Background: New genes generated by retroposition are widespread in humans and other mammalian species. Usually, this process copies a single parental gene and inserts it into a distant genomic location. However, retroposition of two adjacent parental genes, i.e. co-retroposition, had not been reported until the hominoid chimeric gene, PIPSL, was identified recently. It was shown how two genes linked in tandem (phosphatidylinositol-4-phosphate 5-kinase, type I, alpha, PIP5K1A and proteasome 26S subunit, non-ATPase, 4, PSMD4) could be co-retroposed from a single RNA molecule to form this novel chimeric gene. However, understanding of the origination and biological function of PIPSL requires determination of the coding potential of this gene as well as the evolutionary forces acting on its hominoid copies.Results: We tackled these problems by analyzing the evolutionary signature in both within-species variation and between species divergence in the sequence and structure of the gene. We revealed a significant evolutionary signature: the coding region has significantly lower sequence variation, especially insertions and deletions, suggesting that the human copy may encode a protein. Moreover, a survey across five different hominoid species revealed that all adaptive changes of PSMD4-derived regions occurred on branches leading to human and chimp rather than other hominoid lineages. Finally, computational analysis suggests testis-specific transcription of PIPSL is regulated by tissue-dependent methylation rather than some transcriptional leakage.Conclusion: Therefore, this set of analyses showed that PIPSL is an extraordinary co-retroposed protein-coding gene that may participate in the male functions of humans and its close relatives.

NADP(H) is an important cofactor that controls many fundamental cellular processes. We have determined the crystal structure of HSCARG, a novel NADPH sensor, and found that it forms an asymmetrical dimer with only one subunit occupied by an NADPH molecule, and the two subunits have dramatically different conformations. To study the role of NADPH in affecting the structure and function of HSCARG, here, we constructed a series of HSCARG mutants to abolish NADPH binding ability. Protein structures of two mutants, R37A and Y81A, were solved by X-ray crystallography. The dimerization of wild-type and mutant HSCARG was studied by dynamic light scattering. Differences between the function of wild-type and mutant HSCARG were also compared. Our results show that binding of NADPH is necessary for HSCARG to form a stable asymmetric dimer. The conformation of the monomeric mutants was similar to that of NADPHbound Molecule I in wild-type HSCARG, although some conformational changes were found in the NADPH binding site. Furthermore, we also noticed that abolition of NADPH binding ability changes the distribution of HSCARG in the cell and that these mutants without NADPH are more strongly associated with argininosuccinate synthetase as compared with wild-type HSCARG. These data suggest that NADPH functions as an allosteric regulator of the structure and function of HSCARG. In response to the changes in the NADPH/NADP+ ratio within cells, HSCARG, as a redox sensor, associates and dissociates with NADPH to form a new dynamic equilibrium. This equilibrium, in turn, will tip the dimerization balance of the protein molecule and consequently controls the regulatory function of HSCARG.

Molecular patterns in pathogenic RNAs can be recognized by the innate immune system, and a component of this response is the interferon-induced enzyme RNA-activated protein kinase (PKR). The major activators of PKR have been proposed to be long double-stranded RNAs. We report that RNAs with very limited secondary structures activate PKR in a 5'-triphosphate-dependent fashion in vitro and in vivo. Activation of PKR by 5'-triphosphate RNA is independent of RIG-I and is enhanced by treatment with type 1 interferon (IFN-a). Surveillance of molecular features at the 5' end of transcripts by PKR presents a means of allowing pathogenic RNA to be distinguished from self-RNA. The evidence presented here suggests that this form of RNA-based discrimination may be a critical step in mounting an early immune response.

NAD(P) has long been known as an essential energy-carrying molecule in cells. Recent data, however, indicate that NAD(P) also plays critical signaling roles in regulating cellular functions. The crystal structure of a human protein, HSCARG, with functions previously unknown, has been determined to 2.4-Å resolution. The structure reveals that HSCARG can form an asymmetrical dimer with one subunit occupied by one NADP molecule and the other empty. Restructuring of its NAD(P)-binding Rossmann fold upon NADP binding changes an extended loop to an _-helix to restore the integrity of the Rossmann fold. The previously unobserved restructuring suggests that HSCARG may assume a resting state when the level of NADP(H) is normal within the cell. When the NADP(H) level passes a threshold, an extensive restructuring of HSCARG would result in the activation of its regulatory functions. Immunofluorescent imaging shows that HSCARG redistributes from being associated with intermediate filaments in the resting state to being dispersed in the nucleus and the cytoplasm. The structural change of HSCARG upon NADP(H) binding could be a new regulatory mechanism that responds only to a significant change of NADP(H) levels. One of the functions regulated by HSCARG may be argininosuccinate synthetase that is involved in NO synthesis.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a potential target for structure-based inhibitor design for the treatment of parasitic diseases. We created point mutants of Thermoanaerobacter tengcongensis HGPRT and tested their activities to identify side chains that were important for function. Mutating residues Leu160 and Lys133 substantially diminished the activity of HGPRT, confirming their importance in catalysis. All 11 HGPRT mutants were subject to crystallization screening. The crystal structure of one mutant, L160I, was determined at 1.7 A ˚ resolution. Surprisingly, the active site is occupied by a peptide from the N-terminus of a neighboring tetramer. These crystal contacts suggest an alternate strategy for structure-based inhibitor design.

