Substitution Of Leucine Research Articles

Total deletion and a missense mutation of ITPR1 in Japanese SCA15 families

Spinocerebellar ataxia type 15 (SCA15) is a progressive neurodegenerative disorder characterized by pure cerebellar ataxia, very slow progression, and distinct cerebellar atrophy. The locus for SCA15 was first mapped to 3p24.2-3pter in an Australian family. We have subsequently mapped two Japanese families presenting with ataxia and postural tremor of the head, arm, or trunk to the SCA15 locus. Recently, partial deletions involving both the type 1 inositol 1,4,5-triphosphate receptor (ITPR1) and sulfatase modifying factor 1 (SUMF1) genes have been identified in Australian and British families with SCA15. We conducted fine haplotype analysis on the region including ITPR1. To identify the deletion, we conducted gene dosage analysis and array-based comparative genomic hybridization (aCGH) analysis. Gene expression analysis was performed using quantitative real-time reverse transcription PCR. Mutational analyses of ITPR1 and SUMF1 were also performed. We have identified a 414-kb deletion including the entire ITPR1 and exon 1 of SUMF1 in patients in family A. The expression levels of ITPR1 and SUMF1 mRNAs of the patient were half those of the normal control. Furthermore, in family B, we have identified a C-to-T substitution at position 8581 of ITPR1, resulting in the amino acid substitution of leucine for proline at codon 1059, which is highly conserved among species. Our results strongly confirm that ITPR1 is the causative gene for SCA15 and suggest that we need to investigate the point mutation in ITPR1 in the patients with autosomal dominant cerebellar ataxia and tremor.

Epidemiological Cutoffs and Cross-Resistance to Azole Drugs in Aspergillus fumigatus

Antifungal susceptibility testing of molds has been standardized in Europe and in the United States. Aspergillus fumigatus strains with resistance to azole drugs have recently been detected and the underlying molecular mechanisms of resistance characterized. Three hundred and ninety-three isolates, including 32 itraconazole-resistant strains, were used to define wild-type populations, epidemiological cutoffs, and cross-resistance between azole drugs. The epidemiological cutoff for itraconazole, voriconazole, and ravuconazole for the wild-type populations of A. fumigatus was < or =1 mg/liter. For posaconazole, the epidemiological cutoff was < or =0.25 mg/liter. Up till now, isolates susceptible to itraconazole have not yet displayed resistance to other azole drugs. Cross-resistance between azole drugs depends on specific mutations in cyp51A. Thus, a substitution of glycine in position 54 of Cyp51A confers cross-resistance between itraconazole and posaconazole. A substitution of methionine at position 220 or a duplication in tandem of a 34-bp fragment in the cyp51A promoter combined with a substitution of leucine at position 98 for histidine confers cross-resistance to all azole drugs tested. The results obtained in this study will help to develop clinical breakpoints for azole drugs and A. fumigatus.

A Conserved Sequence within the H2 Subunit of the Vaccinia Virus Entry/Fusion Complex Is Important for Interaction with the A28 Subunit and Infectivity

The recently described vaccinia virus entry/fusion complex (EFC) comprises at least eight polypeptides that are conserved in all poxviruses. Neither the structure of the complex nor the roles of individual subunits are known. Here we provide evidence for an interaction between the H2 and A28 subunits in the context of a virus infection as well as in uninfected cells transfected with plasmids expressing the corresponding genes. We focused on a highly conserved 21-amino acid-segment in H2 that is flanked by cysteine residues. The effect of amino acid substitutions within the 21-amino-acid segment was determined by an infectivity complementation assay using a conditional H2-null mutant of vaccinia virus. Mutations that had no, moderate, or large negative effects on complementation were found. The latter group included glutamic acid substitutions of leucine and individual glycines and alanine substitution of both glycines within a LGYSG sequence. Mutations with the most pronounced effect on infectivity disrupted the interaction of H2 with A28 to the greatest extent in both infected and uninfected cells. These data indicate that the LGYSG sequence is important for the interaction of H2 with A28 and suggest that this sequence is buried within the EFC complex.

