The NRT2.1 gene of Arabidopsis thaliana encodes a major component of the root high-affinity NO(3)(-) transport system (HATS) that plays a crucial role in NO(3)(-) uptake by the plant. Although NRT2.1 was known to be induced by NO(3)(-) and feedback repressed by reduced nitrogen (N) metabolites, NRT2.1 is surprisingly up-regulated when NO(3)(-) concentration decreases to a low level (<0.5 mm) in media containing a high concentration of NH(4)(+) or Gln >>or=1 mm). The NRT3.1 gene, encoding another key component of the HATS, displays the same response pattern. This revealed that both NRT2.1 and NRT3.1 are coordinately down-regulated by high external NO(3)(-) availability through a mechanism independent from that involving N metabolites. We show here that repression of both genes by high NO(3)(-) is specifically mediated by the NRT1.1 NO(3)(-) transporter. This mechanism warrants that either NRT1.1 or NRT2.1 is active in taking up NO(3)(-) in the presence of a reduced N source. Under low NO(3)(-)/high NH(4)(+) provision, NRT1.1-mediated repression of NRT2.1/NRT3.1 is relieved, which allows reactivation of the HATS. Analysis of atnrt2.1 mutants showed that this constitutes a crucial adaptive response against NH(4)(+) toxicity because NO(3)(-) taken up by the HATS in this situation prevents the detrimental effects of pure NH(4)(+) nutrition. It is thus hypothesized that NRT1.1-mediated regulation of NRT2.1/NRT3.1 is a mechanism aiming to satisfy a specific NO(3)(-) demand of the plant in relation to the various specific roles that NO(3)(-) plays, in addition to being a N source. A new model is proposed for regulation of the HATS, involving both feedback repression by N metabolites and NRT1.1-mediated repression by high NO(3)(-).

We have studied binding and block of sodium channels by 12 derivatives of the 22-residue peptide mu-conotoxin GIIIA (mu-CTX) in which single amino acids were substituted as follows: Arg or Lys by Gln, Gln-18 by Lys, Asp by Asn, and HO-Pro by Pro. Derivatives were synthesized as described by Becker et al. [(1989) Eur. J. Biochem. 185, 79]. Binding was measured by displacement of labeled saxitoxin from eel electroplax membranes (100 mM choline chloride, 10 mM HEPES-NaOH, pH 7.4). Blocking kinetics were evaluated from steady-state, single-channel recordings from rat skeletal muscle sodium channels incorporated into planar, neutral phospholipid/decane bilayers (200 mM NaCl, 10 mM HEPES-NaOH, pH 7.0). Blocking events generally appeared as periods of seconds to minutes in which current through the single channel was completely eliminated. A notable exception was seen for the substitution Arg-13-Gln for which the "blocked" events showed measurable conductances of about 20-40% of the open state. The substitution of Arg-13 reduced binding to electroplax membranes to undetectable levels and increased the apparent dissociation constant determined for skeletal muscle channels by greater than 80-fold compared with the native peptide. Other substitutions caused smaller decreases in affinity. The decreased potency of the toxin derivatives resulted both from increases in the rates of dissociation from the channel, and from decreases in association rates. Our data support the suggestion by Sato et al. [(1991) J. Biol. Chem. 265, 16989] that Arg-13 associates intimately with the binding site on the channel. In addition, our results suggest that certain residues affect almost exclusively the approach and docking of the toxin with its binding site, others appear to be important only to the strength of the association once binding has taken place, and yet others affect both.

TDP-43 is one of the major components of the neuronal and glial inclusions observed in several neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. These characteristic aggregates are a "landmark" of the disease, but their role in the pathogenesis is still obscure. In previous works, we have shown that the C-terminal Gln/Asn-rich region (residues 321-366) of TDP-43 is involved in the interaction of this protein with other members of the heterogeneous nuclear ribonucleoprotein protein family. Furthermore, we have shown that the interaction through this region is important for TDP-43 splicing inhibition of cystic fibrosis transmembrane regulator exon 9, and there were indications that it was involved in the aggregation process. Our experiments show that in cell lines and primary rat neuronal cultures, the introduction of tandem repeats carrying the 331-369-residue Gln/Asn region from TDP-43 can trigger the formation of phosphorylated and ubiquitinated aggregates that recapitulate many but not all the characteristics observed in patients. These results establish a much needed cell-based TDP-43 aggregation model useful to investigate the mechanisms involved in the formation of inclusions and the gain- and loss-of-function consequences of TDP-43 aggregation within cells. In addition, it will be a powerful tool to test novel therapeutic strategies/effectors aimed at preventing/reducing this phenomenon.

