Detection of heat shock protein 70 in choroidal neovascular membranes secondary to age related macular degeneration

<p>Abstract</p> <p>Background</p> <p>Heat shock proteins are acute phase proteins that are upregulated in inflammation or following thermal stress. We analyzed the presence of the heat shock protein 70 (Hsp 70) in choroidal neovascular (CNV) membranes secondary to AMD after treatment with verteporphin photodynamic therapy (PDT) or transpupillary thermo therapy (TTT) to determine whether treatment correlated with the presence of Hsp70.</p> <p>Results</p> <p>CNV membranes were removed by pars plana vitrectomy (ppV) and subretinal extraction. The membranes were analysed by light microscopy and the presence of Hsp 70 was examined using histochemistry. HeLa Cells served as controls.</p> <p>Of the 14 membranes analysed 11 were Hsp70 positive and 3 negative. In the no pre-treatment group of 8 membranes 6 were Hsp70 positive and 2 negative; in the PTD group all 4 membranes were positive and in the TTT group 1 membrane was positive and 1 membrane was negative for Hsp70.</p> <p>Conclusion</p> <p>Hsp70 is present in the most CNV membranes secondary to AMD. Pre-treatment of the membrane with PTD or TTT does not appear to influence the expression of Hsp70.</p

<i>C-elegans</i> model identifies genetic modifiers of alpha-synuclein inclusion formation during aging

Inclusions in the brain containing alpha-synuclein are the pathological hallmark of Parkinson's disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a &lt;i&gt;C-elegans&lt;/i&gt; model that makes it possible to monitor, in living animals, the formation of alpha-synuclein inclusions. In worms of old age, inclusions contain aggregated alpha-synuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in alpha-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between alpha-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinson's disease and other alpha-synuclein related disorders

Enhanced Hsp70 Expression Protects against Acute Lung Injury by Modulating Apoptotic Pathways

The Acute respiratory distress syndrome (ARDS) is a highly lethal inflammatory lung disorder. Apoptosis plays a key role in its pathogenesis. We showed that an adenovirus expressing the 70 kDa heat shock protein Hsp70 (AdHSP) protected against sepsis-induced lung injury. In this study we tested the hypothesis that AdHSP attenuates apoptosis in sepsis-induced lung injury

Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

BACKGROUND:Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free alpha-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal alpha-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s). METHODOLOGY/PRINCIPAL FINDINGS:Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H(2)O(2); 0.1-0.5 mM range). Results show that A) Cells overexpressing "wild type" human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H(2)O(2) concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H(2)O(2) treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase. CONCLUSION/SIGNIFICANCE:Apoptosis can be modulated by "on/off" switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein

Anti-tumour activity in vitro and in vivo of selective differentiating agents containing hydroxamate

A series of hydroxamates, which are not metalloprotease inhibitors, have been found to be selectively toxic to a range of transformed and human tumour cells without killing normal cells (fibroblasts, melanocytes) at the same concentrations. Within 24 h of treatment, drug action is characterized by morphological reversion of tumour cells to a more normal phenotype (dendritic morphology), and rapid and reversible acetylation of histone H4 in both tumour and normal cells. Two; hydroxamates inhibited growth of xenografts of human melanoma cells in nude mice; resistance did not develop in vivo or in vitro. A third hydroxamate, trichostatin A, was active in vitro but became inactivated and had no anti-tumour activity in vivo. Development of dendritic morphology was found to be dependent upon phosphatase activity, RNA and protein synthesis. Proliferating hybrid clones of sensitive and resistant cells remained sensitive to ABHA, indicating a dominant-negative mechanism of sensitivity. Histone H4 hyperacetylation suggests that these agents act at the chromatin level. This work may lead to new drugs that are potent, and selective anti-tumour agents with low toxicity to normal Cells

Network Analysis of Oyster Transcriptome Revealed a Cascade of Cellular Responses during Recovery after Heat Shock

Oysters, as a major group of marine bivalves, can tolerate a wide range of natural and anthropogenic stressors including heat stress. Recent studies have shown that oysters pretreated with heat shock can result in induced heat tolerance. A systematic study of cellular recovery from heat shock may provide insights into the mechanism of acquired thermal tolerance. In this study, we performed the first network analysis of oyster transcriptome by reanalyzing microarray data from a previous study. Network analysis revealed a cascade of cellular responses during oyster recovery after heat shock and identified responsive gene modules and key genes. Our study demonstrates the power of network analysis in a non-model organism with poor gene annotations, which can lead to new discoveries that go beyond the focus on individual genes

