A mutation within subdomain 1 of the hepatitis C virus (HCV) NS3 helicase (NS3-Q221L) (M. Yi, Y. Ma, J. Yates, and S. M. Lemon, J. Virol. 81:629-638, 2007) rescues a defect in production of infectious virus by an intergenotypic chimeric RNA (HJ3). Although NS3-Gln-221 is highly conserved across HCV genotypes, the Leu-221 substitution had no effect on RNA replication or NS3-associated enzymatic activities. However, while transfection of unmodified HJ3 RNA failed to produce either extracellular or intracellular infectious virus, transfection of HJ3 RNA containing the Q221L substitution (HJ3/QL) resulted in rapid accumulation of intracellular infectious particles with release into extracellular fluids. In the absence of the Q221L mutation, both NS5A and NS3 were recruited to core protein on the surface of lipid droplets, but there was no assembly of core into high-density, rapidly sedimenting particles. Further analysis demonstrated that a Q221N mutation minimally rescued virus production and led to a second-site I399V mutation in subdomain 2 of the helicase. Similarly, I399V alone allowed only low-level virus production and led to selection of an I286V mutation in subdomain 1 of the helicase which fully restored virus production, confirming the involvement of both major helicase subdomains in the assembly process. Thus, multiple mutations in the helicase rescue a defect in an early-intermediate step in virus assembly that follows the recruitment of NS5A to lipid droplets and precedes the formation of dense intracellular viral particles. These data reveal a previously unsuspected role for the NS3 helicase in early virion morphogenesis and provide a new perspective on HCV assembly.

Matriptase is an epithelial-derived, integral membrane serine protease. The enzyme was initially isolated from human breast cancer cells and has been implicated in breast cancer invasion and metastasis. In the current study, using active matriptase isolated from human milk, we demonstrate that matriptase is able to cleave various synthetic substrates with arginine or lysine as their P1 sites and prefers small side chain amino acids, such as Ala and Gly, at P2 sites. For the most reactive substrates, N-tert-butoxycarbonyl (N-t-Boc)-gamma-benzyl-Glu-Ala-Arg-7-amino-4-methylcoumarin (AMC) and N-t-Boc-Gln-Ala-Arg-AMC, the K(m) values were determined to be 3. 81 and 4.89 microm, respectively. We further demonstrated that matriptase can convert hepatocyte growth factor/scattering factor to its active form, which can induce scatter of Madin-Darby canine kidney epithelial cells and can activate c-Met tyrosine phosphorylation in A549 human lung carcinoma cells. In addition, we noted that matriptase can activate urokinase plasminogen activator but has no affect on plasminogen. These results suggest that matriptase could act as an epithelial, upstream membrane activator to recruit and activate stromal-derived downstream effectors important for extracellular matrix degradation and epithelial migration, two major events of tissue remodeling, cancer invasion, and metastasis.

The heterotrimeric G protein G(s) couples hormone receptors (as well as other receptors) to the effector enzyme adenylyl cyclase and is therefore required for hormone-stimulated intracellular cAMP generation. Receptors activate G(s) by promoting exchange of GTP for GDP on the G(s) alpha-subunit (G(s)alpha) while an intrinsic GTPase activity of G(s)alpha that hydrolyzes bound GTP to GDP leads to deactivation. Mutations of specific G(s)alpha residues (Arg(201) or Gln(227)) that are critical for the GTPase reaction lead to constitutive activation of G(s)-coupled signaling pathways, and such somatic mutations are found in endocrine tumors, fibrous dysplasia of bone, and the McCune-Albright syndrome. Conversely, heterozygous loss-of-function mutations may lead to Albright hereditary osteodystrophy (AHO), a disease characterized by short stature, obesity, brachydactyly, sc ossifications, and mental deficits. Similar mutations are also associated with progressive osseous heteroplasia. Interestingly, paternal transmission of GNAS1 mutations leads to the AHO phenotype alone (pseudopseudohypoparathyroidism), while maternal transmission leads to AHO plus resistance to several hormones (e.g., PTH, TSH) that activate G(s) in their target tissues (pseudohypoparathyroidism type IA). Studies in G(s)alpha knockout mice demonstrate that G(s)alpha is imprinted in a tissue-specific manner, being expressed primarily from the maternal allele in some tissues (e.g., renal proximal tubule, the major site of renal PTH action), while being biallelically expressed in most other tissues. Disrupting mutations in the maternal allele lead to loss of G(s)alpha expression in proximal tubules and therefore loss of PTH action in the kidney, while mutations in the paternal allele have little effect on G(s)alpha expression or PTH action. G(s)alpha has recently been shown to be also imprinted in human pituitary glands. The G(s)alpha gene GNAS1 (as well as its murine ortholog Gnas) has at least four alternative promoters and first exons, leading to the production of alternative gene products including G(s)alpha, XLalphas (a novel G(s)alpha isoform that is expressed only from the paternal allele), and NESP55 (a chromogranin-like protein that is expressed only from the maternal allele). A fourth alternative promoter and first exon (exon 1A) located approximately 2.5 kb upstream of the G(s)alpha promoter is normally methylated on the maternal allele and transcriptionally active on the paternal allele. In patients with isolated renal resistance to PTH (pseudohypoparathyroidism type IB), the exon 1A promoter region has a paternal-specific imprinting pattern on both alleles (unmethylated, transcriptionally active), suggesting that this region is critical for the tissue-specific imprinting of G(s)alpha. The GNAS1 imprinting defect in pseudohypoparathyroidism type IB is predicted to decrease G(s)alpha expression in renal proximal tubules. Studies in G(s)alpha knockout mice also demonstrate that this gene is critical in the regulation of lipid and glucose metabolism.

Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7c. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH2-coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. We conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c (or P3-2 and P3-6a) and not in P2-X.

Previous studies have shown that the glutamate/glutamine (Glu/Gln) neurotransmitter cycle and neuronal glucose oxidation are proportional (1:1), with increasing neuronal activity above isoelectricity. GABA, a product of Glu metabolism, is synthesized from astroglial Gln and contributes to total Glu/Gln neurotransmitter cycling, although the fraction contributed by GABA is unknown. In the present study, we used (13)C NMR spectroscopy together with i.v. infusions of [1,6-(13)C(2)]glucose and [2-(13)C]acetate to separately determine rates of Glu/Gln and GABA/Gln cycling and their respective tricarboxylic acid cycles in the rat cortex under conditions of halothane anesthesia and pentobarbital-induced isoelectricity. Under 1% halothane anesthesia, GABA/Gln cycle flux comprised 23% of total (Glu plus GABA) neurotransmitter cycling and 18% of total neuronal tricarboxylic acid cycle flux. In isoelectric cortex, glucose oxidation was reduced >3-fold in glutamatergic and GABAergic neurons, and neurotransmitter cycling was below detection. Hence, in both cell types, the primary energetic costs are associated with neurotransmission, which increase together as cortical activity is increased. The contribution of GABAergic neurons and inhibition to cortical energy metabolism has broad implications for the interpretation of functional imaging signals.

Environmental carcinogens contained in air pollution, such as polycyclic aromatic hydrocarbons, aromatic amines or N-nitroso compounds, predominantly form DNA adducts but can also generate interstrand cross-links and reactive oxygen species. If unrepaired, such lesions increase the risk of somatic mutations and cancer. Our study investigated the relationships between 22 polymorphisms (and their haplotypes) in 16 DNA repair genes belonging to different repair pathways in 1094 controls and 567 cancer cases (bladder cancer, 131; lung cancer, 134; oral-pharyngeal cancer, 41; laryngeal cancer, 47; leukaemia, 179; death from emphysema and chronic obstructive pulmonary disease, 84). The design was a case-control study nested within a prospective investigation. Among the many comparisons, few polymorphisms were associated with the diseases at the univariate analysis: XRCC1-399 Gln/Gln variant homozygotes [odds ratios (OR) = 2.20, 95% confidence intervals (CI) = 1.16-4.17] and XRCC3-241 Met/Met homozygotes (OR = 0.51, 95% CI = 0.27-0.96) and leukaemia. The recessive model in the stepwise multivariate analysis revealed a possible protective effect of XRCC1-399Gln/Gln in lung cancer (OR = 0.22, 95% CI = 0.05-0.98), and confirmed an opposite effect (OR = 2.47, 95% CI = 1.02-6.02) in the leukaemia group. Our results also suggest that the XPD/ERCC1-GAT haplotype may modulate leukaemia (OR = 1.28, 95% CI = 1.02-1.61), bladder cancer (OR = 1.38, 95% CI = 1.06-1.79) and possibly other cancer risks. Further investigations of the combined effects of polymorphisms within these DNA repair genes, smoking and other risk factors may help to clarify the influence of genetic variation in the carcinogenic process.