Carpal tunnel syndrome (CTS) has been associated with amyloid deposits and is now regarded as a major complication in chronic hemodialysis patients. While this new syndrome has been receiving increasing attention, its etiology has not been clarified. We have isolated amyloid fibrils from amyloid laden tissues inside the carpal tunnel in four different hemodialysis patients with CTS. After solubilization in guanidine HCl, a significant amount of the protein was located in a homogeneous, low molecular weight fraction. Each protein was found to be identical to beta 2-microglobulin with regard to its molecular weight of 11,000 on SDS-PAGE, amino acid composition and N-terminal amino acids: Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu. In direct immunofluorescent study, anti-beta 2-microglobulin did react positively with amyloid deposits. These results demonstrate that the amyloid associated with chronic hemodialysis contains as major component a new form of amyloid fibril protein that is homologous to beta 2-microglobulin. It is postulated that beta 2-microglobulin cannot be removed from the blood by conventional hemodialysis, and accumulates in tissues causing the formation of amyloid fibrils, which, having a relatively high affinity to the carpal tunnel area, thus causes CTS.

Signal transduction underlying bacterial chemotaxis involves excitatory phosphorylation and feedback control through deamidation and methylation of sensory receptors. The structure of a complex between the signal-terminating phosphatase, CheC, and the receptor-modifying deamidase, CheD, reveals how CheC mimics receptor substrates to inhibit CheD and how CheD stimulates CheC phosphatase activity. CheD resembles other cysteine deamidases from bacterial pathogens that inactivate host Rho-GTPases. CheD not only deamidates receptor glutamine residues contained within a conserved structural motif but also hydrolyzes glutamyl-methyl-esters at select regulatory positions. Substituting Gln into the receptor motif of CheC turns the inhibitor into a CheD substrate. Phospho-CheY, the intracellular signal and CheC target, stabilizes the CheC:CheD complex and reduces availability of CheD. A point mutation that dissociates CheC from CheD impairs chemotaxis in vivo. Thus, CheC incorporates an element of an upstream receptor to influence both its own effect on receptor output and that of its binding partner, CheD.

OBJECTIVE

XRCC1 and XPD play key roles in the repair of DNA lesions and adducts. Contrasting findings have been reported on the effect of polymorphisms of these genes on the response to platinum-based chemotherapy in advanced non-small-cell lung cancer (NSCLC). This study aimed to investigate the relationship between the XPD Lys751Gln and XRCC1 Arg399Gln genotypes and outcome in lung cancer patients.

METHODS

We genotyped 203 NSCLC and 45 small-cell lung carcinoma (SCLC) patients for the two polymorphisms. Most of the patients (81%) received a platinum-based chemotherapy.

RESULTS

The patients' genotype frequencies did not significantly differ from controls and both groups were in Hardy-Weinberg equilibrium for the two polymorphisms. The XRCC1399 Gln/Gln variant genotype was associated with a higher median survival time (80 weeks versus 54.6 weeks for the Arg/Gln heterozygous and 55.6 weeks for the wild-type Arg/Arg genotype; P=0.09). At the multivariable analysis adjusted for histology, stage of the disease, performance status, age, and gender, the Gln/Gln genotype was associated with a better survival of borderline significance in the subgroup of patients treated with cisplatin (hazard ratio, 0.55; 95% CI, 0.30-1.00); this association became significant for those with grade 3-4 clinical toxicity (hazard ratio, 0.46; 95% CI, 0.22-0.98). No association between XPD Lys751Gln genotype and clinical outcome was found.

CONCLUSIONS

This prospective investigation provides suggestive evidence of a favorable effect of the XRCC1399 Gln/Gln genotype on survival in platinum-treated NSCLC and, for the first time, in SCLC patients also. This contrasts with other authors who did not include non-platinum-treated patients, but it does fit the expectation for a suboptimal ability to remove DNA adducts.