Mutations of carnitine palmitoyltransferase II (CPT II) in Japanese patients with CPT II deficiency

Carnitine palmitoyltransferase II (CPT II) deficiency is an inherited disorder involving beta-oxidation of long-chain fatty acids. CPT II deficiency is a wide-spectrum disorder that includes a lethal neonatal form, an infantile form, and an adult-onset form. However, the ethnic characteristics and the relationship between genotype and clinical manifestation are not well understood. We investigated three non-consanguineous Japanese patients with CPT II deficiency and examined cell lines from 4 unrelated patients and 50 healthy donors. The CPT 2 gene was typed by direct DNA sequencing of polymerase chain reaction-amplified gene products. Case 1 (infantile form) was heterozygous for a phenylalanine to tyrosine substitution at position 383 (p.F383Y) and a novel valine to leucine substitution at 605 (p.V605L). Cases 2, 4, and 5 (infantile form) and case 3 (adult-onset form) were heterozygous for a single mutation at F383Y. Case 6 (adult-onset form) was compound heterozygous at the CPT 2 locus, with deletion of cytosine and thymine at residue 408, resulting in a stop signal at 420 (p.Y408fsX420), and an arginine to cysteine substitution at position 631 (p.R631C). Case 7 (adult-onset form) was homozygous for the p.F383Y mutation. In conclusion, we identified p.F383Y mutations in six of seven patients with CPT II deficiency and two novel variants of the coding gene: p.Y408fsX420 and p.V605L. These mutations differ from those in Caucasian patients, who commonly harbor p.S113L, p.P50H, and p.Q413fsX449 mutations; therefore, our data and those of other Japanese groups suggest that the p.F383Y mutation is significant in Japanese patients with CPT II deficiency.

Role of the carboxyl terminal di-leucine in phosphorylation and internalization of C5a receptor

The carboxyl tail of G protein-coupled receptors contains motifs that regulate receptor interactions with intracellular partners. Activation of the human neutrophil complement fragment C5a receptor (C5aR) is terminated by phosphorylation of the carboxyl tail followed by receptor internalization. In this study, we demonstrated that bulky hydrophobic residues in the membrane-proximal region of the C5aR carboxyl tail play an important role in proper structure and function of the receptor: Substitution of leucine 319 with alanine (L319A) resulted in receptor retention in the endoplasmic reticulum, whereas a L318A substitution allowed receptor transport to the cell surface, but showed slow internalization upon activation, presumably due to a defect in phosphorylation by both PKC and GRK. Normal agonist-induced activation of ERK1/2 and intracellular calcium release suggested that the L318A mutation did not affect receptor signaling. Binding of GRK2 and PKCβII to intracellular loop 3 of C5aR in vitro indicated that mutagenesis of L318 did not affect kinase binding. Limited proteolysis with trypsin revealed a conformational difference between wild type and mutant receptor. Our studies support a model in which the L318/L319 stabilizes an amphipathic helix (Q305–R320) in the membrane-proximal region of C5aR.

Effects of the myostatin F94L substitution on beef traits1

This study investigated the effects of a SNP in the myostatin gene (MSTN or growth differentiation factor 8, GDF8) on birth, growth, carcass, and beef quality traits in Australia (Aust.) and New Zealand (NZ). The SNP is a cytosine to adenine transversion in exon 1, causing an amino acid substitution of leucine for phenylalanine(94) (F94L). The experiment used crosses between the Jersey and Limousin breeds, with the design being a backcross using first-cross bulls of Jersey x Limousin or Limousin x Jersey breeding, mated to Jersey and Limousin cows. Progeny were genotyped for the myostatin SNP and phenotyped in Aust., with finishing on feedlot (366 calves, over 3 birth years) and in NZ with finishing on pasture (416 calves, over 2 birth years). The effect of the F94L allele (A allele) on birth and growth traits was not significant. The F94L allele in Limousin backcross calves was associated with an increase in meat weight (7.3 and 5.9% of the trait mean in Aust. and NZ, respectively, P < 0.001), and a reduction in fat depth (-13.9 and -18.7% of the trait means on live calves (600 d) and carcasses, respectively, Aust. only, P < 0.001), intramuscular fat content (-8.2% of the trait mean in Aust., P < 0.05; -7.1% in NZ, not significant), total carcass fat weight (-16.5 and -8.1% of the trait mean, Aust. and NZ; P < 0.001 and P < 0.05, respectively). Meat tenderness, pH, and cooking loss of the M. longissimus dorsi were not affected by the F94L variant. In the Jersey backcross calves, additive and dominance effects were confounded because the F94L allele was not segregating in the Jersey dams. The combined effects, however, were significant on LM area (4.4% in both Aust., P < 0.05, and NZ, P < 0.01), channel fat (-11.7%, NZ only, P < 0.01), rib fat depth (-11.2%, NZ only, P < 0.05), and carcass fat weight (-7.1%, NZ only, P < 0.05). The results provide strong evidence that this myostatin F94L variant provides an intermediate and more useful phenotype than the more severe double-muscling phenotype caused by knockout mutations in the myostatin gene.

