Transcriptional Modulation of Heat-Shock Protein Gene Expression

Heat-shock proteins (Hsps) are molecular chaperones that are ubiquitously expressed but are also induced in cells exposed to stressful stimuli. Hsps have been implicated in the induction and propagation of several diseases. This paper focuses on regulatory factors that control the transcription of the genes encoding Hsps. We also highlight how distinct transcription factors are able to interact and modulate Hsps in different pathological states. Thus, a better understanding of the complex signaling pathways regulating Hsp expression may lead to novel therapeutic targets

The HPV cellular transactivator Brn-3a can be used to predict cervical adenocarcinoma and squamous carcinoma precancer lesions in the developed and developing worlds

The cellular transactivator Brn-3a has previously been shown to be expressed at elevated levels in the cervix of women with squamous cell carcinoma of the cervix (SCC) and to activate the expression of HPV E6 mRNA. In this study, we show that common and rare cervical precancer lesions, including those of adenocarcinoma (AC), which are usually difficult to diagnose using classical procedures, also expressed high levels of Brn-3a and can be diagnosed by measuring the levels of Brn-3a and E6 mRNAs

Differential regulation of different human papilloma virus variants by the POU family transcription factor Brn-3a

The Brn-3a POU family transcription factor is over-expressed in human cervical carcinoma biopsies and is able to activate expression of the human papilloma virus type 16 (HPV-16) upstream regulatory region (URR), which drives the expression of the E6 and E7 oncoproteins. Inhibition of Brn-3a expression in human cervical cancer cells inhibits HPV gene expression and reduces cellular growth and anchorage independence in vitro as well as the ability to form tumours in vivo. Here we show that Brn-3a differentially regulates different HPV-16 variants that have previously been shown to be associated with different risks of progression to cervical carcinoma. In human cervical material Brn-3a levels correlate directly with HPV E6 levels in individuals infected with a high risk variant of HPV-16 whereas this is not the case for a low risk variant. Moreover, the URRs of high and intermediate risk variants are activated by Brn-3a in transfection assays whereas the URR of a low risk variant is not. The change of one or two bases in a low risk variant URR to their equivalent in a higher risk URR can render the URR responsive to Brn-3a and vice versa. These results help explain why the specific interplay between viral and cellular factors necessary for the progression to cervical carcinoma, only occurs in a minority of those infected with HPV-16

A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli

BACKGROUND: The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (β(2)GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL. METHODS: Using a computer programme called Juniper, sequentially overlapping primers were designed to be used in a recursive polymerase chain reaction (PCR) to produce a synthetic DI gene. Specifically Juniper incorporates 'major' codons preferred by bacteria altering 41 codons out of 61. This was cloned into the expression plasmid pET(26b) and expressed in BL21(DE3) Escherichia coli (E. coli). By virtue of a pelB leader sequence, periplasmic localisation of DI aided disulphide bond formation and toxicity was addressed by tightly regulating expression through the high stringency T7lac promoter. RESULTS: Purified, soluble his-tagged DI in yields of 750 μg/L bacterial culture was obtained and confirmed on Western blot. Expression using the native human cDNA sequence of DI in the same construct under identical conditions yielded significantly less DI compared to the recombinant optimised sequence. This constitutes the first description of prokaryotic expression of soluble DI of β(2)GPI. Binding to murine monoclonal antibodies that recognise conformationally restricted epitopes on the surface of DI and pathogenic human monoclonal IgG aPL was confirmed by direct and indirect immunoassay. Recombinant DI also bound a series of 21 polyclonal IgG samples derived from patients with APS. CONCLUSION: By producing a synthetic gene globally optimised for expression in E. coli, tightly regulating expression and utilising periplasmic product translocation, efficient, soluble E. coli expression of the eukaryotic protein DI of β(2)GPI is possible. This novel platform of expression utilising pan-gene prokaryote codon optimisation for DI production will aid future antigenic studies. Furthermore if DI or peptide derivatives of DI are eventually used in the therapeutic setting either as toleragen or as a competitive inhibitor of pathogenic aPL, then an E. coli production system may aid cost-effective production

Stable expression of a recombinant human antinucleosome antibody to investigate relationships between antibody sequence, binding properties, and pathogenicity