The use of oseltamivir, widely stockpiled as one of the drugs for use in a possible avian influenza pandemic, has been reported to be associated with neuropsychiatric disorders and severe skin reactions, primarily in Japan. Here we identified a nonsynonymous SNP (single nucleotide polymorphism) in dbSNP database, R41Q, near the enzymatic active site of human cytosolic sialidase, a homologue of virus neuraminidase that is the target of oseltamivir. This SNP occurred in 9.29% of Asian population and none of European and African American population. Our structural analyses and Ki measurements using in vitro sialidase assays indicated that this SNP could increase the unintended binding affinity of human sialidase to oseltamivir carboxylate, the active form of oseltamivir, thus reducing sialidase activity. In addition, this SNP itself results in an enzyme with an intrinsically lower sialidase activity, as shown by its increased Km and decreased Vmax values. Theoretically administration of oseltamivir to people with this SNP might further reduce their sialidase activity. We note the similarity between the reported neuropsychiatric side effects of oseltamivir and the known symptoms of human sialidase-related disorders. We propose that this Asian-enriched sialidase variation caused by the SNP, likely in homozygous form, may be associated with certain severe adverse reactions to oseltamivir.

Background: Hepatitis B virus (HBV) infection is a serious health problem worldwide. Treatment recommendation and response are mainly indicated by viral load, e antigen (HBeAg) seroconversion, and ALT levels. The S antigen (HBsAg) seroconversion is much less frequent. Since HBeAg can be negative in the presence of high viral replication, preS antigen (HBpreSAg) might be a useful indicator in management of chronic HBV infection.Results: A new assay of double antibody sandwich ELISA was established to detect preS antigens. Sera of 104 HBeAg-negative and 50 HBeAg-positive chronic hepatitis B patients have been studied and 23 HBeAg-positive patients were enrolled in a treatment follow-up study. 70% of the HbeAgpositive patients and 47% of the HBeAg-negative patients showed HBpreSAg positive. Particularly, in the HBeAg-negative patients, 30 out of 47 HBpreSAg positive patients showed no evidence of viral replication based on HBV DNA copies. A comparison with HBV DNA copies demonstrated that the overall accuracy of the HBpreSAg test could reach 72% for active HBV replication. HBpreSAg changes were well correlated with changes of HBsAg, HBV DNA and ALT levels during the course of IFN-α treatment and follow-up. HBeAg positive patients responded well to treatment when reduction of HBpreSAg levels was more pronounced.Conclusion: Our results suggested that HBpreSAg could be detected effectively, and well correlated with HBsAg and HBV DNA copies. The reduction of HBpreSAg levels in conjunction with the HBV DNA copies appears to be an improved predictor of treatment outcome.

CARP is a novel pro-apoptotic protein that has been cloned and characterized in our previous report. Previous studies showed that suppression of CARP expression results in cell proliferation in several mammalian cell lines and over-expression of CARP leads to apoptosis and inhibition of proliferation in seven tumor cell lines [Liu et al., CARP is a novel caspase recruitment domain containing pro-apoptotic protein, Biochem. Biophys. Res. Commun. 293 (2002) 1396]. To obtain soluble and active form of CARP protein for further functional and structural studies, we have expressed CARP in Escherichia coli by using Gateway cloning system. Optimal induction and expression conditions were also studied. Recombinant histidine-tagged CARP was expressed in E. coli when the carp gene was subcloned into a Gateway expression vector pET21-DEST. The partially soluble recombinant CARP protein was puriWed to near homogeneity by a two-step FPLC procedure, Wrst by Ni2+ aYnity chromatography followed by a gel-Wltration chromatography, which yielded about 10mg protein/L culture with at least 95% purity. Two peaks were detected in the analytical gel-Wltration chromatograph while only one peak corresponding to monomer of the CARP protein was left after adding 2mM dithiothreitol (DTT). The polymers observed are likely due to the formation of intermolecular disulWde bridges. These results suggest that adding DTT is a good solution to prevent the formation of disulWde bonds and to stabilize the protein. Successfully growing crystals of the puriWed CARP protein also proved that we can produce well folded CARP protein in E. coli.

Imidazolonepropionase (EC 3.5.2.7) is the third enzyme of the histidine degradation pathway that has been conserved from bacteria to eukaryotes. The enzyme is the only one with unknown three-dimensional structure in this pathway. In this work, Bacillus subtilis imidazolonepropionase (HutI) was expressed in E. coli and purified to homogeneity. After thrombin digestion, high quality crystals were obtained by hanging-drop vapor diffusion method. The best crystal diffracted to 2.0 A° and belonged to the space group P21 with unit-cell parameters a=57.73A°, b=106.34A°, c=66.47A°, b=89.93.

The adrenal gland protein AD-004 like protein (ADLP) from Caenorhabditis elegans was cloned and expressed in Escherichia coli. Enzyme assays showed that ADLP has special adenylate kinase (AK) properties, with ATP and dATP as the preferred phosphate donors. In contrast to all other AK isoforms, AMP and dAMP were the preferred substrates of ADLP; CMP, TMP and shikimate acid were also good substrates. Subcellular localization studies showed a predominant nuclear localization for this protein, which is different from AK1–AK5, but similar to that of human AK6. These results suggest that ADLP is more likely a member of the AK6 family. Furthermore, RNAi experiments targeting ADLP were conducted and showed that RNAi treatment resulted in the suppression of worm growth.