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Letter↗
Biomedical subjects
Jean-Jacques Diaz
Publications and source records attributed to Jean-Jacques Diaz.
S-adenosyl methionine decarboxylase activity is required for the outcome of herpes simplex virus type 1 infection and represents a new potential therapeutic target.
All the available antiherpetic drugs are directed against viral proteins. Their extensive clinical use has led to the emergence of resistant viral strains. There is a need for the treatment of herpes infections due to resistant strains, especially for immunocompromised patients. To design new kinds of drugs, we have developed a strategy to identify cellular targets. Herpes simplex virus type 1 (HSV-1) infection is concomitant to a repression of most host protein synthesis. However, some cellular proteins continue to be efficiently synthesized. We speculated that some of them could determine the outcome of infection. Since two polyamines, spermidine and spermine, are components of the HSV-1 virions, we investigated whether enzymes involved in their synthesis could be required for viral infection. We show that inhibition of S-adenosyl methionine decarboxylase, a key enzyme of the polyamine metabolic pathway, prevents HSV-1 infection. Inhibition of polyamine synthesis prevents infection of culture cells with HSV-1 laboratory strains as well as clinical isolates that are resistant to the conventional antiviral drugs acyclovir and foscarnet. Our data provide the opportunity to develop molecules with a novel mechanism of action for the treatment of herpes infection.
Acyclovir↗
Testosterone down-regulates ornithine aminotransferase gene and up-regulates arginase II and ornithine decarboxylase genes for polyamines synthesis in the murine kidney.
The enzymes ornithine aminotransferase (OAT) and ornithine decarboxylase (ODC) share L-ornithine as a common substrate and arginase II produces this amino acid. In the murine kidney, testosterone induced ODC gene expression and polyamine production, but it is unknown how OAT gene is expressed under androgen treatment. These experiments were designed to study the influence of testosterone on the renal expression of OAT gene. Pharmacological and physiological doses of testosterone were injected into female and castrated male mice. Total RNA and soluble proteins extracted from whole kidneys were analyzed by Northern and Western blots, respectively. The results clearly indicate that pharmacological doses of testosterone simultaneously down-regulated the level of OAT protein and up-regulated the expression of arginase II and ODC genes. Variations of the levels of OAT protein and arginase II mRNA and protein were strongly correlated with testosteronemia. Orchidectomy increased the renal level of OAT protein and decreased that of ODC and arginase II. These effects were reversed by injecting a physiological dose of testosterone into castrated male mice. In conclusion, OAT and ODC genes are inversely regulated by testosterone in the mouse kidney. Consequently, in kidneys of testosterone-treated mice, L-arginine-derived ornithine produced by arginase II might be preferentially used by ODC for putrescine production rather than by OAT. This metabolic fate of L-ornithine was facilitated by decreasing OAT gene expression. In contrast, in female and castrated male mice devoided of testosterone, OAT gene is highly expressed and L-ornithine is converted into L-glutamate.
Androgens↗
US11 of herpes simplex virus type 1 interacts with HIPK2 and antagonizes HIPK2-induced cell growth arrest.
Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase of the subfamily of dual-specificity Yak1-related kinase proteins. HIPK2 was first described as a homeodomain-interacting protein kinase acting as a corepressor for homeodomain transcription factors. More recently, it was reported that HIPK2 plays a role in p53-mediated cellular apoptosis and could also participate in the regulation of the cell cycle. US11 protein of herpes simplex virus type 1 is a multifunctional protein involved in the regulation of several processes related to the survival of cells submitted to environmental stresses by mechanisms that are not fully elucidated. In an attempt to better understand the multiple functions of US11, we identified cellular binding partners of this protein by using the yeast two-hybrid system. We report that US11 interacts with HIPK2 through the PEST domain of HIPK2 and that this interaction occurs also in human cells. This interaction modifies the subcellular distribution of HIPK2 and protects the cell against the HIPK2-induced cell growth arrest.
Adaptor Proteins, Signal Transducing↗
Influence of testosterone on regulation of ODC, antizyme, and N1-SSAT gene expression in mouse kidney.
Polyamines are involved in the control of the cell cycle and cell growth. In murine kidney, testosterone enhances gene expression of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis. In this study, we document the time course effect of testosterone on 1) gene expression of ODC, antizyme 1 (AZ1), and spermidine/spermine-N1-acetyltransferase (N1-SSAT); 2) ODC activity in proximal convoluted tubules (PCT) and cortical proximal straight tubules (CPST); and 3) renal polyamine levels. Female mice were treated with testosterone for a period of 1, 2, 3, and 5 consecutive days. ODC gene expression was extremely low in kidneys of untreated female mice compared with that of males. Consequently, the renal putrescine level was sevenfold lower in females than in males, whereas spermidine and spermine levels did not differ between sexes. In female kidneys, testosterone treatment sharply increased ODC mRNA and protein levels as well as ODC activity. Testosterone increased the expression of ODC in PCT and CPST over different time courses, which suggests that ODC activity is differentially regulated in distinct tubules. The expression of AZ1 and N1-SSAT mRNA was similar in male and female mouse kidneys. Testosterone treatment enhanced AZ1 and N1-SSAT mRNA levels in a time-dependent manner by unknown molecular mechanisms. Putrescine and spermidine levels increased after testosterone treatment in female kidneys. Surprisingly, although ODC protein and activity were undetectable in female kidneys, the levels of AZ1 mRNA and protein were similar to those in males. Therefore, one may propose that ODC protein could be continuously degraded by AZ1 in female kidneys.