Identification and Functional Analysis of a New WNT4 Gene Mutation among 28 Adolescent Girls with Primary Amenorrhea and Müllerian Duct Abnormalities: A French Collaborative Study

Müllerian duct development depends on gene and hormone interactions. Female Wnt4-knockout mice lack müllerian ducts and are virilized due to the inappropriate expression of the enzymes required for androgen production (normally repressed in female ovary). The WNT4 mutation was recently reported to be associated with failure of müllerian duct formation and virilization in two 46, XX women. This collaborative work was designed to determine whether the WNT4 mutation could be identified in a group of adolescent girls with Mayer-Rokitansky-Küster-Hauser syndrome. We analyzed 28 DNA samples from adolescent girls with primary amenorrhea and failure of müllerian duct formation by direct sequencing and identified a new L12P mutation within exon 1 of the WNT4 gene. The substitution of leucine by proline is crucial for the conformation of the expressed protein. This amino acid substitution is unlikely to be a polymorphism because it was not found in 100 DNAs from control subjects. Functional analysis revealed that the mutation induces significantly increased expression of the enzymes involved in androgen biosynthesis (3beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase). It is interesting to note that the adolescent carrying the mutation was referred to our clinic for primary amenorrhea and hyperandrogenism (severe acne and plasma testosterone: 1.8 vs. 1.2 nmol/liter in controls). She also presented with uterine hypoplasia and follicle depletion. We suggest that in adolescent girls with primary amenorrhea, müllerian duct abnormalities, and hyperandrogenism, a WNT4 mutation should be sought. Moreover, our data confirm that WNT4 is involved in the regulation of müllerian duct development and ovarian androgen biosynthesis. WNT4 may also contribute to human follicle development and/or maintenance.

An investigation into the native‐like properties of de novo designed cavitand‐based four‐helix bundle proteins

We investigated the hydrophobic packing of two previously designed caviteins, LG2 and LG3, which differ by one Gly in the linker regions between the peptide sequence and the cavitand scaffold. We sought to diminish the putative native-like properties of LG2 and LG3, and see if we could diagnose a change in the conformational specificity of the hydrophobic core. We replaced the leucine residues with norleucine residues at the hydrophobic positions in LG2 and LG3, to create NG2 and NG3, respectively. LG2 exhibited more dispersion, but less sharp signals than LG3 in the amide region of its (1)H NMR spectrum. NG3 and NG2 were found to be slightly less helical and significantly less stable toward guanidine hydrochloride compared with their reference caviteins. The (1)H NMR spectrum of NG2 was very similar to that of LG2, whereas there was a noticeable loss in the number and sharpness of the amide signals of NG3 compared with LG3. These data suggest that LG3 is very well packed; a loss in conformational specificity resulted from replacement of the leucine residues with norleucine residues. In contrast, the packing and dynamics of the hydrophobic core in LG2 were similar to those in NG2 (both more modest than LG3), as their (1)H NMR spectra were virtually indistinguishable. Overall, substitution of leucine by norleucine provided an efficient, convenient, and informative probe of the packing and dynamics of our caviteins' hydrophobic cores.