When purified under rigorous conditions, some murine anti-double-stranded-DNA (anti-dsDNA) antibodies actually bind chromatin rather than dsDNA. This suggests that they may actually be antinucleosome antibodies that only appear to bind dsDNA when they are incompletely dissociated from nucleosomes. Experiments in murine models suggest that antibody–nucleosome complexes may play a crucial role in the pathogenesis of glomerulonephritis in systemic lupus erythematosus. Some human monoclonal anti-DNA antibodies are pathogenic when administered to mice with severe combined immunodeficiency (SCID). Our objective was to achieve stable expression of sequence-altered variants of one such antibody, B3, in Chinese hamster ovary (CHO) cells. Purified antibodies secreted by these cells were tested to investigate whether B3 is actually an antinucleosome antibody. The pathogenic effects of the antibodies were tested by implanting CHO cells secreting them into SCID mice. Purified B3 does not bind to dsDNA unless supernatant from cultured cells is added, but does bind to nucleosomes. The strength of binding to dsDNA and nucleosomes is dependent on the sequence of the light chain. Mice that received CHO cells secreting wild-type B3 developed more proteinuria and died earlier than control mice that received nonsecreting CHO cells or mice that received B3 with a single light chain mutation. However, none of the mice had histological changes or deposition of human immunoglobulin G in the kidneys. Sequence changes may alter the pathogenicity of B3, but further studies using different techniques are needed to investigate this possibility

Brn-3b enhances the pro-apoptotic effects of p53 but not its induction of cell cycle arrest by cooperating in trans-activation of bax expression

The Brn-3a and Brn-3b transcription factor have opposite and antagonistic effects in neuroblastoma cells since Brn-3a is associated with differentiation whilst Brn-3b enhances proliferation in these cells. In this study, we demonstrate that like Brn-3a, Brn-3b physically interacts with p53. However, whereas Brn-3a repressed p53 mediated Bax expression but cooperated with p53 to increase p21(cip1/waf1), this study demonstrated that co-expression of Brn-3b with p53 increases trans-activation of Bax promoter but not p21(cip1/waf1). Consequently co-expression of Brn-3b with p53 resulted in enhanced apoptosis, which is in contrast to the increased survival and differentiation, when Brn-3a is co-expressed with p53. For Brn-3b to cooperate with p53 on the Bax promoter, it requires binding sites that flank p53 sites on this promoter. Furthermore, neurons from Brn-3b knock-out (KO) mice were resistant to apoptosis and this correlated with reduced Bax expression upon induction of p53 in neurons lacking Brn-3b compared with controls. Thus, the ability of Brn-3b to interact with p53 and modulate Bax expression may demonstrate an important mechanism that helps to determine the fate of cells when p53 is induced

Anti-factor Xa antibodies in patients with antiphospholipid syndrome and their effects upon coagulation assays

- Introduction: The aim of this study was to examine the prevalence and functional effects of antibodies directed against Factor (F)Xa and other serine proteases (SP) in patients with antiphospholipid syndrome (APS). - Methods: Serum from patients with APS (n = 59), systemic lupus erythematosus (SLE; n = 106), other autoimmune rheumatic disease (ARD; n = 63) and 40 healthy controls (HC) were tested for IgG activity against thrombin (Thr), FXa, FVIIa, phosphatidylserine (PS)/FXa and antithrombin (AT)-III by enzyme-linked immunosorbent assay (ELISA). Anti-FXa positive IgG were purified to measure their avidity by chaotropic ELISA and functional effects upon clotting time (FXa-ACT) and FXa enzymatic activity (± AT-III). - Results: Anti-FXa IgG were found in patients with SLE (49.1%) and APS (33.9%) (P <0.05) but not in ARD controls and HC. In contrast, anti-Thr and anti-PS/FXa IgG were identified in other ARD and anti-FVIIa IgG were low in all groups. The avidity of APS-IgG to FXa was significantly higher than SLE-IgG (P <0.05). Greatest prolongation of FXa-ACT was observed with APS-IgG and greatest inhibitory effect upon FXa enzymatic activity was found with APS-IgG followed by SLE-IgG compared to HC-IgG. ATIII inhibition of FXa was significantly reduced by APS-IgG compared with HC and SLE (P <0.05) and did not correlate with binding to AT-III. - Conclusion: APS anti-FXa IgG have higher avidity to FXa and greater effects upon the enzymatic and coagulant activity of FXa compared with SLE anti-FXa IgG. Further studies of anti-FXa antibodies in APS, SLE and other non-autoimmune thrombotic disease cohorts are now required to evaluate whether targeting FXa with selective inhibitors in patients bearing anti-FXa antibodies may be an effective treatment strategy

Expression of the SmB′ splicing protein in rodent cells capable of following an alternative RNA splicing pathway

AbstractThe expression of the SmB and SmB′ spliceosome proteins in a variety of cell types and tissues has been investigated. Although SmB is found in all cells studied, the SmB′ protein is found only in a small number of rodent cell types. The presence of this protein is correlated with the ability to utilize an alternative pathway of RNA splicing which is not available in most cell types. This is the first demonstration of tissue specific expression of a protein component of the spliceosome and suggests a role for SmB′ in the regulation of some cases of alternative RNA splicing

PINK1 Is Necessary for Long Term Survival and Mitochondrial Function in Human Dopaminergic Neurons

Parkinson's disease (PD) is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson's disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of-function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD