András Orosz

Direct Sensing of Heat and Oxidation by Drosophila Heat Shock Transcription Factor

The heat shock transcription factor HSF activates expression of its target genes in response to elevated temperatures and chemical or physiological stress. A key step in the activation process involves the formation of HSF homotrimers, leading to high-affinity DNA binding. The mechanism by which HSF trimerization and DNA binding is regulated by stress signals has remained elusive. Here, we report that trimerization and DNA binding of purified Drosophila HSF can be directly and reversibly induced in vitro by heat shock temperatures in the physiological range and by an oxidant, hydrogen peroxide. Other inducers of the heat shock response, including salicylate, dinitrophenol, ethanol, and arsenite, have no effect on HSF trimerization in vitro, indicating that these inducers act by indirect mechanisms.

Solution structure of the DNA-binding domain of Drosophila heat shock transcription factor.

The solution structure of the DNA-binding domain of the Drosophila heat shock transcription factor, as determined by multidimensional multinuclear NMR, resembles that of the helix-turn-helix class of DNA-binding proteins. The domain comprises a four-stranded antiparallel β-sheet, packed against a three-helix bundle. The second helix is significantly distorted and is separated from the third helix by an extended turn which is subject to conformational averaging on an intermediate time scale. Helix 3 forms a classical amphipathic helix with polar and charged residues exposed to the solvent. Upon titration with DNA, resonance shifts in the backbone and Asn and Gln side-chain amides indicate that helix 3 acts as the recognition helix of the heat shock transcription factor.

Inhibition of CCAAT/Enhancer Binding Protein Family DNA Binding in Mouse Epidermis Prevents and Regresses Papillomas

The CCAAT/enhancer binding proteins (C/EBP) are a family of B-ZIP DNA binding proteins that act as transcription factors to regulate growth and differentiation of many cell types, including keratinocytes. To examine the consequences of inhibiting the C/EBP family of transcription factors in skin, we generated transgenic mice that use the tetracycline system to conditionally express A-C/EBP, a dominant negative that inhibits the DNA binding of C/EBP family members. We expressed A-C/EBP in the basal layer of the skin epidermis during a two-step skin carcinogenesis protocol. A-C/EBP expression caused hyperplasia of the basal epidermis and increased apoptosis in the suprabasal epidermis. The mice developed fewer papillomas and had systemic hair loss. A-C/EBP expression caused C/EBPbeta protein to disappear whereas C/EBPalpha, p53, Bax, and caspase-3 protein levels were dramatically up-regulated in the suprabasal layer. Primary keratinocytes recapitulate the A-C/EBP induction of cell growth and increase in p53 protein. A-C/EBP expression after papilloma development caused the papillomas to regress with an associated increase in apoptosis and up-regulation of p53 protein. Furthermore, A-C/EBP-expressing mice heterozygous for p53 were more susceptible to papilloma formation, suggesting that the suppression of papilloma formation has a p53-dependent mechanism. These results implicate DNA binding of C/EBP family members as a potential molecular therapeutic target.

Inhibition of CREB Function in Mouse Epidermis Reduces Papilloma Formation

We used a double transgenic tetracycline system to conditionally express A-CREB, a dominant negative protein that prevents the DNA binding and function of cAMP-responsive element binding protein (CREB) family members, in mouse basal epidermis using the keratin 5 promoter. There was no phenotype in the adult. However, following a 7,12-dimethylbenz(a)anthracene (DMBA)/phorbol-12-myristate-13-acetate two-stage skin carcinogenesis experiment, A-CREB–expressing epidermis develop 5-fold fewer papillomas than wild-type controls. However, A-CREB expression one month after DMBA treatment does not prevent papilloma formation, suggesting that CREB functions at an early stage of papilloma formation. Oncogenic H-Ras genes with A→T mutations in codon 61 were found in wild-type skin but not in A-CREB–expressing skin 2 days after DMBA treatment, suggesting that A-CREB either prevents DMBA mutagenesis or kills oncogenic H-Ras cells. In primary keratinocyte cultures, A-CREB expression induced apoptosis of v-RasHa–infected cells and suppressed the expression of cell cycle proteins cyclin B1 and cyclin D1. These results suggest that inhibiting CREB function is a valuable cancer prevention strategy.(Mol Cancer Res 2009;7(5):654–64)

A search for candidate genes for lipodystrophy, obesity and diabetes via gene expression analysis of A-ZIP/F-1 mice

Genome scans for diabetes have identified many regions of the human genome that correlate with the disease state. To identify candidate genes for type 2 diabetes, we examined the transgenic A-ZIP/F-1 mouse. This mouse model has no white fat, resulting in abnormal levels of glucose, insulin, and leptin, making the A-ZIP/F-1 mice a good model for lipodystrophy and insulin resistance. We used cDNA-based microarrays to find differentially expressed genes in four tissues of these mice. We examined these results in the context of human linkage scans for lipodystrophy, obesity, and type 2 diabetes. We combined 199 known human orthologs of the misregulated mouse genes with 33 published human genome scans on a genome map. Integrating expression data with human linkage results permitted us to suggest and prioritize candidate genes for lipodystrophy and related disorders. These genes include a cluster of 3 S100A genes on chromosome 1 and SLPI1 on chromosome 20.

