Itzik Harosh - Academia.edu (original) (raw)

Papers by Itzik Harosh

The Journal of biological chemistry, Jan 5, 1989

The Rad3 ATPase/DNA helicase was purified to physical homogeneity from extracts of yeast cells co... more The Rad3 ATPase/DNA helicase was purified to physical homogeneity from extracts of yeast cells containing overexpressed Rad3 protein. The DNA helicase can unwind duplex regions as short as 11 base pairs in a partially duplex circular DNA substrate and does so by a strictly processive mechanism. On partially duplex linear substrates, the enzyme has a strict 5'----3' polarity with respect to the single strand to which it binds. Nicked circular DNA is not utilized as a substrate, and the enzyme requires single-stranded gaps between 5 and 21 nucleotides long to unwind oligonucleotide fragments from partially duplex linear molecules. The enzyme also requires duplex regions at least 11 base pairs long when these are present at the ends of linear molecules. Rad3 DNA helicase activity is inhibited by the presence of ultraviolet-induced photoproducts in duplex regions of partially duplex circular molecules.

Future medicinal chemistry, 2010

Several hundred genes associated or linked to obesity have been described in the scientific liter... more Several hundred genes associated or linked to obesity have been described in the scientific literature. Whereas many of these genes are potential targets for the treatment of obesity and associated conditions, none of them have permitted the developement of an efficient drug therapy. As proposed by the 'thrifty genotype' theory, obesity genes may have conferred an evolutionary advantage in times of food shortage through efficient energy exploitation, while 'lean' or 'energy expenditure' genes may have become very rare during the same periods. It is therefore a challenge to identify 'energy expenditure genes' or 'energy absorption genes,' whose mutations or single nucleotide polymorphisms do result in reduced energy intake. We submit that such 'energy absorption' or 'energy expenditure' genes (crucial genes) are potential new targets for the treatment of obesity. These genes can be identified in rare genetic diseases that produc...

The Journal of biological chemistry, Jan 5, 1988

Hypoxanthine-DNA glycosylase from Escherichia coli was partially purified by ammonium sulfate fra... more Hypoxanthine-DNA glycosylase from Escherichia coli was partially purified by ammonium sulfate fractionation and by chromatography on Sephacryl S-200, DEAE-cellulose, and phosphocellulose P-11 columns. Analysis of the enzymatic reaction products was carried out on a minicolumn of DEAE-cellulose and/or by paper chromatography, by following the release of the free base [3H]hypoxanthine from [3H]dIMP-containing phi X174 DNA. In native conditions, the enzyme has a molecular mass of 60 +/- 4 kDa, as determined by gel filtration on Sephadex G-150 and Sephacryl S-200 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed a major polypeptide band of an apparent molecular mass of 56 kDa, and glycerol gradient centrifugation indicated a sedimentation coefficient of 4.0 S. Hypoxanthine-DNA glycosylase from E. coli has an obligatory requirement for Mg2+ and is totally inhibited in the presence of EDTA. Co2+ can only partially replace Mg2+. The enzyme is inhibited by...

The Journal of biological chemistry, Jan 15, 1992

Rad3 protein from the yeast Saccharomyces cerevisiae is a single-stranded DNA-dependent ATPase wh... more Rad3 protein from the yeast Saccharomyces cerevisiae is a single-stranded DNA-dependent ATPase which catalyzes the unwinding of DNA.DNA duplexes. In the present studies we have demonstrated that the purified enzyme additionally catalyzes the displacement of RNA fragments annealed to complementary DNA. Quantitative comparisons using otherwise identical partially duplex DNA.DNA and DNA.RNA substrates indicate a significant preference for the latter. Competition for ATPase or DNA helicase activity by various homopolymers suggests that Rad3 protein does not discriminate between ribonucleotide and deoxyribonucleotide homopolymers with respect to binding. However, neither single-stranded RNA nor various ribonucleotide homopolymers supported the hydrolysis of nucleoside 5'-triphosphates. Additionally, Rad3 protein was unable to catalyze the displacement of oligo(dA) annealed to poly(U), suggesting that the catalytic domain of the enzyme is exquisitely sensitive to chemical and/or or co...

Current Pharmaceutical Biotechnology, 2014

Treatments of obesity and type II diabetes target often gene functions involved in appetite-satie... more Treatments of obesity and type II diabetes target often gene functions involved in appetite-satiety, fat and carbohydrate metabolism or thermogenesis. None of these, have provided efficient drug therapy due to a large number of genes involved in weight and energy management, the redundancy of biochemical pathways and the environmental factors. Here I discuss a new approach based on studies of genes/proteins that are associated with human "lean or starvation" phenotype that became very rare in the course of evolution. This approach has led to the identification of the congenital enteropeptidase deficiency gene and the Andersonʼs Disease gene as a potential targets for obesity and type II diabetes treatment. The advantages of these targets are: 1) they are expressed exclusively in the intestine; 2) they are peripheral targets as opposed to systemic targets; 3) they are not redundant targets. These targets open new hopes for the development of novel drugs for the treatment of common metabolic syndrome.