Conserved Leucines in N-Terminal Heptad Repeat HR1 of Envelope Fusion Protein F of Group II Nucleopolyhedroviruses Are Important for Correct Processing and Essential for Fusogenicity

The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common features to class I fusion proteins, such as proteolytic cleavage and the presence of an N-terminal open fusion peptide and multiple HR domains on the transmembrane subunit F(1). Similar to many vertebrate viral fusion proteins, a conserved leucine zipper motif is predicted in this HR region proximal to the fusion peptide in baculovirus F proteins. To facilitate our understanding of the functional role of this leucine zipper-like HR1 domain in baculovirus F protein synthesis, processing, and viral infectivity, key leucine residues (Leu209, Leu216, and Leu223) were replaced by alanine (A) or arginine (R), respectively. By using Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) as a pseudotype expression system, we demonstrated that all mutant F proteins incorporated into budded virus, indicating that leucine substitutions did not affect intercellular trafficking of F. Furin-like protease cleavage was not affected by any of the leucine substitutions; however, the disulfide bridging and N-linked glycosylation patterns were partly altered. Single substitutions in HR1 showed that the three leucine residues were critical for F fusogenicity and the rescue of AcMNPV infectivity. Our results support the view that the leucine zipper-like HR1 domain is important to safeguard the proper folding, glycosylation, and fusogenicity of baculovirus F proteins.

Congenital Insensitivity to Pain with Anhydrosis in a Malaysian Family: A Genetic Analysis

A Malaysian family with congenital insensitivity to pain with anhydrosis was diagnosed based on clinical symptoms of chronic ulcers, joint deformities, malunited fractures, anhydrosis, and learning disabilities. We detected a compound heterozygous mutation in exon 16: V709L from the mother and G718S from the father. Two novel mutations were identified: at amino acid 709, a change of G to C at nucleotide 2209 (approximately 2209G to C) causing a valine to leucine substitution (V709L), and at amino acid 718, a change of G to A at nucleotide 2236 (approximately 2236G to A) causing a glycine to serine substitution (G718S). Polymorphisms identified were at nucleotides approximately 2113G to C and approximately 2176T to C.

The W148L substitution in the Escherichia coli ammonium channel AmtB increases flux and indicates that the substrate is an ion.

The Amt/Mep ammonium channels are trimers in which each monomer contains a long, narrow, hydrophobic pore. Whether the substrate conducted by these pores is NH(3) or NH(4)(+) remains controversial. Substitution of leucine for the highly conserved tryptophan 148 residue at the external opening to Escherichia coli AmtB pores allowed us to address this issue. A strain carrying AmtB(W148L) accumulates much larger amounts of both [(14)C]methylammonium and [(14)C]methylglutamine in a washed cell assay than a strain carrying wild-type AmtB. Accumulation of methylammonium occurs within seconds and appears to reflect channel conductance, whereas accumulation of methylglutamine, which depends on the ATP-dependent activity of glutamine synthetase, increases for many minutes. Concentration of methylammonium was most easily studied in strains that lack glutamine synthetase. It is eliminated by the protonophore carbonyl cyanide m-chlorophenyl hydrazone and is approximately 10-fold higher in the strain carrying AmtB(W148L) than wild-type AmtB. The results indicate that AmtB allows accumulation of CH(3)NH(3)(+) ion in response to the electrical potential across the membrane and that the rate of flux through AmtB(W148L) is approximately 10 times faster than through wild-type AmtB. We infer that both mutant and wild-type proteins also carry NH(4)(+). Contrary to our previous views, we assess that E. coli AmtB does not differ from plant Amt proteins in this regard; both carry ions. We address the role of W148 in decreasing the activity and increasing the selectivity of AmtB and the implications of our findings with respect to the function of Rh proteins, the only known homologues of Amt/Mep proteins.

Mutant TIRAP Gene Polymorphism Is Associated with Outcomes in HLA Genoidentical Bone Marrow Transplants Recipients with Leukemia.

