Ornithine transcarbamylase (OTC) deficiency is the most common inherited disorder of the urea cycle and is transmitted as an X-linked trait. Defects in the OTC gene cause a block in ureagenesis resulting in hyperammonemia, which can lead to brain damage and death. Three previous mutation updates for the OTC gene have been published, in 1993, 1995, and 2002. The most recent comprehensive update, in 2002, contained 244 mutations including 13 nondisease-causing mutations and polymorphisms. This current update reports 341 mutations, of which 93 have not been previously reported, and an additional 29 nondisease-causing mutations and polymorphisms. Out of the 341 mutations, 149 were associated with neonatal onset of hyperammonemia (within the first week of life), 70 were seen in male patients with later onset of hyperammonemia, and 121 were found in heterozygous females (one unknown). Along with the reported mutations, residual enzyme activities and other pertinent clinical information are included whenever available. Most mutations in the OTC gene are specific to a particular family ("private" mutations). They are distributed throughout the gene, with a significant paucity of mutations in the 32 first codons encoding the "leader" peptide (exon 1 and the beginning of exon 2). Almost all mutations in consensus splice sites confer a neonatal onset phenotype. Using the current molecular screening methods, mutations are found in about 80% of the patients. The remaining patients may have mutations in regulatory domains or mutations deep in the introns, which constitute 98.5% of the genomic sequence. In addition, a phenocopy of OTC deficiency caused by mutations in another unknown gene cannot be excluded.

SummaryRegulation of the bacterial phage-shock-protein (Psp) system involves communication between integral (PspBC) and peripheral (PspA) cytoplasmic membrane proteins and a soluble transcriptional activator (PspF). In this study protein subcellular localization studies were used to distinguish between spatial models for this putative signal transduction pathway in Yersinia enterocolitica. In non-inducing conditions PspA and PspF were almost exclusively in the soluble fraction, consistent with them forming an inhibitory complex in the cytoplasm. However, upon induction PspA, but not PspF, mainly associated with the membrane fraction. This membrane association was dependent on PspBC but independent of increased PspA concentration. Analysis of psp null, overexpression and altered function mutants further supported a model where PspA is predominantly membrane associated only when the system is induced. Activation of the Psp system normally leads to a large increase in PspA concentration and we found that this provided a second mechanism for its membrane association, which did not require PspBC. These data suggest that basal PspFABC protein levels constitute a regulatory switch that moves some PspA to the membrane when an inducing trigger is encountered. Once this switch is activated PspA concentration increases, which might then allow it to directly contact the membrane for its physiological function.

Summary PspA, -B and -C regulate the bacterial phage shock protein stress response by controlling the PspF transcription factor. Here, we have developed complementary approaches to study the behavior of these proteins at their endogenous levels in Yersinia enterocolitica. First, we observed GFP-tagged versions with an approach that resolves individual protein complexes in live cells. This revealed that PspA, -B and -C share common behaviors, including a striking contrast before and after induction. In uninduced cells, PspA, -B and -C were highly mobile and widely distributed. However, induction reduced mobility and the proteins became more organized. Combining mCherry- and GFP-tagged proteins also revealed that PspA colocalizes with PspB and PspC into large stationary foci, often located close to the pole of induced cells. In addition, co-immunoprecipitation assays provided the first direct evidence supporting the model that PspA switches binding partners from PspF to PspBC upon induction. Together, these data suggest that PspA, -B and -C do not stably interact and are highly mobile before induction, perhaps sampling the status of the membrane and each other. However, an inducing signal promotes PspABC complex formation and their relocation to discrete parts of the membrane, which might then be important for mitigating envelope stress.

