Active site mutants of human secreted Group IIA Phospholipase A(2) lacking hydrolytic activity retain their bactericidal effect

The Human Secreted Group IIA Phospholipase A(2) (hsPIA2GIIA) presents potent bactericidal activity, and is considered to contribute to the acute-phase immune response. Hydrolysis of inner membrane phospholipids is suggested to underlie the bactericidal activity, and we have evaluated this proposal by comparing catalytic activity with bactericidal and liposome membrane damaging effects of the G30S, H48Q and D49K h5PLA2GIIA mutants. All mutants showed severely impaired hydrolytic activities against mixed DOPC:DOPG liposome membranes, however the bactericidal effect against Micrococcus luteus was less affected, with 50% killing at concentrations of 1, 3, 7 and 9 mu g/mL for the wild-type, D49K, H48Q and G30S mutants respectively. Furthermore, all proteins showed Ca2+-independent damaging activity against Liposome membranes demonstrating that in addition to the hydrolysis-dependent membrane damage, the hsPLA2GIIA presents a mechanism for permeabilization of phospholipid bilayers that is independent of catalytic activity, which may play a role in the bactericidal function of the protein (C) 2011 Elsevier Masson SAS. All rights reserved.

Order-Disorder Transitions Govern Kinetic Cooperativity and Allostery of Monomeric Human Glucokinase

Glucokinase (GCK) catalyzes the rate-limiting step of glucose catabolism in the pancreas, where it functions as the body's principal glucose sensor. GCK dysfunction leads to several potentially fatal diseases including maturity-onset diabetes of the young type II (MODY-II) and persistent hypoglycemic hyperinsulinemia of infancy (PHHI). GCK maintains glucose homeostasis by displaying a sigmoidal kinetic response to increasing blood glucose levels. This positive cooperativity is unique because the enzyme functions exclusively as a monomer and possesses only a single glucose binding site. Despite nearly a half century of research, the mechanistic basis for GCK's homotropic allostery remains unresolved. Here we explain GCK cooperativity in terms of large-scale, glucose-mediated disorder-order transitions using 17 isotopically labeled isoleucine methyl groups and three tryptophan side chains as sensitive nuclear magnetic resonance (NMR) probes. We find that the small domain of unliganded GCK is intrinsically disordered and samples a broad conformational ensemble. We also demonstrate that small-molecule diabetes therapeutic agents and hyperinsulinemia-associated GCK mutations share a strikingly similar activation mechanism, characterized by a population shift toward a more narrow, well-ordered ensemble resembling the glucose-bound conformation. Our results support a model in which GCK generates its cooperative kinetic response at low glucose concentrations by using a millisecond disorder-order cycle of the small domain as a "time-delay loop," which is bypassed at high glucose concentrations, providing a unique mechanism to allosterically regulate the activity of human GCK under physiological conditions.

Inactivating Mutations of the Human Luteinizing Hormone Receptor in Both Sexes

The human luteinizing hormone/chorionic gonadotropin receptor (LHCGR) plays a fundamental role in male and female reproductive physiology. Over the past 15 years, several homozygous or compound heterozygous loss-of-function mutations in the LHCGR gene have been described in males and females. In genetic males, mutations in LHCGR were associated with distinct degrees of impairment in pre- and postnatal testosterone secretion resulting in a phenotypic spectrum. Patients with the severe form of LH resistance have predominantly female external genitalia and absence of secondary sex differentiation at puberty. Patients with milder forms have predominantly male external genitalia with micropenis and/or hypospadias or only infertility without ambiguity. The undermasculization is associated with low basal, as well as human CG-stimulated, testosterone levels and elevated LH levels after pubertal age, without abnormal step-up in testosterone biosynthesis precursors. The testes have only slightly reduced size but mature Leydig cells are absent or scarce (Leydig cell hypoplasia). Genetic females with inactivating LHCGR mutations have female external genitalia, spontaneous breast and pubic hair development at puberty, and normal or late menarche followed by oligoamenorrhea and infertility. Estradiol and progesterone levels are normal for the early to midfollicular phase, but do not reach ovulatory or luteal phase levels. Serum LH levels are high whereas follicle-stimulating hormone levels are normal or only slightly increased. Pelvic ultrasound has demonstrated a small or normal uterus and normal or enlarged ovaries with cysts. The inactivating mutations of the LHCGR have provided important insights into distinct physiological roles of LH in reproduction of both sexes.