Toll-like receptors (TLRs) and members of their signaling pathway are important in the initiation of the innate immune response to a wide variety of pathogens. The adaptor protein TIRAP, mediates downstream signaling of TLR2 and TLR4. A leucine (Leu) substitution at position 180 serine (Ser) of TIRAP was shown to protect from infections. We hypothesized that the incidence of infections and outcome after bone marrow transplantation (BMT) could be influenced by the TIRAP gene polymorphisms. Genotyping for TIRAP at Ser180 was performed in 107 donor/recipients pairs: 64% of recipients and 63% of donors were homozygote wild type (wt) Ser180/Ser180, 20% and 19% were Ser180/Leu180 and 17% and 18% Leu180/Leu180. Severe bacterial (pneumonia, septicemia and septic shock), viral or invasive fungal infections during 180 days after BMT, TRM (transplant related mortality), acute GVHD and survival were studied retrospectively. An allogeneic HLA-identical BMT was performed in 107 patients (median age was 35 years), with acute (n=39) or chronic Leukemia (n=68). Conditioning regimen consisted of Busulfan+Cyclophosphamide±others (BU-CY) in 73 (68%) patients and TBI+CY in 15 (14%) or TBI+Melphalan in 17 (16%). GVHD prophylaxis consisted of CsA+MTX in 94%. Median follow-up time was 77 months (18–147). Cumulative incidence of neutrophil recovery was 94%. Acute GVHD (II-IV) was observed in 42%, and chronic GVHD in 50 out of 98 at risk. Estimate 5-year survival was 54%. The cumulative incidence of any first infection by day 180 was 56%, being 28% for at least one bacterial infection, 39% for viral infection and 12% for invasive fungal infection (8 invasive aspergillosis, 4 disseminated candidiosis and 1 disseminated Malassezia furfur fungal infection). TRM cumulative incidence at 1 year was 35%. Recipient's TIRAP gene polymorphisms were not associated with outcome. However, patients transplanted from a mutant TIRAP donor (Leu/Ser or Leu/Leu) had a trend to lower incidence of fungal infections (p=0.08); lower incidence of acute GVHD (p=0.03), decreased TRM (p=0.02) and improved overall survival (p=0.02). In a multivariate analysis, adjusting for other prognostic factors, TIRAP donor polymorphisms were associated with decreased TRM (HR=0.49, p=0.02): TRM at 1 year was 43% when donors were wt (Ser/Ser) as compared to 20% when donors were mutants. There was also a trend to decreased incidence of acute GVHD in patients with a mutant TIRAP donor as compared to the others (30% versus 49%; HR 1.88 p=0.06) and better overall survival (67% versus 44%, respectively HR 1.82; p=0.07). Recipients of bone marrow (BM) from wt TIRAP donors seemed to die more frequently from infection (1 yr cumulative incidence 21% vs 8%, p=0.07). In conclusion, the presence of a Leu substitution at position 180 in the TIRAP gene of BM donors decreases the incidence of TRM in HLA-identical BMT, probably reflecting better immune reconstitution and protection against infections. This finding can help defining better surveillance and prophylaxis of bacterial infections in BMT recipients from donors with the wt TIRAP (Ser/Ser) genotype.

Transmembrane Domain of Myelin Protein Zero Can Form Dimers: Possible Implications for Myelin Construction

Myelin protein zero (MPZ) is the major integral membrane protein of peripheral nerve myelin in higher vertebrates, mediating homoadhesion of the multiple, spiraling wraps of the myelin sheath. Previous studies have shown that full-length MPZ can form dimers and tetramers, and biochemical studies on the extracellular domain (ECD) indicate that it can form a tetramer, albeit very weakly. On the basis of cross-linking studies and equilibrium sedimentation of a transmembrane (TM) domain peptide (MPZ-TM), we find that the MPZ-TM can form homodimers. We further characterized the dimer by measuring the effects of alanine and leucine substitutions on the ability of the TM to dimerize in Escherichia coli membranes. Our results indicate that the primary packing interface for the MPZ TM homodimer is a glycine zipper (GxxxGxxxG) motif. We also find that the G134R mutation, which lies within the glycine zipper packing interface and causes Charcot-Marie-Tooth disease type 1B, severely inhibits dimerization, suggesting that dimerization of the TM domain may be important for the normal functioning of MPZ. By combining our new results with prior work, we suggest a new model for an MPZ lattice that may form during the construction of myelin.

