The role of arachidonic acid mobilisation in myeloid cells

Haemopoietic growth factors (GF) are important for regulating the proliferation and differentiation of immature cells, but granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) also have a role in regulating the function of mature phagocytic cells. The study presented in this thesis investigated the GF regulation of phospholipase A2 (PLA2) in immature and mature haemopoietic cells. In mature neutrophils, respiratory burst (NADPH oxidase) and PLA2 activity stimulated by the agonist FMLP can be enhanced (primed) by GF. A comparison was made between the mechanism of priming of both PLA2 and NADPH oxidase by GM-CSF and TNF. GM-CSF and TNF both stimulate the phosphorylation and activation of p42ERK2 and p38 MAP kinase (MAPK) in neutrophils. To investigate the role of these MAPKs in priming PLA2 and NADPH oxidase, inhibitors of p42ERK2 (PD098059) and p38 MAPK (SB203580) were used. Inhibition of p42ERK2 blocked neither superoxide generation nor cytokine-mediated priming, but partially inhibited cytokine-mediated priming of PLA2. In contrast inhibition of p38 MAPK blocked primed and unprimed NADPH oxidase activity, but only partially inhibited primed PLA2 activity. The dissociation of the priming of these two enzymes systems indicates that they may activated by different mechanisms. Inflammation of the vascular endothelium is pail of the pathogenic process in the crisis phase of sickle cell disease (SCD). This study investigated whether the priming of neutrophil PLA2 and NADPH oxidase activity in response to in vitro GM-CSF and TNF was altered in both the steady state and crisis of SCD. The data showed raised resting levels of neutrophil PLA2 even in the steady state, indicating an ongoing activation of these cells. But there were reduced responses of PLA2 and NADPH oxidase to GF priming in both steady state and crisis, and this may contribute to the susceptibility of these patients to infection. Immature cells were also studied. A range of myeloid and lymphocytic cell lines were screened for the presence of PLA2 activity measured by arachidonate release stimulated by calcium ionophore. Immature myeloid cells (HL-60 and K562) had extremely low PLA2 activity which was not enhanced by GF. Immature lymphocytic cells. Daudi and IL-2-dependent CTLL cells also released little arachidonic acid, and PLA2 activity was not further enhanced by stimulation with IL-2. However, the GF dependent myeloid cell lines (TF-1, THP-1) and purified human CD34+ stem cells showed higher levels of PLA2 activity which was increased by GM-CSF and IL-3. This suggests that PLA2 activity may be important for mediating the effects of myeloid growth factors

Post-translational modifications of Hsp90 and their contributions to chaperone regulation

AbstractMolecular chaperones, as the name suggests, are involved in folding, maintenance, intracellular transport, and degradation of proteins as well as in facilitating cell signaling. Heat shock protein 90 (Hsp90) is an essential eukaryotic molecular chaperone that carries out these processes in normal and cancer cells. Hsp90 function in vivo is coupled to its ability to hydrolyze ATP and this can be regulated by co-chaperones and post-translational modifications. In this review, we explore the varied roles of known post-translational modifications of cytosolic and nuclear Hsp90 (phosphorylation, acetylation, S-nitrosylation, oxidation and ubiquitination) in fine-tuning chaperone function in eukaryotes. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90)

c-Abl mediated tyrosine phosphorylation of Aha1 activates its co-chaperone function in cancer cells

The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific "client" proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1), promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo

Expressed in the yeast Saccharomyces cerevisiae, human ERK5 is a client of the Hsp90 chaperone that complements loss of the Slt2p (Mpk1p) cell integrity stress-activated protein kinase

ERK5 is a mitogen-activated protein (MAP) kinase regulated in human cells by diverse mitogens and stresses but also suspected of mediating the effects of a number of oncogenes. Its expression in the slt2Delta Saccharomyces cerevisiae mutant rescued several of the phenotypes caused by the lack of Slt2p (Mpk1p) cell integrity MAP kinase. ERK5 is able to provide this cell integrity MAP kinase function in yeast, as it is activated by the cell integrity signaling cascade that normally activates Slt2p and, in its active form, able to stimulate at least one key Slt2p target (Rlm1p, the major transcriptional regulator of cell wall genes). In vitro ERK5 kinase activity was abolished by Hsp90 inhibition. ERK5 activity in vivo was also lost in a strain that expresses a mutant Hsp90 chaperone. Therefore, human ERK5 expressed in yeast is an Hsp90 client, despite the widely held belief that the protein kinases of the MAP kinase class are non-Hsp90-dependent activities. Two-hybrid and protein binding studies revealed that strong association of Hsp90 with ERK5 requires the dual phosphorylation of the TEY motif in the MAP kinase activation loop. These phosphorylations, at positions adjacent to the Hsp90-binding surface recently identified for a number of protein kinases, may cause a localized rearrangement of this MAP kinase region that leads to creation of the Hsp90-binding surface. Complementation of the slt2Delta yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5

Pitting Corrosion Behavior of CUSTOM 450 Stainless Steel Using Electrochemical Characterization