A seven amino acid yeast prion sup-35 fragment (GNNQQNY) forms amyloid fibrils. The availability of its detailed atomic oligomeric structure makes it a good model for studying the early stage of aggregation. Here we perform long all-atom explicit solvent molecular simulations of various sizes and arrangements of oligomer seeds of the wild-type and its mutants to study its stability and dynamics. Previous studies have suggested that the early stage rate-limiting step of oligomer formation occurs in high-order oligomers. Our simulations show that with the increase in the number of strands even from a dimer to a trimer, oligomer stability increases dramatically. This suggests that the minimal nucleus seed for GNNQQNY fibril formation could be small and is likely three or four peptides, in agreement with experiment, and that higher-order oligomers do not dissociate quickly since they have small diffusion coefficients and thus slow kinetics. Further, for the hydrophilic polar GNNQQNY, there are no hydrogen bonds and no hydrophobic interactions between adjacent beta-sheets. Simulations suggest that within the sheet, the driving forces to associate and stabilize are interstrand backbone-backbone and side chain-side chain hydrogen bonds, whereas between the sheets, shape-complementary by the dry polar steric zipper via the side chains of Asn-2, Gln-4, and Asn-6 holds the sheets together, as proposed in an earlier study. Since the polar side chains of Asn-2, Gln-4, and Asn-6 act as a hook to bind two neighboring sheets together, these geometric restraints reduce the conformational search for the correct side chain packing to a two-dimensional problem of intersheet side chain interactions. Mutant simulations show that substitution of Asn-2, Gln-4, or Asn-6 by Ala would disrupt this steric zipper, leading to unstable oligomers.

Escherichia coli has eight genes predicted to encode sulfurtransferases having the active site consensus sequence Cys-Xaa-Xaa-Gly. One of these genes, ybbB, is frequently found within bacterial operons that contain selD, the selenophosphate synthetase gene, suggesting a role in selenium metabolism. We show that ybbB is required in vivo for the specific substitution of selenium for sulfur in 2-thiouridine residues in E. coli tRNA. This modified tRNA nucleoside, 5-methylaminomethyl-2-selenouridine (mnm(5)se(2)U), is located at the wobble position of the anticodons of tRNA(Lys), tRNA(Glu), and tRNA(1)(Gln). Nucleoside analysis of tRNAs from wild-type and ybbB mutant strains revealed that production of mnm(5)se(2)U is lost in the ybbB mutant but that 5-methylaminomethyl-2-thiouridine, the mnm(5)se(2)U precursor, is unaffected by deletion of ybbB. Thus, ybbB is not required for the initial sulfurtransferase reaction but rather encodes a 2-selenouridine synthase that replaces a sulfur atom in 2-thiouridine in tRNA with selenium. Purified 2-selenouridine synthase containing a C-terminal His(6) tag exhibited spectral properties consistent with tRNA bound to the enzyme. In vitro mnm(5)se(2)U synthesis is shown to be dependent on 2-selenouridine synthase, SePO(3), and tRNA. Finally, we demonstrate that the conserved Cys(97) (but not Cys(96)) in the rhodanese sequence motif Cys(96)-Cys(97)-Xaa-Xaa-Gly is required for 2-selenouridine synthase in vivo activity. These data are consistent with the ybbB gene encoding a tRNA 2-selenouridine synthase and identifies a new role for the rhodanese homology domain in enzymes.

Glutamine synthetase brings nitrogen into metabolism by condensing ammonia and glutamate, with the aid of ATP, to yield glutamine, ADP, and inorganic phosphate. Here we present five crystal structures of GS complexed with each of two substrates, Glu and AMPPNP (an ATP analog), with a transition-state analogue, L-methionine-S-sulfoximine, and with each of two products, Gln and ADP. GS of the present study is from Salmonella typhimurium, has Mn2+ bound, and is fully unadenylylated. Protein-metal-substrate interactions and small but significant conformational changes induced by substrate binding are defined by Fourier maps. On the basis of these maps, we propose a tentative structure-based enzymatic mechanism of glutamine synthesis with these steps: (1) ATP binds first at the top of the funnel-shaped active site cavity, adjacent to the n2 Mn2+; Arg 359 moves toward the Glu binding site. (2) Glu binds adjacent to the n1 Mn2+ at the bottom of the active site near a flexible loop (residues 324-328). As proposed earlier by Meister and others, Glu attacks the gamma-phosphorus atom of ATP to produce gamma-glutamyl phosphate and ADP. (3) The presence of ADP (but not ATP) moves Arg 339 toward the Pi site, perhaps stabilizing the gamma-glutamyl phosphate, and moves Asp 50' of the adjacent subunit toward a putative ammonium ion site, enhancing binding of this third substrate. Deprotonation of the ammonium ion, perhaps by Asp 50', permits the resulting active species, ammonia, to attack the gamma-glutamyl phosphate, forming a tetrahedral intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