Multiple ALS Mutations Confer Herbicide Resistance in Waterhemp (Amaranthus tuberculatus)

In a survey of herbicide responses among Illinois waterhemp half-sib populations, several were observed with differential responses to imazethapyr and thifensulfuron, two acetolactate synthase (ALS)–inhibiting herbicides. Plants from two waterhemp populations were verified resistant to imazethapyr, but susceptible to chlorimuron, using a nondestructive leaf-disc assay. Sequencing of the ALS gene revealed that imazethapyr-resistant waterhemp plants from both populations had inferred amino acid substitutions at position 653 of ALS. Depending on the population, the serine at position 653 of ALS was substituted with either asparagine (S653N) or threonine (S653T). Waterhemp lines were derived from each population to create uniformly imidazolinone-resistant (IR) waterhemp biotypes, designated IR-62 and IR-101. ALS-inhibitor responses of each IR biotype were compared with a previously identified ALS inhibitor–resistant biotype with a tryptophan to leucine substitution at position 574 (W574L) and an herbicide-susceptible control. Whole-plant dose–response experiments with waterhemp biotypes containing W574L, S653N, or S653T mutations indicated that each biotype was resistant to imazethapyr, but only the biotype with a W574L mutation was resistant to thifensulfuron. In vitro ALS-activity assays revealed unique patterns of cross-resistance among protein extracts derived from each biotype in response to imazethapyr, thifensulfuron, cloransulam, and pyrithiobac. In conclusion, three different forms of target-site–based resistance to ALS inhibitors have been identified in waterhemp.

SRD5A2 and HSD3B2 polymorphisms are associated with prostate cancer risk and aggressiveness

Dihydrotestosterone (DHT) is believed to play an important role in prostate carcinogenesis. Five alpha reductase type II (SRD5A2) and 3 beta-hydroxysteroid dehydrogenase type II (HSD3B2) are responsible for the biosynthesis and degradation of DHT in the prostate. Two polymorphisms, a valine (V) for leucine (L) substitution at the 89 codon of the SRD5A2 gene and a (TG)n,(TA)n,(CA)n repeat polymorphism within the third intron of the HSD3B2 gene were evaluated with regard to prostate cancer risk. Blood samples were collected for 637 prostate cancer cases and 244 age and race frequency matched controls. In analysis, the SRD5A2 VL and LL genotypes were combined into one group and the HSD3B2 repeat polymorphism was dichotomized into short (<283) and long (> or =283) alleles. The SRD5A2 V89L polymorphism was not independently associated with prostate cancer risk. Carriage of at least one HSD3B2 intron 3 intron 3 short allele was associated with a significant increased risk for prostate cancer among all subjects (OR = 2.07, 95% CI = 1.08-3.95, P = 0.03) and Caucasians (OR = 2.80, CI = 2.80-7.43, P = 0.04), but not in African Americans (OR = 1.50, CI = 0.62-3.60, P = 0.37). Stratified analyses revealed that most of the prostate cancer risk associated with the intron 3 HSD3B2 short allele was confined to the SRD5A2 89L variant subgroup and indicated that in combination these polymorphisms may be associated with increased risk of aggressive (Gleason >7) disease (Gleason >7). In Caucasians, the HSD3B2 (TG)n,(TA)n,(CA)n intron 3 length polymorphism is associated with both prostate cancer risk and aggressiveness and the SRD5A2 V89L polymorphism may modify the risk conferred by this polymorphism.