Acetyltransferases↗
Alteration of ribosomal protein maps in herpes simplex virus type 1 infection.
At present, the effect of herpes simplex virus infection on the entire proteomes of infected cells is very poorly documented. Following several studies performed over the past few years, the modifications of a sub-cellular fraction induced by herpes simplex virus type 1 can be documented. These studies were performed in order to characterize the virally-induced modifications of a major component of the translational apparatus, the ribosomes. The very basic nature of most of the ribosomal proteins renders them very difficult to separate using isoelectric focusing (IEF). Therefore these studies were achieved using several different but related two-dimensional electrophoretic systems which allowed several two-dimensional ribosomal protein maps to be built. Comparison of the ribosomal protein maps built from non-infected cells with those built from infected cells demonstrated that infection by herpes simplex virus type 1 (HSV-1) induces important modifications of ribosomes: (i) non-reversible phosphorylation of ribosomal protein S6; (ii) unusual phosphorylation of several proteins of the small and the large subunits; and (iii) association of viral and cellular proteins to the ribosomal fraction. An overview of these published studies is presented in this review.
Herpes Simplex↗
Hydrogen/deuterium exchange for higher specificity of protein identification by peptide mass fingerprinting.
Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences. Such amounts of material continuously increase protein database sizes. At present, 22 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT. One of the methods of choice for protein identification makes use of specific endoproteolytic cleavage followed by matrix-assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the digested product. Since 1993, when this technique was first demonstrated, the conditions required for a correct identification have changed dramatically. Whilst 4-5 peptides with an uncertainty of 2-3 Da were sufficient for a correct identification in 1993, 10-13 peptides with less than 60 ppm mass error are now required for human and E. coli proteins. This evolution is directly related to the continuous increase in protein database sizes, which causes an increase in the number of false positive matches in identification results. Use of an information complement deduced from the primary protein sequence, in the process of identification by peptide mass fingerprints, can help to increase confidence in the identification results. In this article, we propose the exchange of labile hydrogen atoms with deuterium atoms to provide an alternative information complement. The exchange reaction with optimised techniques has shown an average 95% of hydrogen/deuterium (H/D) exchange on tryptic peptides. This level of exchange was sufficient to single out one or more peptides from a list of potential candidate proteins due to the dependence of H/D exchange on the peptide primary structure. This technique also has clear advantages in the identification of small proteins where direct protein identification is impaired by the limited number of endoproteolytic peptides. Then, information related to primary sequence obtained with this technique could help to identify proteins with high confidence without any expensive tandem mass spectrometry instruments.
Amino Acid Sequence↗
Functional proteomic analysis of human nucleolus.
The notion of a "plurifunctional" nucleolus is now well established. However, molecular mechanisms underlying the biological processes occurring within this nuclear domain remain only partially understood. As a first step in elucidating these mechanisms we have carried out a proteomic analysis to draw up a list of proteins present within nucleoli of HeLa cells. This analysis allowed the identification of 213 different nucleolar proteins. This catalog complements that of the 271 proteins obtained recently by others, giving a total of approximately 350 different nucleolar proteins. Functional classification of these proteins allowed outlining several biological processes taking place within nucleoli. Bioinformatic analyses permitted the assignment of hypothetical functions for 43 proteins for which no functional information is available. Notably, a role in ribosome biogenesis was proposed for 31 proteins. More generally, this functional classification reinforces the plurifunctional nature of nucleoli and provides convincing evidence that nucleoli may play a central role in the control of gene expression. Finally, this analysis supports the recent demonstration of a coupling of transcription and translation in higher eukaryotes.
Cell Nucleolus↗
Unique motif for nucleolar retention and nuclear export regulated by phosphorylation.
By microinjecting purified glutathione S-transferase linked to all or parts of herpes simplex virus type 1 US11 protein into either the nucleus or the cytoplasm, we have demonstrated that this nucleolar protein exhibits a new type of localization signal controlling both retention in nucleoli and export to the cytoplasm. Saturated mutagenesis combined with computer modeling allowed us to draw the fine-structure map of this domain, revealing a new proline-rich motif harboring both activities, which are temperature dependent and regulated by phosphorylation. Finally, crossing the nuclear pore complex from the cytoplasm to the nucleus is an energy-dependent process for US11 protein, while getting to nucleoli through the nucleoplasm is energy independent.
Active Transport, Cell Nucleus↗
Deciphering the human nucleolar proteome.
Nucleoli are plurifunctional nuclear domains involved in the regulation of several major cellular processes such as ribosome biogenesis, the biogenesis of non-ribosomal ribonucleoprotein complexes, cell cycle, and cellular aging. Until recently, the protein content of nucleoli was poorly described. Several proteomic analyses have been undertaken to discover the molecular bases of the biological roles fulfilled by nucleoli. These studies have led to the identification of more than 700 proteins. Extensive bibliographic and bioinformatic analyses allowed the classification of the identified proteins into functional groups and suggested potential functions of 150 human proteins previously uncharacterized. The combination of improvements in mass spectrometry technologies, the characterization of protein complexes, and data mining will assist in furthering our understanding of the role of nucleoli in different physiological and pathological cell states.
Cell Nucleolus↗