Glucosamine (GLN) and chondroitin sulfate (CS) are widely used to alleviate symptoms of osteoarthritis (OA). However, the mechanism(s) of action of these nutraceuticals remains unresolved. In the present study, we determined the effect of physiologically relevant concentrations of GLN and CS on gene expression and synthesis of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in cytokine-stimulated articular cartilage explants.

METHODS

Using bovine articular cartilage explants in culture stimulated with IL-1, the effects of physiologically relevant concentrations of GLN and CS on gene expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGEs1) were assessed with quantitative real-time polymerase chain reaction (Q-RT-PCR). The production of NO and PGE(2) was also quantified.

RESULTS

CS and the GLN and CS combination at concentrations attainable in the blood down-regulated IL-1 induced mRNA expression of iNOS at 24 and 48 h post-culture. Up-regulated iNOS expression at 24h by IL-1 was also suppressed by GLN. GLN and CS transiently repressed the cytokine-stimulated mPGEs1 transcript. Synthesis of NO was reduced with CS alone and the combination after 24h of culture. Repression of COX-2 transcripts by GLN and CS was accompanied by concomitant reduction in PGE(2).

CONCLUSIONS

Our results indicate that physiologically relevant concentrations of GLN and CS can regulate gene expression and synthesis of NO and PGE(2), providing a plausible explanation for their purported anti-inflammatory properties.

BACKGROUND

It is unclear whether single nucleotide polymorphisms (SNPs) in the gene for IL-13 (IL13) influence asthma severity and/or asthma morbidity.

OBJECTIVE

To examine the relation between IL13 SNPs and asthma-related phenotypes in 2 independent populations.

METHODS

We used family-based methods to test for association between SNPs in IL13 and asthma-related phenotypes in Costa Rican children with asthma. We attempted to reproduce significant findings in white (non-Hispanic) children with asthma in the Childhood Asthma Management Program (CAMP).

RESULTS

In Costa Rica and in CAMP, the A allele (Gln) of IL13 coding SNP (rs20541) was significantly associated with increased eosinophil count (P < .011 in both studies) and increased serum total IgE (P < .054 in both studies). The T allele of IL13 promoter SNP (rs1800925) was inversely associated with asthma exacerbations in Costa Rica (P = .069). Although this SNP (rs1800925) was not associated with asthma exacerbations among all white children in CAMP, it was associated with increased risk of asthma exacerbations among children on inhaled corticosteroids (P = .02).

CONCLUSIONS

Polymorphisms in IL13 were significantly associated with serum total IgE and eosinophil count in 2 populations. IL13 polymorphisms may also be associated with asthma exacerbations, and this effect may be dependent on medication use. Our study is the first to report a potential negative interaction between a genetic polymorphism and response to inhaled corticosteroids.

CONCLUSIONS

Polymorphisms in IL13 are associated with serum total IgE and eosinophil count and may be associated with asthma exacerbations.