Mutations in BRCA1 account for 45% of families with high incidence of breast cancer and for 80-90% of families with both breast and ovarian cancer. BRCA1 protein includes an amino-terminal zinc finger motif as well as an excess of negatively charged amino acids near the C terminus. In addition, BRCA1 contains two nuclear localization signals and localizes to the nucleus of normal cells. While these features suggest a role in transcriptional regulation, no function has been assigned to BRCA1. Here, we show that the C-terminal region, comprising exons 16-24 (aa 1560-1863) of BRCA1 fused to GAL4 DNA binding domain can activate transcription both in yeast and mammalian cells. Furthermore, we define the region comprising exons 21-24 (aa 1760-1863) as the minimal transactivation domain. Any one of four germ-line mutations in the C-terminal region found in patients with breast or ovarian cancer (Ala-1708->>Glu, Gln-1756 C+, Met-1775->>Arg, Tyr-1853 >>Stop), had markedly impaired transcription activity. Together these data underscore the notion that one of the functions of BRCA1 may be the regulation of transcription.

Understanding how nutrients affect gene expression will help us to understand the mechanisms controlling plant growth and development as a function of nutrient availability. Nitrate has been shown to serve as a signal for the control of gene expression in Arabidopsis. There is also evidence, on a gene-by-gene basis, that downstream products of nitrogen (N) assimilation such as glutamate (Glu) or glutamine (Gln) might serve as signals of organic N status that in turn regulate gene expression. To identify genome-wide responses to such organic N signals, Arabidopsis seedlings were transiently treated with ammonium nitrate in the presence or absence of MSX, an inhibitor of glutamine synthetase, resulting in a block of Glu/Gln synthesis. Genes that responded to organic N were identified as those whose response to ammonium nitrate treatment was blocked in the presence of MSX. We showed that some genes previously identified to be regulated by nitrate are under the control of an organic N-metabolite. Using an integrated network model of molecular interactions, we uncovered a subnetwork regulated by organic N that included CCA1 and target genes involved in N-assimilation. We validated some of the predicted interactions and showed that regulation of the master clock control gene CCA1 by Glu or a Glu-derived metabolite in turn regulates the expression of key N-assimilatory genes. Phase response curve analysis shows that distinct N-metabolites can advance or delay the CCA1 phase. Regulation of CCA1 by organic N signals may represent a novel input mechanism for N-nutrients to affect plant circadian clock function.

The forces stabilizing the three-dimensional structures of membrane proteins are currently not well understood. Previously, it was shown that a single Asn side chain in a transmembrane segment can mediate the dimerization and trimerization of a variety of hydrophobic helices. Here, we examine the tendencies of a representative set of amino acids (Asn, Gln, Asp, Glu, Lys, Ala, Val, Leu, Ser, Thr) to direct the oligomerization of a model transmembrane helix. The model peptide is entirely hydrophobic throughout a 20-residue segment and contains a single central site for the introduction of various amino acid "guests." Analytical ultracentrifugation and gel electrophoresis were used to determine the stoichiometry and free energy of association of the entire set of peptides within micelles. Variants with two polar atoms at the guest site-Asn, Gln, Asp, and Glu-formed stable trimers, whereas residues with one or fewer polar atoms showed a much weaker tendency to associate. The data are examined in light of the frequencies of occurrence of various amino acid side chains in membrane proteins and provide insight into the role of polar interactions in directing transmembrane helix association. These data also suggest an approach to the design of variants of natural single-span transmembrane proteins with various potentials to associate in the bilayer.

L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.

DNA fragments of 8.5 kb containing the gyrA gene were cloned from Escherichia coli KL-16 and from four spontaneous gyrA mutants which showed various levels of resistance to quinolones. The gyrA gene was situated at about 4 kb in front of the nrdA gene and transcribed counterclockwise on the E. coli chromosome. It encoded a polypeptide of 875 amino acids with a molecular weight of about 97,000. The four gyrA mutations were located strikingly close to one another within a small region near the N-terminus of the gyrA polypeptide, i.e., nucleotide changes from C to T, from C to G, from G to T and from G to T at nucleotides 248, 248, 318 and 199, respectively, resulting in amino acid changes from Ser to Leu, from Ser to Trp, from Gln to His and from Ala to Ser at amino acids 83, 83, 106 and 67, respectively. These mutations were situated in the relatively hydrophilic regions of the GyrA polypeptide and close to Tyr at amino acid 122 which has been shown to be the site covalently bound to DNA.