In this study, the electrochemical polarization tests were performed on tensioned and non-tensioned CUSTOM 450 specimens in a 3.5 wt% NaCl solution to investigate pitting potential and stable pit initiation time. A potentiodynamic test was conducted to determine the exact amount of pitting potentials. According to the potentiostatic tests, a relation between applied potential and the stable pit initiation time was obtained. Concerning this relation, stable pitting time can be predicted without experimental works. Optical microscopy was used to evaluate the shape of the pits. Tensile stress led the pit to experience the “pit to crack” step. The corrosion rate of samples was studied by the determination of mass loss. Mass loss measurements and current density–time curve in potentiostatic tests demonstrated the rate of pitting corrosion decreased as time passed. Finally, the depth of the pits was measured by the eddy current technique. The results showed that tensile stress facilitated deeper pit development

Mutation of the co-chaperone Tsc1 in bladder cancer diminishes Hsp90 acetylation and reduces drug sensitivity and selectivity

The molecular chaperone Heat shock protein 90 (Hsp90) is essential for the folding, stability, and activity of several drivers of oncogenesis. Hsp90 inhibitors are currently under clinical evaluation for cancer treatment, however their efficacy is limited by lack of biomarkers to optimize patient selection. We have recently identified the tumor suppressor tuberous sclerosis complex 1 (Tsc1) as a new co-chaperone of Hsp90 that affects Hsp90 binding to its inhibitors. Highly variable mutations of TSC1 have been previously identified in bladder cancer and correlate with sensitivity to the Hsp90 inhibitors. Here we showed loss of TSC1 leads to hypoacetylation of Hsp90-K407/K419 and subsequent decreased binding to the Hsp90 inhibitor ganetespib. Pharmacologic inhibition of histone deacetylases (HDACs) restores acetylation of Hsp90 and sensitizes Tsc1-mutant bladder cancer cells to ganetespib, resulting in apoptosis. Our findings suggest that TSC1 status may predict response to Hsp90 inhibitors in patients with bladder cancer, and co-targeting HDACs can sensitize tumors with Tsc1 mutations to Hsp90 inhibitors

Mps1 mediated phosphorylation of Hsp90 confers renal cell carcinoma sensitivity and selectivity to Hsp90 inhibitors

The molecular chaperone Hsp90 protects deregu- lated signaling proteins that are vital for tumor growth and survival. Tumors generally display sensi- tivity and selectivity toward Hsp90 inhibitors; however, the molecular mechanism underlying this phenotype remains undefined. We report that the mitotic checkpoint kinase Mps1 phosphorylates a conserved threonine residue in the amino-domain of Hsp90. This, in turn, regulates chaperone function by reducing Hsp90 ATPase activity while fostering Hsp90 association with kinase clients, including Mps1. Phosphorylation of Hsp90 is also essential for the mitotic checkpoint because it confers Mps1 stability and activity. We identified Cdc14 as the phosphatase that dephosphorylates Hsp90 and dis- rupts its interaction with Mps1. This causes Mps1 degradation, thus providing a mechanism for its inactivation. Finally, Hsp90 phosphorylation sensi- tizes cells to its inhibitors, and elevated Mps1 levels confer renal cell carcinoma selectivity to Hsp90 drugs. Mps1 expression level can potentially serve as a predictive indicator of tumor response to Hsp90 inhibitors

Mechanisms of Hsp90 regulation

Heat shock protein 90 (Hsp90) is a molecular chaperone that is involved in the activation of disparate client proteins. This implicates Hsp90 in diverse biological processes that require a variety of co-ordinated regulatory mechanisms to control its activity. Perhaps the most important regulator is heat shock factor 1 (HSF1), which is primarily responsible for upregulating Hsp90 by binding heat shock elements (HSEs) within Hsp90 promoters. HSF1 is itself subject to a variety of regulatory processes and can directly respond to stress. HSF1 also interacts with a variety of transcriptional factors that help integrate biological signals, which in turn regulate Hsp90 appropriately. Because of the diverse clientele of Hsp90 a whole variety of co-chaperones also regulate its activity and some are directly responsible for delivery of client protein. Consequently, co-chaperones themselves, like Hsp90, are also subject to regulatory mechanisms such as post translational modification. This review, looks at the many different levels by which Hsp90 activity is ultimately regulated

A Systematic Protocol for the Characterization of Hsp90 Modulators

This is the author's accepted manuscript. Made available by the permission of the publisher.Several Hsp90 modulators have been identified including the N-terminal ligand geldanamycin (GDA), the C-terminal ligand novobiocin (NB), and the co-chaperone disruptor celastrol. Other Hsp90 modulators elicit a mechanism of action that remains unknown. For example, the natural product gedunin and the synthetic anti-spermatogenic agent H2-gamendazole, recently identified Hsp90 modulators, manifest biological activity through undefined mechanisms. Herein, we report a series of biochemical techniques used to classify such modulators into identifiable categories. Such studies provided evidence that gedunin and H2-gamendazole both modulate Hsp90 via a mechanism similar to celastrol, and unlike NB or GDA