Characterization of suramin binding sites on the human group IIA secreted phospholipase A(2) by site-directed mutagenesis and molecular dynamics simulation

Suramin is a polysulphonated naphthylurea with inhibitory activity against the human secreted group IIA phospholipase A(2) (hsPLA2GIIA), and we have investigated suramin binding to recombinant hsPLA2GIIA using site-directed mutagenesis and molecular dynamics (MD) simulations. The changes in suramin binding affinity of 13 cationic residue mutants of the hsPLA2GIIA was strongly correlated with alterations in the inhibition of membrane damaging activity of the protein. Suramin binding to hsPLA2GIIA was also studied by MD simulations, which demonstrated that altered intermolecular potential energy of the suramin/mutant complexes was a reliable indicator of affinity change. Although residues in the C-terminal region play a major role in the stabilization of the hsPLA2GIIA/suramin complex, attractive and repulsive hydrophobic and electrostatic interactions with residues throughout the protein together with the adoption of a bent suramin conformation, all contribute to the stability of the complex. Analysis of the h5PLA2GIIA/suramin interactions allows the prediction of the properties of suramin analogues with improved binding and higher affinities which may be candidates for novel phospholipase A(2) inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

Genome mapping and expression analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in genes related to regulation of transcription

Abstract Background RNAs transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression. However, the complement of human genes in which introns are transcribed, and the number of intronic transcriptional units and their tissue expression patterns are not known. Results A survey of mRNA and EST public databases revealed more than 55,000 totally intronic noncoding (TIN) RNAs transcribed from the introns of 74% of all unique RefSeq genes. Guided by this information, we designed an oligoarray platform containing sense and antisense probes for each of 7,135 randomly selected TIN transcripts plus the corresponding protein-coding genes. We identified exonic and intronic tissue-specific expression signatures for human liver, prostate and kidney. The most highly expressed antisense TIN RNAs were transcribed from introns of protein-coding genes significantly enriched (p = 0.002 to 0.022) in the 'Regulation of transcription' Gene Ontology category. RNA polymerase II inhibition resulted in increased expression of a fraction of intronic RNAs in cell cultures, suggesting that other RNA polymerases may be involved in their biosynthesis. Members of a subset of intronic and protein-coding signatures transcribed from the same genomic loci have correlated expression patterns, suggesting that intronic RNAs regulate the abundance or the pattern of exon usage in protein-coding messages. Conclusion We have identified diverse intronic RNA expression patterns, pointing to distinct regulatory roles. This gene-oriented approach, using a combined intron-exon oligoarray, should permit further comparative analysis of intronic transcription under various physiological and pathological conditions, thus advancing current knowledge about the biological functions of these noncoding RNAs.

Movement of NH3 through the human urea transporter B: a new gas channel

Aquaporins and Rh proteins can function as gas (CO2 and NH3) channels. The present study explores the urea, H2O, CO2, and NH3 permeability of the human urea transporter B (UT-B) (SLC14A1), expressed in Xenopus oocytes. We monitored urea uptake using [14C]urea and measured osmotic water permeability (Pf) using video microscopy. To obtain a semiquantitative measure of gas permeability, we used microelectrodes to record the maximum transient change in surface pH (∆pHS) caused by exposing oocytes to 5% CO2/33 mM HCO3- (pHS increase) or 0.5 mM NH3/NH4+ (pHS decrease). UT-B expression increased oocyte permeability to urea by >20-fold, and Pf by 8-fold vs. H2O-injected control oocytes. UT-B expression had no effect on the CO2-induced ∆pHS but doubled the NH3-induced ∆pHS. Phloretin reduced UT-B-dependent urea uptake (Jurea * ) by 45%, Pf * by 50%, and (- ∆pHS * )NH3 by 70%. p-Chloromercuribenzene sulfonate reduced Jurea * by 25%, Pf * by 30%, and (∆pHS * )NH3 by 100%. Molecular dynamics (MD) simulations of membrane-embedded models of UT-B identified the monomeric UT-B pores as the main conduction pathway for both H2O and NH3 and characterized the energetics associated with permeation of these species through the channel. Mutating each of two conserved threonines lining the monomeric urea pores reduced H2O and NH3 permeability. Our data confirm that UT-B has significant H2O permeability and for the first time demonstrate significant NH3 permeability. Thus the UTs become the third family of gas channels. Inhibitor and mutagenesis studies and results of MD simulations suggest that NH3 and H2O pass through the three monomeric urea channels in UT-B.