Ras-GAP SH3 domain binding protein (G3BP) is a modulator of USP10, a novel human ubiquitin specific protease.

Degradation of cellular proteins through ubiquitination is a fundamental strategy for regulating biological pathways. De-ubiquitination, i.e. the removal of ubiquitin from proteins and peptides to which ubiquitin is attached, is catalyzed by processing proteases known as de-ubiquitinating enzymes. We are studying the biology of a family of de-ubiquitinating enzymes, the mammalian ubiquitin-specific proteases (USPs), some of which appear to play a role in growth control. Given the fact that the modes of regulation of USPs and of their substrate specificity are poorly understood, we decided to attempt the identification of USP interacting proteins. Using the yeast two-hybrid system (2HS), we have isolated a cDNA clone whose product specifically interacts with USP10 but not with other USP baits tested. The isolated clone encodes a protein known to interact with the Ras-GTPase activating protein (G3BP). This interaction was further confirmed by performing a 2HS with G3BP, which led to the isolation of USP10 encoding cDNAs. We validated the interaction between the two proteins by performing in vitro binding assays and immunoprecipitations in human cells. G3BP does not appear to be a substrate of USP10; it rather inhibits the ability of USP10 to disassemble ubiquitin chains. The USP10/G3BP complex appears to co-immunoprecipitate with ubiquitinated species that could be substrates of USP10.

Gamma-hydroxybutyric acid in the treatment of alcohol and heroin dependence.

We briefly review two double-blind, placebo-controlled surveys conducted in this laboratory with the aim of evaluating the efficacy of gamma-hydroxybutyric acid in the treatment of alcohol withdrawal syndrome as well as alcohol craving and consumption in alcoholics. In the first study, acute administration of 50 mg/kg gamma-hydroxybutyric acid, a nonhypnotic dose in alcoholic patients, resulted in a rapid and significant reduction of the severity score of alcohol withdrawal signs and symptoms that lasted as long as 7 hours. In the second study, treatment with 50 mg/kg/day gamma-hydroxybutyric acid for 3 consecutive months (1) reduced the number of daily drinks by approximately 50%, (2) increased the days of abstinence approximately threefold, and (3) reduced the alcohol craving score by up to 60%. These results feature gamma-hydroxybutyric acid as an effective agent for the treatment of alcohol dependence. Data on the effect of gamma-hydroxybutyric acid on opiate withdrawal syndrome also are reviewed. Administration of 25 mg/kg induced a marked reduction of opiate withdrawal score in both heroin- and methadone-dependent subjects. Finally, we report the cases of adverse reactions to and abuse of gamma-hydroxybutyric acid revealed in a retrospective analysis of patients recruited in this laboratory over a 10-year period.

The accumulation of a cold-regulated chloroplastic protein is light-dependent.

The protein encoded by cDNA clone pt59 and induced in barley (Hordeum vulgare L.) by cold was over-expressed in coli to produce the matching antibody, which in vivo recognized a cold-induced protein of 14 kDa (COR14) that was found in the chloroplast stroma. The accumulation of COR14 occurred only at low temperatures after even a brief exposure of the plants to light. Plants grown and fully hardened in the dark accumulated a reduced amount of pt59-corresponding mRNA and only traces of COR14. Light exposure for as short as 5 min was enough to normalize the expression of pt59-corresponding mRNA and increase the accumulation of COR14. These findings indicate that one or more light-dependent factors are involved in transcription of the gene and accumulation of the protein. The COR14 protein was stored in amounts only slightly greater in the resistant barley cultivar. Onice than in the susceptible cultivar Gitane, although the former had a higher induction-temperature threshold for COR14 than the latter. This fact is an evolutionary advantage, enabling the resistant varieties in the field to prepare the cold well ahead of the susceptible ones.

High level expression in E. coli and purification of yeast transcription factor IIIA.

Saccharomyces cerevisiae transcription factor IIIA, a sequence-specific DNA binding protein that is required for transcription of 5S rRNA genes by RNA polymerase III, has been expressed in Escherichia coli in a full length, native form. High level expression was achieved through the combined use of a T7 RNA polymerase expression system and of a multicopy plasmid carrying an E. coli gene, argU, which codes for a minor Arg(AGA/AGG) tRNA species. Recombinant yeast transcription factor IIIA was purified to 95% homogeneity, at a final yield of 8 mg/liter of bacterial culture, by three chromatographic steps, and it was shown to be at least 55% active by quantitative in vitro transcription assays.