We have determined the three-dimensional structure of two crystal forms of an antilysozyme Fab-lysozyme complex by x-ray crystallography. The epitope on lysozyme consists of three sequentially separated subsites, including one long, nearly continuous, site from Gln-41 through Tyr-53 and one from Gly-67 through Pro-70. Antibody residues interacting with lysozyme occur in each of the six complementarity-determining regions and also include one framework residue. Arg-45 and Arg-68 form a ridge on the surface of lysozyme, which binds in a groove on the antibody surface. Otherwise the surface of interaction between the two proteins is relatively flat, although it curls at the edges. The surface of interaction is approximately 26 X 19 A. No water molecules are found in the interface. The positive charge on the two arginines is complemented by the negative charge of Glu-35 and Glu-50 from the heavy chain of the antibody. The backbone structure of the antigen, lysozyme, is mostly unperturbed, although there are some changes in the epitope region, most notably Pro-70. One side chain not in the epitope, Trp-63, undergoes a rotation of approximately 180 degrees about the C beta--C gamma bond. The Fab elbow bends in the two crystal forms differ by 7 degrees.

Although many polar residues are directly involved in transmembrane protein functions, the extent to which they contribute to more general structural features is still unclear. Previous studies have demonstrated that asparagine residues can drive transmembrane helix association through interhelical hydrogen bonding [Choma, C., Gratkowski, H., Lear, J. D. & DeGrado, W. F. (2000) Nat. Struct. Biol. 7, 161-166; and Zhou, F. X., Cocco, M. J., Russ, W. P., Brunger, A. T. & Engelman, D. M. (2000) Nat. Struct. Biol. 7, 154-160]. We have studied the ability of other polar residues to promote helix association in detergent micelles and in biological membranes. Our results show that polyleucine sequences with Asn, Asp, Gln, Glu, and His, residues capable of being simultaneously hydrogen bond donors and acceptors, form homo- or heterooligomers. In contrast, polyleucine sequences with Ser, Thr, and Tyr do not associate more than the polyleucine sequence alone. The results therefore provide experimental evidence that interactions between polar residues in the helices of transmembrane proteins may serve to provide structural stability and oligomerization specificity. Furthermore, such interactions can allow structural flexibility required for the function of some membrane proteins.

Autophagy is a tightly regulated catabolic process that plays key roles in normal cellular homeostasis and survival during periods of extracellular nutrient limitation and stress. The environmental signals that regulate autophagic activity are only partially understood. Here, we report a direct link between glutamine (Gln) metabolism and autophagic activity in both transformed and nontransformed human cells. Cells cultured for more than 2 days in Gln-containing medium showed increases in autophagy that were not attributable to nutrient depletion or to inhibition of mammalian target of rapamycin. Conditioned medium from these cells contained a volatile factor that triggered autophagy in secondary cell cultures. We identified this factor as ammonia derived from the deamination of Gln by glutaminolysis. Gln-dependent ammonia production supported basal autophagy and protected cells from tumor necrosis factor-alpha (TNF-alpha)-induced cell death. Thus, Gln metabolism not only fuels cell growth but also generates an autocrine- and paracrine-acting regulator of autophagic flux in proliferating cells.

Oncogenic Myc alters mitochondrial metabolism, making it dependent on exogenous glutamine (Gln) for cell survival. Accordingly, Gln deprivation selectively induces apoptosis in MYC-overexpressing cells via unknown mechanisms. Using MYCN-amplified neuroblastoma as a model, we identify PUMA, NOXA, and TRB3 as executors of Gln-starved cells. Gln depletion in MYC-transformed cells induces apoptosis through ATF4-dependent, but p53-independent, PUMA and NOXA induction. MYC-transformed cells depend on both glutamate-oxaloacetate transaminase and glutamate dehydrogenase to maintain Gln homeostasis and suppress apoptosis. Consequently, either ATF4 agonists or glutaminolysis inhibitors potently induce apoptosis in vitro and inhibit tumor growth in vivo. These results reveal mechanisms whereby Myc sensitizes cells to apoptosis, and validate ATF4 agonists and inhibitors of Gln metabolism as potential Myc-selective cancer therapeutics.

The X-ray structures of the guanine nucleotide binding domains (amino acids 1-166) of five mutants of the H-ras oncogene product p21 were determined. The mutations described are Gly-12----Arg, Gly-12----Val, Gln-61----His, Gln-61----Leu, which are all oncogenic, and the effector region mutant Asp-38----Glu. The resolutions of the crystal structures range from 2.0 to 2.6 A. Cellular and mutant p21 proteins are almost identical, and the only significant differences are seen in loop L4 and in the vicinity of the gamma-phosphate. For the Gly-12 mutants the larger side chains interfere with GTP binding and/or hydrolysis. Gln-61 in cellular p21 adopts a conformation where it is able to catalyze GTP hydrolysis. This conformation has not been found for the mutants of Gln-61. Furthermore, Leu-61 cannot activate the nucleophilic water because of the chemical nature of its side chain. The D38E mutation preserves its ability to bind GAP.

The Escherichia coli heat shock proteins DnaK and DnaJ function cooperatively as molecular chaperones. Central to their biochemical functions is the ability of DnaJ to interact with DnaK and to stimulate its ATPase activity. Here, we report the genetic isolation of dnaJ12, which has a nonsense mutation at codon 109, yet was able to support lambda growth at 30 degrees C. The 12-kDa DnaJ12 protein was purified to homogeneity and shown to be active in an in vitro lambda-DNA replication system and to be capable of stimulating DnaK's ATPase activity, specifically at the step of ATP hydrolysis. The previously well studied and characterized dnaJ259 mutation was also cloned and sequenced, revealing a single His->>Gln amino acid change at codon 33. The purified DnaJ259 protein was inactive in an in vitro lambda-DNA replication system and was unable to stimulate DnaK's ATPase activity. Consistent with this, an NH2-terminal deletion of the first 34 amino acids or an Asp insertion at residue 35 of DnaJ resulted in a protein that completely lacked DnaJ activity. Collectively, these results demonstrate that the highly conserved NH2-terminal region of DnaJ, the so-called J region, is necessary and sufficient for stimulating both DnaK's ATPase activity and lambda-DNA replication. These results may be applicable to other eukaryotic proteins that contain this conserved J domain as proteins that interact and stimulate the hydrolysis of ATP by their cognate HSP70 proteins.

Breast cancer resistance protein (BCRP) confers multidrug resistance to cancer cells against agents such as SN-38 (an active metabolite of irinotecan), mitoxantrone, and topotecan. Among 59 human tumor cell lines tested, 6 cell lines, A549, NCI-H460, KM-12, HT-29, OVCAR-5, and RPMI8226, showed high BCRP expression. BCRP cDNA was isolated from 11 cancer cell lines and three variant cDNAs [G34A substituting Met for Val-12 (V12M), C421A substituting Lys for Gln-141 (Q141K), and 944-949 deletion lacking Ala-315 and Thr-316 (delta315-6)] were identified. G34A and C421A variants were polymorphisms, and 944-949 deletion was a splicing variant. C421A BCRP-transfected PA317 cells showed markedly decreased protein expression and low-level drug resistance compared with wild-type BCRP-transfected cells when transfectants expressed similar levels of BCRP mRNA. G34A or 944-949-deleted BCRP-transfected PA317 cells showed similar or somewhat lower protein expression and drug resistance compared with wild-type BCRP-transfected cells. Of 124 healthy Japanese volunteers, 67 were wild-type, 48 were heterozygous, and 9 were homozygous for the C421A allele. These results suggest that some people possess the C421A polymorphic BCRP gene and express low amounts of Q141K BCRP. In addition to that, C376T polymorphism in exon 4 substituting stop codon for Gln-126 was found in 3 of the 124 general Japanese population. This C376T polymorphism may also have high impact because active BCRP protein will not be expressed from the C376T allele. Therefore, people with C376T and/or C421A polymorphisms may express low amounts of BCRP, and this low BCRP expression might result in hypersensitivity of normal cells to such anticancer drugs as irinotecan and mitoxantrone.

We have isolated a cDNA from rat small intestine that encodes a novel Na+-independent neutral amino acid transporter with distinctive characteristics in substrate selectivity and transport property. The encoded protein, designated L-type amino acid transporter-2 (LAT-2), shows amino acid sequence similarity to the system L Na+-independent neutral amino acid transporter LAT-1 (Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., and Endou, H. (1998) J. Biol. Chem. 273, 23629-23632) (50% identity) and the system y+L transporters y+LAT-1 (47%) and KIAA0245/y+LAT-2 (45%) (Torrents, D., Estevez, R., Pineda, M., Fernandez, E., Lloberas, J., Shi, Y.-B., Zorzano, A., and Palacin, M. (1998) J. Biol. Chem. 273, 32437-32445). LAT-2 is a nonglycosylated membrane protein. It requires 4F2 heavy chain, a type II membrane glycoprotein, for its functional expression in Xenopus oocytes. LAT-2-mediated transport is not dependent on Na+ or Cl- and is inhibited by a system L-specific inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), indicating that LAT-2 is a second isoform of the system L transporter. Compared with LAT-1, which prefers large neutral amino acids with branched or aromatic side chains, LAT-2 exhibits remarkably broad substrate selectivity. It transports all of the L-isomers of neutral alpha-amino acids. LAT-2 exhibits higher affinity (Km = 30-50 microM) to Tyr, Phe, Trp, Thr, Asn, Ile, Cys, Ser, Leu, Val, and Gln and relatively lower affinity (Km = 180-300 microM) to His, Ala, Met, and Gly. In addition, LAT-2 mediates facilitated diffusion of substrate amino acids, as distinct from LAT-1, which mediates amino acid exchange. LAT-2-mediated transport is increased by lowering the pH level, with peak activity at pH 6.25, because of the decrease in the Km value without changing the Vmax value. Because of these functional properties and a high level of expression of LAT-2 in the small intestine, kidney, placenta, and brain, it is suggested that the heterodimeric complex of LAT-2 and 4F2 heavy chain is involved in the trans-cellular transport of neutral amino acids in epithelia and blood-tissue barriers.

Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast. Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p. Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim. By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC). Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p. In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm. Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p. To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes. A mutant protein containing a Glu->>Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs. Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus.

The beta2-adrenergic receptor (beta2AR) agonists are the most widely used agents in the treatment of asthma, but the genetic determinants of responsiveness to these agents are unknown. Two polymorphic loci within the coding region of the beta2AR have been recently described at amino acids 16 and 27. It has been reported that glycine at codon 16 (Gly-16) is associated with increased agonist-promoted downregulation of the beta2AR as compared with arginine-16 (Arg-16). The form of the receptor with glutamic acid at codon 27 (Glu-27), on the other hand, has been shown to be resistant to downregulation when compared with glutamine-27 (Gln-27), but only when coexpressed with Arg-16. To assess if different genotypes of these two polymorphisms would show differential responses to inhaled beta2AR agonists, we genotyped 269 children who were participants in a longitudinal study of asthma. Spirometry was performed before and after administration of 180 microg of albuterol, and a positive response was considered an increase of >15.3% predicted FEV1. There was marked linkage disequilibrium between the two polymorphisms, with 97.8% of all chromosomes that carried Arg-16 also carrying Gln-27. When compared to homozygotes for Gly-16, homozygotes for Arg-16 were 5.3 times (95% confidence interval 1.6-17.7) and heterozygotes for beta2AR-16 were 2.3 times (1.3-4.2) more likely to respond to albuterol, respectively. Similar trends were observed for asthmatic and nonasthmatic children, and results were independent of baseline lung function, ethnic origin, and previous use of antiasthma medication. No association was found between the beta2AR-27 polymorphism and response to albuterol. These results may explain some of the variability in response to therapeutic doses of albuterol in children.

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

A new NMR experiment is presented for the measurement of micros-ms time scale dynamics of Asn and Gln side chains in proteins. Exchange contributions to the (15)N line widths of side chain residues are determined via a relaxation dispersion experiment in which the effective nitrogen transverse relaxation rate is measured as a function of the number of refocusing pulses in constant-time, variable spacing CPMG intervals. The evolution of magnetization from scalar couplings and dipole-dipole cross-correlations, which has limited studies of exchange in multi-spin systems in the past, does not affect the extraction of accurate exchange parameters from relaxation profiles of NH(2) groups obtained in the present experiment. The utility of the method is demonstrated with an application to a Leu ->> Ala cavity mutant of T4 lysozyme, L99A. It is shown that many of the side chain amide groups of Asn and Gln residues in the C-terminal domain of the protein are affected by a chemical exchange process which may be important in facilitating the rapid binding of hydrophobic ligands to the cavity.

The structure of a complex between a peptide inhibitor with the sequence N-acetyl-Thr-Ile-Nle-psi[CH2-NH]-Nle-Gln-Arg.amide (Nle, norleucine) with chemically synthesized HIV-1 (human immunodeficiency virus 1) protease was determined at 2.3 A resolution (R factor of 0.176). Despite the symmetric nature of the unliganded enzyme, the asymmetric inhibitor lies in a single orientation and makes extensive interactions at the interface between the two subunits of the homodimeric protein. Compared with the unliganded enzyme, the protein molecule underwent substantial changes, particularly in an extended region corresponding to the "flaps" (residues 35 to 57 in each chain), where backbone movements as large as 7 A are observed.