a role in airway remodeling in asthma. CXCL1 chemokine has been linked to tissue growth and metastasis. In this study, we present a detailed examination of the inhibitory effect of CXCL1 on human primary ASMC migration and the role of the decoy receptor, Duffy AgR for chemokines (DARC), in this inhibition. Western blots and pathway inhibitors showed that this phenomenon was mediated by activation of the ERK-1/2 MAPK pathway, but not p38 MAPK or PI3K, suggesting a biased selection in the signaling mechanism. Despite being known as a nonsignaling receptor, small interference RNA knockdown of DARC showed that ERK-1/2 MAPK activation was significantly dependent on DARC functionality, which, in turn, was dependent on the presence of heat shock protein 90 subunit a. Interestingly, DARC- or heat shock protein 90 subunit a-deficient ASMCs responded to CXCL1 stimulation by enhancing p38 MAPK activation and ASMC migration through the CXCR2 receptor. In conclusion, we demonstrated DARC's ability to facilitate CXCL1 inhibition of ASMC migration through modulation of the ERK-1/2 MAPK-signaling pathway.

To understand the mechanism underlying the sudden animal death caused by acute heart failure during heat stress, the relationships among the heat-induced pathological changes and apoptosis and the variations in the levels of protective Hsp90a and its mRNA in the heat-stressed primary myocardial cells of neonatal rats in vitro were studied by cytopathological observation, immunoblotting, RT-PCR, and analysis of the related enzymes. After a period of adaptive cell culture, the myocardial cells were immediately exposed to heat stress at 42°C for 10, 20, 40, 60, 120, 240, 360, and 480 min. Levels of creatine kinase increased from the beginning of heat stress, and the cells exposed to heat stress showed acute cellular lesions characterized by vacuolar degeneration and necrosis after 40 min of heat stress, suggesting that the myocardial cells in vitro were obviously stressed and damaged by higher temperature. The levels of cleaved caspase-3 and cytochrome C, which were related to apoptosis, increased significantly after 40 min of heat stress while the Hsp90a protein level significantly decreased. In contrast, after 6 h of exposure to heat stress, the levels of cleaved caspase-3 and cytochrome C decreased while those of Hsp90a significantly increased, suggesting that early depletion of Hsp90a coincides with a high rate of necrosis and apoptosis in heat-stressed myocardial cells, while the Hsp90a level in surviving cells increases again with significantly less apoptosis after 6 h of heat stress. These findings also indicate that apoptosis of myocardial cells occurs through the activation of the cytochrome C and caspase-3 pathway. The cell repair capacity of Hsp90a is overstrained in the early phase of heat treatment and needs some hours to stabilize. As a result, in the primary myocardial cells in vitro, Hsp90a shows protective activity against damage at the end period of the heat exposure.

Heat shock proteins (HSPs) are highly regulated proteins that are involved in normal cellular activity and are up-regulated when the cell is exposed to stress such as heat or excess reactive oxygen species (ROS) production. HSPs are molecular chaperones that mediate the proper folding of proteins and promote recovery of the native conformations of proteins lost due to stress. Improperly folded or denatured proteins tend to aggregate and accumulate in cells. A number of neurodegenerative diseases such as Parkinson disease (PD) and Alzheimer disease (AD) have been called "protein misfolding disorders" due their characteristic pathology. Until now the exact mechanism(s) of AD progression and pathogenesis largely remains unknown. Reasoning that stress is present in brain in AD, we tested the suggestion that HSP levels would be increased in amnestic mild cognitive impairment (aMCI), a transition stage between normal aging and AD. Accordingly, in the present study we measured the levels of HSPs in hippocampus, inferior parietal lobule (IPL) and cerebellum of subjects with aMCI. The results show a general induction of HSPs and decreased levels of Thioredoxin 1 in aMCI brain suggesting that alteration in the chaperone protein systems might contribute to the pathogenesis and progression of AD. The results also are consistent with the notion that targeting HSP could be a therapeutic approach to delay the progression of aMCI to AD.