Hsp-90 and the biology of nematodes

Background  

Hsp-90 from the free-living nematode Caenorhabditis elegans is unique in that it fails to bind to the specific Hsp-90 inhibitor, geldanamycin (GA). Here we surveyed 24 different free-living or parasitic nematodes with the aim of determining whether C. elegans Hsp-90 was the exception or the norm amongst the nematodes. We combined these data with codon evolution models in an attempt to identify whether hsp-90 from GA-binding and non-binding species has evolved under different evolutionary constraints.     

The stress responsive and morphologically regulated <Emphasis Type="Italic">hsp90</Emphasis> gene from <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> is essential to cell viability

Background  

Paracoccidioides brasiliensis is a dimorphic fungus that causes the most prevalent systemic mycosis in Latin America. The response to heat shock is involved in pathogenesis, as this pathogen switches from mycelium to yeast forms in a temperature dependent fashion that is essential to establish infection. HSP90 is a molecular chaperone that helps in the folding and stabilization of selected polypeptides. HSP90 family members have been shown to present important roles in fungi, especially in the pathogenic species, as an immunodominant antigen and also as a potential antifungal therapeutic target.     

Scoring functions and enrichment: a case study on Hsp90

Background  

The need for fast and accurate scoring functions has been driven by the increased use of in silico virtual screening twinned with high-throughput screening as a method to rapidly identify potential candidates in the early stages of drug development. We examine the ability of some the most common scoring functions (GOLD, ChemScore, DOCK, PMF, BLEEP and Consensus) to discriminate correctly and efficiently between active and non-active compounds among a library of ~3,600 diverse decoy compounds in a virtual screening experiment against heat shock protein 90 (Hsp90).     

Alteration in Endometrial Proteins during Early- and Mid-Secretory Phases of the Cycle in Women with Unexplained Infertility

Background

Compromised receptivity of the endometrium is a major cause of unexplained infertility, implantation failure and subclinical pregnancy loss. In order to investigate the changes in endometrial protein profile as a cause of unexplained infertility, the current study was undertaken to analyze the differentially expressed proteins of endometrium from early-secretory (LH+2) to mid-secretory phase (LH+7), in women with unexplained infertility.

Methods

2-D gel electrophoresis was performed to analyze the proteomic changes between early- (n = 8) and mid-secretory (n = 8) phase endometrium of women with unexplained infertility. The differentially expressed protein spots were identified by LC-MS analysis and validated by immunoblotting and immuno-histochemical analysis in early- (n = 4) and mid-secretory (n = 4) phase endometrium of infertile women. Validated proteins were also analyzed in early- (n = 4) and mid-secretory (n = 4) phase endometrium of fertile women.

Results

Nine proteins were found to be differentially expressed between early- and mid- secretory phases of endometrium of infertile women. The expression of Ras-related protein Rap-1b, Protein disulfide isomerase A3, Apolipoprotein-A1 (Apo-A1), Cofilin-1 and RAN GTP-binding nuclear protein (Ran) were found to be significantly increased, whereas, Tubulin polymerization promoting protein family member 3, Superoxide dismutase [Cu-Zn], Sorcin, and Proteasome subunit alpha type-5 were significantly decreased in mid- secretory phase endometrium of infertile women as compared to early-secretory phase endometrium of infertile women. Validation of 4 proteins viz. Sorcin, Cofilin-1, Apo-A1 and Ran were performed in separate endometrial biopsy samples from infertile women. The up-regulated expression of Sorcin and down-regulated expression of Cofilin-1 and Apolipoprotein-A1, were observed in mid-secretory phase as compared to early-secretory phase in case of fertile women.

Conclusions

De-regulation of the expression of Sorcin, Cofilin-1, Apo-A1 and Ran, during early- to mid-secretory phase may have physiological significance and it may be one of the causes for altered differentiation and/or maturation of endometrium, in women with unexplained infertility.     