In translation, separate aminoacyl-tRNA synthetases attach the 20 different amino acids to their cognate tRNAs, with the exception of glutamine. Eukaryotes and some bacteria employ a specific glutaminyl-tRNA synthetase (GlnRS) which other Bacteria, the Archaea (archaebacteria), and organelles apparently lack. Instead, tRNA(Gln) is initially acylated with glutamate by glutamyl-tRNA synthetase (GluRS), then the glutamate moiety is transamidated to glutamine. Lamour et al. [(1994) Proc Natl Acad Sci USA 91:8670-8674] suggested that an early duplication of the GluRS gene in eukaryotes gave rise to the gene for GlnRS-a copy of which was subsequently transferred to proteobacteria. However, questions remain about the occurrence of GlnRS genes among the Eucarya (eukaryotes) outside of the "crown" taxa (animals, fungi, and plants), the distribution of GlnRS genes in the Bacteria, and their evolutionary relationships to genes from the Archaea. Here, we show that GlnRS occurs in the most deeply branching eukaryotes and that putative GluRS genes from the Archaea are more closely related to GlnRS and GluRS genes of the Eucarya than to those of Bacteria. There is still no evidence for the existence of GlnRS in the Archaea. We propose that the last common ancestor to contemporary cells, or cenancestor, used transamidation to synthesize Gln-tRNA(Gln) and that both the Bacteria and the Archaea retained this pathway, while eukaryotes developed a specific GlnRS gene through the duplication of an existing GluRS gene. In the Bacteria, GlnRS genes have been identified in a total of 10 species from three highly diverse taxonomic groups: Thermus/Deinococcus, Proteobacteria gamma/beta subdivision, and Bacteroides/Cytophaga/Flexibacter. Although all bacterial GlnRS form a monophyletic group, the broad phyletic distribution of this tRNA synthetase suggests that multiple gene transfers from eukaryotes to bacteria occurred shortly after the Archaea-eukaryote divergence.

Amyloids are proteinaceous fibers commonly associated with neurodegenerative diseases and prion-based encephalopathies. Many different polypeptides can form amyloid fibers, leading to the suggestion that amyloid is a primitive main chain-dominated structure. A growing body of evidence suggests that amino acid side chains dramatically influence amyloid formation. The specific role fulfilled by side chains in amyloid formation, especially in vivo, remains poorly understood. Here, we determined the role of internally conserved polar and aromatic residues in promoting amyloidogenesis of the functional amyloid protein CsgA, which is the major protein component of curli fibers assembled by enteric bacteria such as Escherichia coli and Salmonella spp. In vivo CsgA polymerization into an amyloid fiber requires the CsgB nucleator protein. The CsgA amyloid core region is composed of five repeating units, defined by regularly spaced Ser, Gln and Asn residues. The results of a comprehensive alanine scan mutagenesis screen showed that Gln and Asn residues at positions 49, 54, 139 and 144 were critical for curli assembly. Alanine substitution of Q49 or N144 impeded the ability of CsgA to respond to CsgB-mediated heteronucleation, and the ability of CsgA to self-polymerize in vitro. However, CsgA proteins harboring these mutations were still seeded by preformed wild-type CsgA fibers in vitro. This suggests that CsgA-fibril-mediated seeding and CsgB-mediated heteronucleation have distinguishable mechanisms. Remarkably, Gln residues at positions 49 and 139 could not be replaced by Asn residues without interfering with curli assembly, suggesting that the side chain requirements were especially stringent at these positions. This analysis demonstrates that bacterial amyloid formation is driven by specific side chain contacts, and provides a clear illustration of the essential roles of specific side chains in promoting amyloid formation.

Eubacterial leucyl/phenylalanyl-tRNA protein transferase (LF-transferase) catalyses peptide-bond formation by using Leu-tRNA(Leu) (or Phe-tRNA(Phe)) and an amino-terminal Arg (or Lys) of a protein, as donor and acceptor substrates, respectively. However, the catalytic mechanism of peptide-bond formation by LF-transferase remained obscure. Here we determine the structures of complexes of LF-transferase and phenylalanyl adenosine, with and without a short peptide bearing an N-terminal Arg. Combining the two separate structures into one structure as well as mutation studies reveal the mechanism for peptide-bond formation by LF-transferase. The electron relay from Asp 186 to Gln 188 helps Gln 188 to attract a proton from the alpha-amino group of the N-terminal Arg of the acceptor peptide. This generates the attacking nucleophile for the carbonyl carbon of the aminoacyl bond of the aminoacyl-tRNA, thus facilitating peptide-bond formation. The protein-based mechanism for peptide-bond formation by LF-transferase is similar to the reverse reaction of the acylation step observed in the peptide hydrolysis reaction by serine proteases.

The human ATP-binding cassette half-transporter ABCG2 is a 72 kDa plasma membrane protein that can confer multidrug resistance to cells in culture when overexpressed. Both transiently and stably expressed ABCG2 are glycosylated, and treatment with peptide N-glycosidase F reduces the apparent molecular mass on SDS-PAGE gels to approximately 60 kDa. Sequence analysis revealed three potential N-linked glycosylation sites in human ABCG2 at amino acids 418, 557, and 596. Site-directed mutagenesis experiments, in which each Asn was changed to Gln independently, revealed that only asparagine 596 is N-linked glycosylated. These data provide the first direct identification of the modified residue in ABCG2 and evidence for the localization of loop 5 to the extracellular space, previously only predicted from hydropathy analysis. Immunoblot and pulse-chase analyses revealed that the glycosylation-deficient ABCG2 (N596Q) variant and the glycosylated parent transporter are expressed equivalently at steady state and have similar half-lives. Cell surface analysis of ABCG2 expression showed comparable amounts of the N596Q variant present at the plasma membrane compared to the glycosylated ABCG2 protein. The ABCG2 (N596Q) variant is also functional, demonstrating rhodamine 123 transport in intact cells comparable to that in cells expressing glycosylated ABCG2. Furthermore, in crude membrane preparations, neither the basal nor the prazosin-stimulated ( approximately 2-fold) ATPase activities of ABCG2 (N596Q) were affected compared to glycosylated ABCG2. Although subtle defects in transporter trafficking and function may exist, these data taken together suggest that N-glycosylation at arginine 596 is not essential for the expression, trafficking to the plasma membrane, or the overall function of ABCG2.

Factor V, the precursor of factor Va, circulates in plasma with little or no procoagulant activity. Activity is generated following limited proteolysis indicating that the conversion of factor V to factor Va results in appropriate structural changes, which impart cofactor function. We have produced recombinant partial B-domain-truncated derivatives of factor V (FV(des811-1491) and FV(des811-1491) with Arg(709) and Arg(1545) mutated to Gln) to investigate whether discrete proteolysis within the B-domain followed by a conformational transition is responsible for activation. Direct binding fluorescence measurements as well as steady-state kinetic assays were employed to assess the ability of these factor V derivatives to assemble and function in prothrombinase. In contrast to human factor V, single-chain B-domain-truncated factor V bound to FXa membranes with an affinity that was identical to factor Va. Additionally, it was found that, once this modified derivative was assembled in prothrombinase, it functioned in an equivalent manner to factor Va. Taken together these data support the hypothesis that proteolysis within the B-domain of factor V, although necessary, is incidental to the mechanism by which cofactor function is realized. Instead, our results are more consistent with the interpretation that proteolytic activation of factor V simply eliminates steric and/or conformational constraints contributed by the B-domain that otherwise interfere with discrete binding interactions that govern the eventual function of factor Va.

Extremely halophilic microorganisms that accumulate KCl for osmotic balance (the Halobacteriaceae, Salinibacter) have a large excess of acidic amino acids in their proteins. This minireview explores the occurrence of acidic proteomes in halophiles of different physiology and phylogenetic affiliation. For fermentative bacteria of the order Halanaerobiales, known to accumulate KCl, an acidic proteome was predicted. However, this is not confirmed by genome analysis. The reported excess of acidic amino acids is due to a high content of Gln and Asn, which yield Glu and Asp upon acid hydrolysis. The closely related Halorhodospira halophila and Halorhodospira halochloris use different strategies to cope with high salt. The first has an acidic proteome and accumulates high KCl concentrations at high salt concentrations; the second does not accumulate KCl and lacks an acidic proteome. Acidic proteomes can be predicted from the genomes of some moderately halophilic aerobes that accumulate organic osmotic solutes (Halomonas elongata, Chromohalobacter salexigens) and some marine bacteria. Based on the information on cultured species it is possible to understand the pI profiles predicted from metagenomic data from hypersaline environments.

Most chemical techniques used to produce antibody-drug conjugates (ADCs) result in a heterogeneous mixture of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between <em>Gln</em> and Lys residues. We have previously shown that MTGase solely recognizes <em>Gln</em>295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, one-step attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymatic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-derivative. To this aim, we investigated two different chemical approaches, namely, thiol-maleimide and strain-promoted azide-alkyne cycloaddition (SPAAC). Direct enzymatic attachment of MMAE-spacer derivatives at an 80 molar excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymatic approach only required a 2.5 molar excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymatic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC50 of 89.0 pM and 21.7 pM, respectively. Thus, the chemo-enzymatic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.

Oxidative damage is implicated in several chronic diseases including cancer. 8-Hydroxyguanine (8-oxoG) is one of the major promutagenic DNA lesions, which is produced by reactive oxygen species, causes G:C to T:A transversions and is excised by OGG1, an 8-oxoG specific DNA glycosylase/AP-Lyase. In a nested case-control study, gDNA from 105 Caucasian primary non-small cell lung cancer cases and 105 matched controls was screened for 6 possible new polymorphic sites in the human OGG1 gene, detected previously mainly in tumour tissue. The previously described Ser(326)Cys polymorphism was found to be common (allele frequency 0.22) in Caucasians. However, no major difference in Ser(326)Cys genotype distribution could be detected between cases and controls. Two 5;-end polymorphisms previously found in Japanese as well as Arg(131)Gln could not be detected in this population. An Ala(85)Ser polymorphism was found in 2 controls, whereas Arg(46)Gln was detected in only 1 case. As the hOGG1 gene is mapped (3p26.2) to a region frequently lost in primary lung tumours, the frequency of loss of heterozygosity (LOH) was investigated. Forty-three percent of the studied lung tumours exhibited loss of one of the hOGG1 alleles. The wt Ser(326) allele was not predominantly lost in our sample set, which suggests a minor role of this polymorphism in tumourgenesis. Our results show that LOH at the hOGG1 gene locus is a very common occurrence in lung tumourgenesis, possibly leading to increased mutational damage due to ROS in smokers. However, the hOGG1 polymorphisms studied are probably not major contributors to individual lung cancer susceptibility in Caucasians.

Neuroglobin is a recently discovered member of the globin superfamily. Combined electron paramagnetic resonance and optical measurements show that, in Escherichia coli cell cultures with low O(2) concentration overexpressing wild-type mouse recombinant neuroglobin, the heme protein is mainly in a hexacoordinated deoxy ferrous form (F8His-Fe(2+)-E7His), whereby for a small fraction of the protein the endogenous protein ligand is replaced by NO. Analogous studies for mutated neuroglobin (mutation of E7-His to Leu, Val, or Gln) reveal the predominant presence of the nitrosyl ferrous form. After sonication of the cells wild-type neuroglobin oxidizes rapidly to the hexacoordinated ferric form, whereas NO ligation initially protects the mutants from oxidation. Flash photolysis studies of wild-type neuroglobin and its E7 mutants show high recombination rates (k(on)) and low dissociation rates (k(off)) for NO, indicating a high intrinsic affinity for this ligand similar to that of other hemoglobins. Since the rate-limiting step in ligand combination with the deoxy-hexacoordinated wild-type form involves the dissociation of the protein ligand, NO binding is slower than for the related mutants. Structural and kinetic characteristics of neuroglobin and its mutants are analyzed. NO production in rapidly growing E. coli cell cultures is discussed.

Glu-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Purified mutant rhodopsin pigments were prepared in which Glu-113 was replaced individually by Gln (E113Q), Asp (E113D), Asn (E113N), or Ala (E113A). E113Q, E113N, and E113A existed as pH-dependent equilibrium mixtures of unprotonated and protonated Schiff base (PSB) forms. The Schiff base pKa values determined by spectrophotometric titration were 6.00 (E113Q), 6.71 (E113N), and 5.70 (E113A). Thus, mutation of Glu-113 markedly reduced the Schiff base pKa. The addition of NaCl promoted the formation of a PSB in E113Q and E113A. An exogenously supplied solute anion replaced Glu-113 to compensate for the positive charge of the PSB in these mutants. The lambda max values of the PSB forms of the mutants in NaCl were 496 nm (E113Q), 506 nm (E113A), 510 nm (E113D), and 520 nm (E113N). To evaluate the effect of different types of solute anions on lambda max values, mutants were prepared in sodium salts of halides, perchlorate, and a series of carboxylic acids of various sizes and acidity. The lambda max values of E113Q and E113A depended on the solute anion present and ranged from 488 nm to 522 nm for E113Q and from 486 nm to 528 nm for E113A. The solute anion affected the lambda max values of E113N and E113D to lesser degrees. The reactivities of the mutants to hydroxylamine were also studied. Whereas rhodopsin was stable to hydroxylamine in the dark, E113N reacted slowly and E113Q reacted rapidly under these conditions, indicating structural differences in the Schiff base environments. The lambda max values and solute anion dependencies of the Glu-113 mutants indicate that interactions between Schiff base and its counterion play a significant role in determining the lambda max of rhodopsin.