European Journal of Biochemistry, 1991

Deoxyribonuclease I1 has been purified through five fractionation steps from the human lymphoblas... more Deoxyribonuclease I1 has been purified through five fractionation steps from the human lymphoblast cell line K562. Isolation included DEAE-cellulose and heparinagarose chromatography followed by fractionation on Mono-S, Mono-Q and Superose-12 FPLC columns. In an extension of previous studies, deoxyribonuclease I1 was found to introduce a much higher proportion of single-strand nicks relative to double-strand breaks into supercoiled DNA than has been reported for linear DNA. Application of DNA sequencing techniques has further revealed a unique resistance of 3' termini to hydrolysis by this enzyme.

PLoS ONE, 2012

Background: Obesity research focuses essentially on gene targets associated with the obese phenot... more Background: Obesity research focuses essentially on gene targets associated with the obese phenotype. None of these targets have yet provided a viable drug therapy. Focusing instead on genes that are involved in energy absorption and that are associated with a ''human starvation phenotype'', we have identified enteropeptidase (EP), a gene associated with congenital enteropeptidase deficiency, as a novel target for obesity treatment. The advantages of this target are that the gene is expressed exclusively in the brush border of the intestine; it is peripheral and not redundant.

Nucleic Acids Research, 1991

The Rad3 protein from Saccharomyces cerevisiae is a DNA repair protein required for the incision ... more The Rad3 protein from Saccharomyces cerevisiae is a DNA repair protein required for the incision of ultraviolet light-damaged DNA and has a second function, yet unknown, which is essential for viability of the cell (1). The gene was cloned and sequenced and its gene product was purified to homogeneity. The Rad3 protein is a DNA-dependent ATPase and a DNA helicase with a 5' to 3' polarity and requires either ATP or dATP to drive the DNA helicase activity (2). Disruption of the RAD3 gene renders the haploid yeast cells inviable (1). Site directed mutagenesis study has shown that opal, amber and ochre mutations at positions 554, 564 and 577 respectively are lethal to the cell and substitution of the amino acids Gly and Ala at positions 595 and 596, respectively, results in cell death (all mutations in box no. VII). It should be noted that the essential function of the gene is independent from the DNA repair activity and each activity can be mutated independently (1). However, the expanding list of DNA helicases associated with RNA helicase activity or with RNA helicase-like motifs bearing the DEAD or DEAH box has prompted us to look whether the Rad3 DNA helicase contains this box as well as other conserved motifs and if the essential function for viability lies within these motifs.

Molecular Microbiology, 1993

The Rad3 protein from Saccharomyces cerevisiae is a DNA helicase which participates in the repair... more The Rad3 protein from Saccharomyces cerevisiae is a DNA helicase which participates in the repair of ultraviolet-irradiated DNA and is inhibited in the presence of DNA containing thymine dimers. This protein is also involved in mitotic recombination and spontaneous mutagenesis and is essential for cell viability in the absence of DNA damage. Furthermore, the Rad3 protein also exhibits a DNA:RNA helicase activity in which there is a significant preference for a partial DNA:RNA hybrid rather than a partial duplex DNA substrate, which suggests that this protein might be involved in DNA repair within transcriptionally active genes. Finally, the Rad3 protein contains the DEAH motif and shares high amino acid sequence similarity with the DEAD family of RNA helicase proteins, suggesting that it might also possess an RNA helicase activity.

Journal of Biological Chemistry, 1999

In the search of new strategies to fight against obesity, we targeted a gene pathway involved in ... more In the search of new strategies to fight against obesity, we targeted a gene pathway involved in energy uptake. We have thus investigated the APOB mRNA editing protein (APOBEC1) gene pathway that is involved in fat absorption in the intestine. The APOB gene encodes two proteins, APOB100 and APOB48, via the editing of a single nucleotide in the APOB mRNA by the APOBEC1 enzyme. The APOB48 protein is mandatory for the synthesis of chylomicrons by intestinal cells to transport dietary lipids and cholesterol. We produced transgenic rabbits expressing permanently and ubiquitously a small hairpin RNA targeting the rabbit APOBEC1 mRNA. These rabbits exhibited a moderately but significantly reduced level of APOBEC1 gene expression in the intestine, a reduced level of editing of the APOB mRNA, a reduced level of synthesis of chylomicrons after a food challenge, a reduced total mass of body lipids and finally presented a sustained lean phenotype without any obvious physiological disorder. Interestingly, no compensatory mechanism opposed to the phenotype. These lean transgenic rabbits were crossed with transgenic rabbits expressing in the intestine the human APOBEC1 gene. Double transgenic animals did not present any lean phenotype, thus proving that the intestinal expression of the human APOBEC1 transgene was able to counterbalance the reduction of the rabbit APOBEC1 gene expression. Thus, a moderate reduction of the APOBEC1 dependent editing induces a lean phenotype at least in the rabbit species. This suggests that the APOBEC1 gene might be a novel target for obesity treatment. Funding: This study was funded by Agence Nationale de la Recherche (ANR-06-RIB-FATSTOP) to ObeTherapy as leader of the program and to INRA. Sandrine Braud and Itzik Harosh are employees of and shareholders in ObeTherapy Biotechnology. ObeTherapy Biotechnology provided support in the form of salaries for authors SB and IH, had a direct role in the study design, and analysis, decision to publish, and preparation of the manuscript. Competing Interests: Sandrine Braud and Itzik Harosh are employees of and shareholders in ObeTherapy Biotechnology. There are no products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. * Email: genevieve.jolivet@jouy.inra.fr (GJ); harosh@obetherapy.com(IH)