Monoclonal antibody 4C5 prevents activation of MMP2 and MMP9 by disrupting their interaction with extracellular HSP90 and inhibits formation of metastatic breast cancer cell deposits

Background  

Heat shock protein 90 (HSP90) is a molecular chaperone that is considered a new target for the treatment of cancer. Increasing data reveal an extracellular chaperoning activity for HSP90. Here we investigate the interaction of the secreted isoforms of HSP90 with matrix metalloproteinases (MMP) MMP2 and MMP9. Moreover we examine the role of a monoclonal antibody (mAb) against HSP90, mAb 4C5, regarding these interactions and its value as a potential inhibitor of human breast cancer cell invasion and metastasis.     

Modulation of cytokines and transcription factors (T-Bet and GATA3) in CD4 enriched cervical cells of Chlamydia trachomatis infected fertile and infertile women upon stimulation with chlamydial inclusion membrane proteins B and C

Background  

Chlamydial Inclusion membrane proteins (Incs), are involved in biochemical interactions with host cells and infecting Chlamydiae. We have previously reported the role of two Chlamydia trachomatis (CT) Incs, namely IncB and IncC in generating host immunity in CT infected women. Emerging data shows involvement of Inc stimulated CD4 positive T cells in aiding host immunity in infected fertile and infertile women through the secretion of interferon gamma. However the lack of data on the intra-cytokine interplay to these Incs in infected cell milieu prompted us to investigate further.     

Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin inhibits the proliferation of ARPE-19 cells

Background  

The antiproliferative effect of the Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin) on human retinal pigment epithelial cells is investigated.     

Predictors of Irrational Parenthood Cognitions in an Iranian Group of Infertile Women

Objectives

The aim of this study was to investigate possible predictors of irrational parenthood cognitions among infertile women seeking treatment.

Methods

In a cross-sectional study, 300 women who visited an Infertility Center in Iran during 2010 were studied. A pre-validated inventory was used to assess irrational parenthood cognitions. Potential predictors of the total irrational parenthood cognitions score were assessed.

Results

Mean irrational parenthood cognition score was 39.7(Range 0–56). Through bivariate analysis, the score on irrational parenthood cognition was inversely correlated with age and positively correlated with length of time seeking for infertility treatment and length of time expecting pregnancy. In a multivariate model, infertile women with higher education, especially academic education, or those with higher economic status were less likely to have irrational parenthood cognitions. However, higher motherhood motivation, no previous experience of pregnancy and being under social pressure, from others around, increased the likelihood of having irrational parenthood cognitions.

Conclusions

Some variables such as female spouse’s educational level and being under social pressure can independently predict irrational parenthood cognitions among infertile women that may be of use in designing health promotion plans in order to target the vulnerable women.     

Use of Endo-Ovarian Tissue Biopsy and Pelvic Aspirated Fluid for the Diagnosis of Female Genital Tuberculosis by Conventional versus Molecular Methods

Background

Til date, none of the diagnostic techniques available for the detection of female genital tuberculosis (FGTB) are 100% accurate. We therefore, proposed to use the endometrial tissue biopsies (ETBs), ovarian tissue biopsies (OTBs) and pelvic aspirated fluids (PAFs) for the diagnosis of FGTB among infertile women by conventional versus molecular methods.

Methodology/Principal Findings

A total of 302 specimens were collected both from 202 infertile women highly suspected of having FGTB on laparoscopy examination and 100 control women of reproductive age. Out of 302 specimens, 150 (49.67%) were ETBs, 95 (31.46%) were OTBs and 57 (18.87%) were PAFs. All specimens were tested by conventional techniques, later compared with multi-gene PCR for the detection of Mycobacterium tuberculosis (MTB) and correlated with laparoscopic findings. The presence of MTB DNA was observed in 49.5% of ETBs, 33.17% of OTBs and 5.44% of PAF specimens collected from highly suspected FGTB patients. All women of control group were confirmed as negative for tuberculosis. The conventional methods showed 99% to 100% specificity with a low sensitivity, ranging from 21.78% to 42.08% while hematoxylin and eosin staining showed a sensitivity of 51.48%. Multi-gene PCR was found to have much higher sensitivity of 70.29% with MTB64 gene, 86.63% with 19 kDa antigen gene at species and TRC4 element at regional MTB complex and 88.12% with 32 kDa protein gene at genus level. The specificity of multi-gene PCR was 100%. Compared with culturing and Ziehl-Neelsen''s staining, multi-gene PCR demonstrated improvement in the detection of FGTB (χ² = 214.612, 1 df, McNemar''s test value <0.0001).