The Anti-Apoptotic Activity of BAG3 Is Restricted by Caspases and the Proteasome

Caspase-mediated cleavage and proteasomal degradation of ubiquitinated proteins are two independent mechanisms for the regulation of protein stability and cellular function. We previously reported BAG3 overexpression protected ubiquitinated clients, such as AKT, from proteasomal degradation and conferred cytoprotection against heat shock. We hypothesized that the BAG3 protein is regulated by proteolysis. caspase-resistant mutant. Caspase and proteasome inhibition resulted in partial and independent protection of BAG3 whereas inhibitors of both blocked BAG3 degradation. STS-induced apoptosis was increased when BAG3 was silenced, and retention of BAG3 was associated with cytoprotection.BAG3 is tightly controlled by selective degradation during STS exposure. Loss of BAG3 under STS injury required sequential caspase cleavage followed by polyubiquitination and proteasomal degradation. The need for dual regulation of BAG3 in apoptosis suggests a key role for BAG3 in cancer cell resistance to apoptosis

Impaired Heat Shock Response in Cells Expressing Full-Length Polyglutamine-Expanded Huntingtin

The molecular mechanisms by which polyglutamine (polyQ)-expanded huntingtin (Htt) causes neurodegeneration in Huntington's disease (HD) remain unclear. The malfunction of cellular proteostasis has been suggested as central in HD pathogenesis and also as a target of therapeutic interventions for the treatment of HD. We present results that offer a previously unexplored perspective regarding impaired proteostasis in HD. We find that, under non-stress conditions, the proteostatic capacity of cells expressing full length polyQ-expanded Htt is adequate. Yet, under stress conditions, the presence of polyQ-expanded Htt impairs the heat shock response, a key component of cellular proteostasis. This impaired heat shock response results in a reduced capacity to withstand the damage caused by cellular stress. We demonstrate that in cells expressing polyQ-expanded Htt the levels of heat shock transcription factor 1 (HSF1) are reduced, and, as a consequence, these cells have an impaired a heat shock response. Also, we found reduced HSF1 and HSP70 levels in the striata of HD knock-in mice when compared to wild-type mice. Our results suggests that full length, non-aggregated polyQ-expanded Htt blocks the effective induction of the heat shock response under stress conditions and may thus trigger the accumulation of cellular damage during the course of HD pathogenesis

Phase I Evaluation of STA-1474, a Prodrug of the Novel HSP90 Inhibitor Ganetespib, in Dogs with Spontaneous Cancer

The novel water soluble compound STA-1474 is metabolized to ganetespib (formerly STA-9090), a potent HSP90 inhibitor previously shown to kill canine tumor cell lines in vitro and inhibit tumor growth in the setting of murine xenografts. The purpose of the following study was to extend these observations and investigate the safety and efficacy of STA-1474 in dogs with spontaneous tumors.This was a Phase 1 trial in which dogs with spontaneous tumors received STA-1474 under one of three different dosing schemes. Pharmacokinetics, toxicities, biomarker changes, and tumor responses were assessed. Twenty-five dogs with a variety of cancers were enrolled. Toxicities were primarily gastrointestinal in nature consisting of diarrhea, vomiting, inappetence and lethargy. Upregulation of HSP70 protein expression was noted in both tumor specimens and PBMCs within 7 hours following drug administration. Measurable objective responses were observed in dogs with malignant mast cell disease (n = 3), osteosarcoma (n = 1), melanoma (n = 1) and thyroid carcinoma (n = 1), for a response rate of 24% (6/25). Stable disease (>10 weeks) was seen in 3 dogs, for a resultant overall biological activity of 36% (9/25).This study provides evidence that STA-1474 exhibits biologic activity in a relevant large animal model of cancer. Given the similarities of canine and human cancers with respect to tumor biology and HSP90 activation, it is likely that STA-1474 and ganetespib will demonstrate comparable anti-cancer activity in human patients

Analysis of the cytochrome c-dependent apoptosis apparatus in cells from human pancreatic carcinoma

Defects in the apoptotic system are likely to play a role in tumorigenesis. Pancreatic carcinoma cells are extremely resistant to apoptosis induction by chemotherapy suggesting that the apoptosis machinery is faulty. We investigated the integrity of the cytochrome c-dependent apoptotic apparatus in 10 human pancreatic carcinoma cell lines. Expression of Apaf-1, caspase-3, -6, -7, -8 and -9, Hsp-70 and XIAP was detected in all cell lines. The expression levels of Apaf-1 and caspase-8 were homogenous in all cell lines whereas differences in expression of other caspases were seen. In cytosolic fractions, all investigated caspases were processed in response to cytochrome c but the extent of processing varied between the cell lines. No stringent correlation between the amount of processing of caspase-9 and effector caspases was seen. Cytochrome c-induced effector caspase activity was quantitated by enzyme assay. Especially at low concentrations of added cytochrome c, this response varied greatly between the cell lines. These data demonstrate that the apoptotic system downstream of the mitochondria is qualitatively intact in pancreatic carcinoma. They further show that the response to cytochrome c can be quantitated in a cell-free system and that determinants other than mere expression of apoptotic molecules can regulate cytochrome c-induced apoptosis