Inhibiting activator protein-1 activity alters cocaine-induced gene expression and potentiates sensitization.

We have expressed A-FOS, an inhibitor of activator protein-1 (AP-1) DNA binding, in adult mouse striatal neurons. We observed normal behavior including locomotion and exploratory activities. Following a single injection of cocaine, locomotion increased similarly in both the A-FOS expressing and littermate controls. However, following repeated injections of cocaine, the A-FOS expressing mice showed increased locomotion relative to littermate controls, an increase that persisted following a week of withdrawal and subsequent cocaine administration. These results indicate that AP-1 suppresses this behavioral response to cocaine. We analyzed mRNA from the striatum before and 4 and 24 h after a single cocaine injection in both A-FOS and control striata using Affymetrix microarrays (430 2.0 Array) to identify genes mis-regulated by A-FOS that may mediate the increased locomotor sensitization to cocaine. A-FOS expression did not change gene expression in the basal state or 4 h following cocaine treatment relative to controls. However, 24 h after an acute cocaine treatment, 84 genes were identified that were differentially expressed between the A-FOS and control mice. Fifty-six genes are down-regulated while 28 genes are up-regulated including previously identified candidates for addiction including brain-derived neurotrophic factor and period homolog 1. Using a random sample of identified genes, quantitative PCR was used to verify the microarray studies. The chromosomal location of these 84 genes was compared with human genome scans of addiction to identify potential genes in humans that are involved in addiction.

Purification of heat shock transcription factor of Drosophila

Publisher Summary This chapter discusses the purification of heat-shock transcription factor of Drosophila. All living organisms respond to elevated temperatures, and to a variety of chemical and physiological stresses by a rapid and transient increase in the synthesis of heat-shock proteins. These proteins function as molecular chaperones involved in protein folding, protein translocation, higher order assembly, and protein degradation. In eukaryotes, transcriptional regulation of the heat-shock genes is under the control of a conserved regulator referred to as the heat-shock transcription factor (HSF). HSF acts through the highly conserved heat-shock response element (HSE), composed of three inverted repeats of the 5-bp sequence nGAAn. HSF is present in a latent state under normal conditions and becomes activated as a consequence of heat stress. Activation of HSF generally occurs through two stages: (1) the induction of high-affinity binding to heat-shock promoters, accomplished by trimerization and cooperative interactions between HSF trimers and (2) the exposure of one or more activator domains. The chapter provides protocols for the purification of the endogenous and recombinant HSF proteins.

Alcohol, Altered Protein Homeostasis, and Cancer

Epidemiological studies provide convincing evidence that alcohol consumption is an etiological inducer of human cancers originated primarily in the upper aerodigestive tract, liver, colorectum and female breast, while association is suggested for cancers of the lung and pancreas. The underlying mechanism of alcohol induced tumorigenesis is incompletely understood, but plausible hypotheses suggest mechanisms that are intimately intertwined with alcohol metabolism including the genotoxic effect of acetaldehyde, increased production of reactive oxygen and nitrogen species, altered folate availability and increased estrogen secretion. Alcohol also induces the evolutionally highly conserved cellular stress pathways: the Heat Shock Response (HSR) in the cytoplasm and the Unfolded Protein Response (UPR) in the endoplasmic reticulum, which lead to massive up-regulation of Heat Shock Protein (HSP) expression in both subcellular compartments. HSPs are molecular chaperones that guard against illicit and promiscuous interaction between proteins and serve cytoprotective and anti-apoptotic roles to safeguard and restore normal protein homeostasis or proteostasis following proteotoxic insults (Fig. 8.1). Surprisingly, while HSPs and their master transcriptional regulator Heat Shock Factor 1 (HSF1) increase fitness, subsistence and longevity under most circumstances, recent studies suggest that in cancer cells their augmented expression promote tumorigenesis and rapid somatic evolution under the hostile acidic, nutrient-limiting tumor environment and compromise survival. Elevated level of HSPs is vital to support many types of tumor growth, thus, inhibiting the chaperone function of HSP70 and HSP90 in cancer cells leads to apoptosis and tumor regression. Consequently, HSPs and HSF1 have become the target of rational anticancer drug design representing a new class of tumorigenesis regulators beside oncogenes and tumor suppressors that can be pharmacologically modulated for anticancer treatment. Hence, studying the role of HSF1 and HSPs in alcohol-induced carcinogenesis is critical to gain understanding to their contribution to the oncogenesis process and develop new strategies for prevention, diagnosis and treatment.