A Novel Point Mutation in Helix 11 of the Ligand-Binding Domain of the Human Glucocorticoid Receptor Gene Causing Generalized Glucocorticoid Resistance

Generalized glucocorticoid resistance is a rare condition characterized by partial, end-organ insensitivity to glucocorticoids, compensatory elevations in adrenocorticotropic hormone and cortisol secretion, and increased production of adrenal steroids with androgenic and/or mineralocorticoid activity. We have identified a new case of glucocorticoid resistance caused by a novel mutation of the human glucocorticoid receptor (hGR) gene and studied the molecular mechanisms through which the mutant receptor impairs glucocorticoid signal transduction. We identified a novel, single, heterozygous nucleotide (T --> C) substitution at position 2209 (exon 9alpha) of the hGR gene, which resulted in phenylalanine (F) to leucine (L) substitution at amino acid position 737 within helix 11 of the ligand-binding domain of the protein. Compared with the wild-type receptor, the mutant receptor hGRalphaF737L demonstrated a significant ligand-exposure time-dependent decrease in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone and displayed a 2-fold reduction in the affinity for ligand, a 12-fold delay in nuclear translocation, and an abnormal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. The mutant receptor preserved its ability to bind to DNA and exerted a dominant-negative effect on the wild-type hGRalpha only after a short duration of exposure to the ligand. The mutant receptor hGRalphaF737L causes generalized glucocorticoid resistance because of decreased affinity for the ligand, marked delay in nuclear translocation, and/or abnormal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. These findings confirm the importance of the C terminus of the ligand-binding domain of the receptor in conferring transactivational activity.

Notch-induced T cell development requires phosphoinositide-dependent kinase 1

Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development.

Isolation and structural analysis of bamylocin A, novel lipopeptide from Bacillus amyloliquefaciens LP03 having antagonistic and crude oil-emulsifying activity

Bacillus amyloliquefaciens strain LP03 isolated from soil, produced an antagonistic compound that strongly inhibited the growth of plant-pathogenic fungi and a lipopeptide biosurfactant. Also, isolated strain LP03 had a marked crude oil-emulsifying activity as it developed a clear zone around the colony after incubation for 24 h at 37 degrees C. LP03 was identified as Bacillus amyloliquefaciens by analysis of partial 16 S rRNA gene and partial gyrA gene sequence. The lipopeptide was purified by acid precipitation of cell-free culture broth, extraction of the precipitates with methanol, silica gel column chromatography, and reverse-phase, high-pressure liquid chromatography. The purified biosurfactant was analyzed biochemical structure by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). The masses of the two peaks were observed by HPLC chromatography. Their masses were determined to be 1,044 and 1,058 m/z with MALDI-TOF mass spectrometry. As constituents of the peptide and lipophilic part of the m/z 1,022.6, seven amino acids (Glu-Leu-Met-Leu-Pro-Leu-Leu) and beta-hydroxy-C13 fatty acid were determined by ESI-MS/MS. The lipopeptide of 1,022.6 Da differed from surfactins in the substitution of leucine, valine and aspartic acid in positions 3, 4, and 5 by methionine, leucine, and proline, respectively. Novel lipopeptide was designated as bamylocin A.

Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase,

The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140 degrees rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.

The Polymorphism P315L of Human Toll-Like Receptor 1 Impairs Innate Immune Sensing of Microbial Cell Wall Components

As a pattern recognition receptor, TLR1 mediates innate immune responses to a variety of microbial cell wall components including bacterial lipoproteins. We have previously shown that the central region of the extracellular domain of human TLR1, comprising leucine-rich repeat (LRR) motifs 9-12, is required for the sensing of bacterial lipopeptides. In this study, we have investigated three nonsynonymous single nucleotide polymorphisms (SNPs) located in this region of TLR1 by generating these variants and examining receptor function. We have found that a variant of TLR1 based upon the SNP P315L, located in the loop of LRR motif 11 (LRR11), is greatly impaired in mediating responses to lipopeptides and a variety of other bacterial agonists for this receptor. Despite normal cell surface expression, the P315L variant also fails to bind to GD2.F4, a commonly used anti-TLR1 mAb. Although a number of amino acid substitutions at position 315 impair receptor function, the leucine substitution has the strongest deleterious effect. GD2.F4 inhibits agonist-induced activation of TLR1, supporting a crucial role for the loop of LRR11 in receptor function. These results also suggest that the P315L SNP may predispose certain individuals to infectious diseases for which the sensing of microbial cell components by TLR1 is critical to innate immune defense.