The crystal structure of a complex between chemically synthesized human immunodeficiency virus type 1 (HIV-1) protease and an octapeptide inhibitor has been refined to an R factor of 0.138 at 2.5-A resolution. The substrate-based inhibitor, H-Val-Ser-Gln-Asn-Leu psi [CH(OH)CH2]Val-Ile-Val-OH (U-85548e) contains a hydroxyethylene isostere replacement at the scissile bond that is believed to mimic the tetrahedral transition state of the proteolytic reaction. This potent inhibitor has Ki less than 1 nM and was developed as an active-site titrant of the HIV-1 protease. The inhibitor binds in an extended conformation and is involved in beta-sheet interactions with the active-site floor and flaps of the enzyme, which form the substrate/inhibitor cavity. The inhibitor diastereomer has the S configuration at the chiral carbon atom of the hydroxyethylene insert, and the hydroxyl group is within H-bonding distance of the two active-site carboxyl groups in the enzyme dimer. The two subunits of the enzyme are related by a pseudodyad, which superposes them at a 178 degrees rotation. The main difference between the subunits is in the beta turns of the flaps, which have different conformations in the two monomers. The inhibitor has a clear preferred orientation in the active site and the alternative conformation, if any, is a minor one (occupancy of less than 30%). A new model of the enzymatic mechanism is proposed in which the proteolytic reaction is viewed as a one-step process during which the nucleophile (water molecule) and electrophile (an acidic proton) attack the scissile bond in a concerted manner.

mu-Conotoxins (mu-CTXs) specifically inhibit Na(+) flux by occluding the pore of voltage-gated Na(+) channels. Although the three-dimensional structures of mu-CTXs are well defined, the molecular configuration of the channel receptor is much less certain; even the fundamental question of whether the four homologous Na(+) channel domains are arranged in a clockwise or counter-clockwise configuration remains unanswered. Residues Asp(762) and Glu(765) from domain II and Asp(1241) from domain III of rat skeletal muscle Na(+) channels are known to be critical for mu-CTX binding. We probed toxin-channel interactions by determining the potency of block of wild-type, D762K, E765K, and D1241C channels by wild-type and point-mutated mu-CTXs (R1A, Q14D, K11A, K16A, and R19A). Individual interaction energies for different toxin-channel pairs were quantified from the half-blocking concentrations using mutant cycle analysis. We find that Asp(762) and Glu(765) interact strongly with Gln(14) and Arg(19) but not Arg(1) and that Asp(1241) is tightly coupled to Lys(16) but not Arg(1) or Lys(11). These newly identified toxin-channel interactions within adjacent domains, interpreted in light of the known asymmetric toxin structure, fix the orientation of the toxin with respect to the channel and reveal that the four internal domains of Na(+) channels are arranged in a clockwise configuration as viewed from the extracellular surface.

Wild type (wt) p21 Bax was cleaved to generate p18 Bax during apoptotic processes by calpain, which was suggested to recognize a certain motif around amino acids 30-33 Phe-Ile-<em>Gln</em>-Asp (FIQD). In the present study, analysis of protein sequencing revealed that the cleavage site was between <em>Gln</em>28 and Gly29. The fragment lacking the NH(2)-terminal amino acids 1-28 (tBax(29)) was more apoptotic than wt Bax. The tBax(29)-induced apoptotic cell death was substantially resistant to Bcl-x(L)-mediated rescue, compared with wt Bax, in spite of the complex formation between these two molecules. Together, the tBax(29) would be valuable for the treatment of tumors with high levels of Bcl-x(L) as well as the understanding of Bax-mediated apoptotic processes.