Conclusions Significance

We suggest site specific sampling, irrespective of sample type and amplification of the 19 kDa antigen gene in combination with TRC4 element as a successful multi-gene PCR for the diagnosis of FGTB and differentiation of mycobacterial infection among endo-ovarian tissue biopsies and PAFs taken from infertile women.     

The hexameric structures of human heat shock protein 90

Background

The human 90-kDa heat shock protein (HSP90) functions as a dimeric molecular chaperone. HSP90 identified on the cell surface has been found to play a crucial role in cancer invasion and metastasis, and has become a validated anti-cancer target for drug development. It has been shown to self-assemble into oligomers upon heat shock or divalent cations treatment, but the functional role of the oligomeric states in the chaperone cycle is not fully understood.

Principal Findings

Here we report the crystal structure of a truncated HSP90 that contains the middle segment and the carboxy-terminal domain, termed MC-HSP90. The structure reveals an architecture with triangular bipyramid geometry, in which the building block of the hexameric assembly is a dimer. In solution, MC-HSP90 exists in three major oligomer states, namely dimer, tetramer and hexamer, which were elucidated by size exclusion chromatography and analytical ultracentrifugation. The newly discovered HSP90 isoform HSP90N that lacks the N-terminal ATPase domain also exhibited similar oligomerization states as did MC-HSP90.

Conclusions

While lacking the ATPase domain, both MC-HSP90 and HSP90N can self-assemble into a hexameric structure, spontaneously. The crystal structure of MC-HSP90 reveals that, in addition to the C-terminal dimerization domain, the residue W320 in the M domain plays a critical role in its oligomerization. This study not only demonstrates how the human MC-HSP90 forms a hexamer, but also justifies the similar formation of HSP90N by using 3D modeling analysis.     

The mRNA expression of SATB1 and SATB2 in human breast cancer

Background  

SATB1 is a nuclear protein that has been recently reported to be a 'genome organizer' which delineates specific epigenetic modifications at target gene loci, directly up-regulating metastasis-associated genes while down-regulating tumor-suppressor genes. In this study, the level of mRNA expression of SATB1 and SATB2 were assessed in normal and malignant breast tissue in a cohort of women with breast cancer and correlated to conventional clinico-pathological parameters.     

Immunoproteomics based identification of thioredoxin reductase GliT and novel <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> antigens for serologic diagnosis of invasive aspergillosis

Background  

There has been a rising incidence of invasive aspergillosis (IA) in critically ill patients, even in the absence of an apparent predisposing immunodeficiency. The diagnosis of IA is difficult because clinical signs are not sensitive and specific, and serum galactomannan has relatively low sensitivity in this group of patients. Therefore, more prompt and accurate disease markers for early diagnosis are needed. To establish disease markers demands a thorough knowledge of fungal antigens which may be detected in the serum or other body fluids of patients. Herein we report novel immunodominant antigens identified from extracellular proteins of Aspergillus fumigatus.     

Sperm cells induce distinct cytokine response in peripheral mononuclear cells from infertile women with serum anti-sperm antibodies

Background and Aims

Anti-sperm antibodies in can markedly reduce the likelihood of natural conception. The etiology of this anti-sperm immunity in human females is unknown. We compared the cytokine response of peripheral blood mononuclear cells (PBMCs) from infertile patients with or without anti-sperm antibodies (ASA) and fertile women.

Methodology/Principal Findings

We cultivated the PBMCs together with sperm antigens (whole cells or cell lysate), and screened the supernatants for 40 cytokines by antibody array. When stimulated with whole sperm cells, the PBMCs from patients with ASA produce less IL-3, IL-11, IL-13, ICAM-1, GCSF and more IL-2, IL-4 and IL-12p70 as compared to healthy women. PBMCs from patients with ASA produce typically less IL-13, IL-7, IL-17 and MIG, and more MIP-1β and IL-8, as compared to PBMCs from patients without ASA. In response to sperm cell lysate, PBMCs from infertile women without ASA respond initially by increase in production of growth factors (GCSF, GM-CSF and PDGF-BB) followed by increase in chemokines (e.g. IL-8, MCP-1 and MIP-1β).