The hepatitis C virus (HCV) is a major cause of chronic liver disease. Here, we report a new and effective strategy for inhibiting HCV replication using 17-allylaminogeldanamycin (17-AAG), an inhibitor of heat-shock protein 90 (Hsp90). Hsp90 is a molecular chaperone with a key role in stabilizing the conformation of many oncogenic signaling proteins. We examined the inhibitory effects of 17-AAG on HCV replication in an HCV replicon cell culture system. In HCV replicon cells treated with 17-AAG, we found that HCV RNA replication was suppressed in a dose-dependent manner, and interestingly, the only HCV protein degraded in these cells was NS3 (nonstructural protein 3). Immunoprecipitation experiments showed that NS3 directly interacted with Hsp90, as did proteins expressed from ⌬NS3 protease expression vectors. These results suggest that the suppression of HCV RNA replication is due to the destabilization of NS3 in disruption of the Hsp90 chaperone complex by 17-AAG. Infection by the hepatitis C virus (HCV)2 is a major public health problem, with 170 million chronically infected people worldwide (1, 2). The current treatment by combined interferon-ribavirin therapy fails to cure the infection in 30 -50% of cases (3, 4), particularly those with HCV genotypes 1 and 2. Chronic infection with HCV results in liver cirrhosis and can lead to hepatocellular carcinoma (5, 6). Although an effective combined interferon-␣-ribavirin therapy is available for about 50% of the patients with HCV, better therapies are needed, and preventative vaccines have not yet been developed.HCV is a member of the Flaviviridae family and has a positive strand RNA genome (7, 8) that encodes a large precursor polyprotein, which is cleaved by host and viral proteases to generate at least 10 functional viral proteins: core, E1 (envelope 1), E2, p7, NS2 (nonstructural protein 2), NS3, NS4A, NS4B, NS5A, and NS5B (9, 10). NS2 and the amino terminus of NS3 comprise the NS2-3 protease responsible for cleavage between NS2 and NS3 (9, 11), whereas NS3 is a multifunctional protein consisting of an amino-terminal protease domain required for processing NS3 to NS5B (12, 13). NS4A is a cofactor that activates the NS3 protease function by forming a heterodimer (14 -17), and the hydrophobic protein NS4B induces the formation of a cytoplasmic vesicular structure, designated the membranous web, which is likely to contain the replication complex of HCV (18,19). NS5A is a phosphoprotein that appears to play an important role in viral replication (20 -23), and NS5B is the RNA-dependent RNA polymerase of HCV (24, 25). The 3Ј-untranslated region consists of a short variable sequence, a poly(U)-poly(UC) tract, and a highly conserved X region and is critical for HCV RNA replication and HCV infection (26 -29).Hsp90 (heat-shock protein 90) is a molecular chaperone that plays a key role in the conformational maturation of many cellular proteins. Hsp90 normally functions in association with other co-chaperone proteins, which together play an important role in folding newly ...

Ornithine transcarbamylase is a highly conserved enzyme in arginine biosynthesis and the urea cycle. In Xanthomonas campestris, the protein annotated as ornithine transcarbamylase, and encoded by the argF gene, is unable to synthesize citrulline directly from ornithine. We cloned and overexpressed this X. campestris gene in Escherichia coli and show that it catalyzes the formation of N-acetyl-L-citrulline from N-acetyl-L-ornithine and carbamyl phosphate. We now designate this enzyme as an acetylornithine transcarbamylase. The K m values for N-acetylornithine and carbamyl phosphate were 1.05 mM and 0.01 mM, respectively. Additional putative transcarbamylases that might also be misannotated were found in the genomes of members of other xanthomonads, Cytophaga, and Bacteroidetes as well as in DNA sequences of bacteria from environmental isolates. It appears that these different paths for arginine biosynthesis arose very early in evolution and that the canonical ornithine transcarbamylase-dependent pathway became the prevalent form. A potent inhibitor, N ␣ -acetyl-N ␦ -phosphonoacetyl-L-ornithine, was synthesized and showed a midpoint of inhibition at approximately 22 nM; this compound may prove to be a useful starting point for designing inhibitors specific to this novel family of transcarbamylases.The enzymatic biosynthesis of L-arginine is accomplished by a complex, highly interconnected pathway with at least eight steps to produce arginine from L-glutamate via a series of acetylated amino acid intermediates (Fig. 1). One of the essential enzymes in the pathway, ornithine transcarbamylase (OTCase) (EC 2.1.3.3), catalyzes the formation of citrulline from ornithine and carbamyl phosphate. Protein sequences within the OTCase family show strong conservation across diverse phylogenetic domains ranging from archaea to mammals (25,26). This suggests that the role of OTCase in arginine biosynthesis was established at a very early stage of evolution, an assertion that is further supported by phylogenetic comparison of the OTCase sequences to the paralogous aspartate transcarbamylases (ATCases) involved in pyrimidine synthesis. Thus, it appears that these two enzyme families already existed at the point of the last universal common ancestor (7).While exploring the arginine biosynthesis pathway in the anaerobe Bacteroides fragilis, we identified and solved the crystal structure of a transcarbamylase-like protein (the product of a gene denoted argFЈ [GI:22218874]) that is essential for arginine biosynthesis (16). However, repeated attempts to detect enzymatic carbamylation of a variety of substrates using the native or recombinant ArgFЈ protein were unsuccessful. The B. fragilis ArgFЈ protein shares only limited sequence homology with other OTCases (38% and 34% similarity to Escherichia coli ArgF and human OTCase, respectively) (16). By comparison, the amino acid sequence of the B. fragilis aspartate transcarbamylase (ATCase) catalytic subunit shares a much higher similarity (70.3%) with E. coli ATCase. Further analysis of t...