Conclusions

Cellular immune responses to sperm antigens, measured by production of cytokines, differ among infertile women with ASA, infertile women without ASA and healthy women. This difference could play an important role in the initial steps of the infertility pathogenesis.     

Oocyte-targeted deletion reveals that hsp90b1 is needed for the completion of first mitosis in mouse zygotes

Background

Hsp90b1 is an endoplasmic reticulum (ER) chaperone (also named Grp94, ERp99, gp96,Targ2, Tra-1, Tra1, Hspc4) (MGI:98817) contributing with Hspa5 (also named Grp78, BIP) (MGI:95835) to protein folding in ER compartment. Besides its high protein expression in mouse oocytes, little is known about Hsp90b1 during the transition from oocyte-to-embryo. Because the constitutive knockout of Hsp90b1 is responsible for peri-implantation embryonic lethality, it was not yet known whether Hsp90b1 is a functionally important maternal factor.

Methodology/Findings

To circumvent embryonic lethality, we established an oocyte-specific conditional knockout line taking advantage of the more recently created floxed Hsp90b1 line (Hsp90b1^flox, MGI:3700023) in combination with the transgenic mouse line expressing the cre recombinase under the control of zona pellucida 3 (ZP3) promoter (Zp3-cre, MGI:2176187). Altered expression of Hsp90b1 in growing oocytes provoked a limited, albeit significant reduction of the zona pellucida thickness but no obvious anomalies in follicular growth, meiotic maturation or fertilization. Interestingly, mutant zygotes obtained from oocytes lacking Hsp90b1 were unable to reach the 2-cell stage. They exhibited either a G2/M block or, more frequently an abnormal mitotic spindle leading to developmental arrest. Despite the fact that Hspa5 displayed a similar profile of expression as Hsp90b1, we found that HSPA5 and HSP90B1 did not fully colocalize in zygotes suggesting distinct function for the two chaperones. Consequently, even if HSPA5 was overexpressed in Hsp90b1 mutant embryos, it did not compensate for HSP90B1 deficiency. Finally, further characterization of ER compartment and cytoskeleton revealed a defective organization of the cytoplasmic region surrounding the mutant zygotic spindle.

Conclusions

Our findings demonstrate that the maternal contribution of Hsp90b1 is critical for the development of murine zygotes. All together our data indicate that Hsp90b1 is involved in unique and specific aspects of the first mitosis, which brings together the maternal and paternal genomes on a single spindle.     

Cartilage oligomeric matrix protein specific antibodies are pathogenic

Introduction

Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP-specific monoclonal antibodies (mAbs).

Methods

B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated and the pathogenicity of mAbs was investigated by passive transfer experiments.

Results

B cell immunodominant epitopes were localized within 4 antigenic domains of the COMP but with preferential response to the epidermal growth factor (EGF)-like domain. Some of our anti-COMP mAbs showed interactions with the native form of COMP, which is present in cartilage and synovium. Passive transfer of COMP-specific mAbs enhanced arthritis when co-administrated with a sub-arthritogenic dose of a mAb specific to collagen type II. Interestingly, we found that a combination of 5 COMP mAbs was capable of inducing arthritis in naive mice.

Conclusions

We have identified the specificities of mAbs to COMP and their contribution to the development of arthritis. These findings will further improve our understanding of the autoantibody mediated immunopathologies occurring widely in rheumatoid arthritis (RA), as well as in other autoimmune disorders.     

Gene trap mutation of murine Outer dense fiber protein-2 gene can result in sperm tail abnormalities in mice with high percentage chimaerism

Background  

Outer dense fiber protein 2, Odf2, is a major component of the outer dense fibers, ODF, in the flagellum of spermatozoa. ODF are associated with microtubule doublets that form the axoneme. We recently demonstrated that tyrosine phosphorylation of Odf2 is important for sperm motility. In the course of a study of Odf2 using Odf2 mouse knockout lines we observed that males of a high percentage chimaerism, made using XL169 embryonic stem cells, were infertile, whereas mice of low-medium percentage chimaerism were fertile.     

Tamoxifen Enhances the Hsp90 Molecular Chaperone ATPase Activity

Background

Hsp90 is an essential molecular chaperone that is also a novel anti-cancer drug target. There is growing interest in developing new drugs that modulate Hsp90 activity.

Methodology/Principal Findings

Using a virtual screening approach, 4-hydroxytamoxifen, the active metabolite of the anti-estrogen drug tamoxifen, was identified as a putative Hsp90 ligand. Surprisingly, while all drugs targeting Hsp90 inhibit the chaperone ATPase activity, it was found experimentally that 4-hydroxytamoxifen and tamoxifen enhance rather than inhibit Hsp90 ATPase.

Conclusions/Significance

Hence, tamoxifen and its metabolite are the first members of a new pharmacological class of Hsp90 activators.     

The NS1 protein of influenza a virus interacts with heat shock protein Hsp90 in human alveolar basal epithelial cells: Implication for virus-induced apoptosis

Background

Our previous study showed that the NS1 protein of highly pathogenic avian influenza A virus H5N1 induced caspase-dependent apoptosis in human alveolar basal epithelial cells (A549), supporting its function as a proapoptotic factor during viral infection, but the mechanism is still unknown.

Results

To characterize the mechanism of NS1-induced apoptosis, we used a two-hybrid system to isolate the potential NS1-interacting partners in A549 cells. We found that heat shock protein 90 (Hsp90) was able to interact with the NS1 proteins derived from both H5N1 and H3N2 viruses, which was verified by co-immunoprecitation assays. Significantly, the NS1 expression in the A549 cells dramatically weakened the interaction between Apaf-1 and Hsp90 but enhanced its interaction with cytochrome c (Cyt c), suggesting that the competitive binding of NS1 to Hsp90 might promote the Apaf-1 to associate with Cyt c and thus facilitate the activation of caspase 9 and caspase 3.

Conclusions

The present results demonstrate that NS1 protein of Influenza A Virus interacts with heat hock protein Hsp90 and meidates the apoptosis induced by influenza A virus through the caspase cascade.

     

UreB immunodominant epitope-specific CD8+ T-cell responses were beneficial in reducing gastric symptoms in Helicobacter pylori-infected individuals

Background and aims

Although Helicobacter pylori is recognized as an extracellular infection bacterium, it can lead to an increase in the number of CD8⁺ T cells after infection. At present, the characteristics of H. pylori antigen-specific CD8⁺ T cells and the epitope response have not been elucidated. This study was focused on putative protective antigen UreB to detect specific CD8⁺ T-cell responses in vitro and screen for predominant response epitopes.

Methods

The PBMCs collected from H. pylori-infected individuals were stimulated by UreB peptide pools in vitro to identify the immunodominant CD8⁺ T-cell epitopes. Furthermore, their HLA restriction characteristics were detected accordingly by NGS. Finally, the relationship between immunodominant responses and appearance of gastric symptoms after H. pylori infection was conducted.

Results

UreB-specific CD8⁺ T-cell responses were detected in H. pylori-infected individuals. Three of UreB dominant epitopes (A-2 (UreB_443–451: GVKPNMIIK), B-4 (UreB_420–428: SEYVGSVEV), and C-1 (UreB_5–13: SRKEYVSMY)) were firstly identified and mainly presented by HLA-A*1101, HLA-B*4001 and HLA-C*0702 alleles, respectively. C-1 responses were mostly occurred in H. pylori-infected subjects without gastric symptoms and may alleviate the degree of gastric inflammation.

Conclusions

The UreB dominant epitope-specific CD8⁺ T-cell response was closely related to the gastric symptoms after H. pylori infection, and the C-1 (UreB_5-13) dominant peptides may be protective epitopes.