Kinetics of CO and NO ligation with the Cys(331)-->Ala mutant of neuronal nitric-oxide synthase

Nitric-oxide synthases (NOS) catalyze the conversion of l-arginine to NO, which then stimulates many physiological processes. In the active form, each NOS is a dimer; each strand has both a heme-binding oxygenase domain and a reductase domain. In neuronal NOS (nNOS), there is a conserved cysteine motif (CX(4)C) that participates in a ZnS(4) center, which stabilizes the dimer interface and/or the flavoprotein-heme domain interface. Previously, the Cys(331) --> Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding of l-Arg and tetrahydrobiopterin (BH(4)). Because binding l-Arg and BH(4) to wild type nNOS profoundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has behavior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding l-Arg and BH(4) has little effect immediately but substantial effect after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS. Such incubation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is related to a structural rather than a catalytic role in maintaining active nNOS.     

Evidence for the autocrine induction of capacitation of mammalian spermatozoa

Mammalian spermatozoa require a maturational event after ejaculation that allows them to acquire the capacity for fertilization. This process, known as capacitation, occurs spontaneously in simple defined medium implicating a potential role of autocrine induction. This study shows that the ether phospholipid 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF) meets the criteria for an autocrine mediator of capacitation. Sperm released PAF after their dilution into capacitation medium and expressed a receptor for PAF on their membranes. PAF stimulated changes in the motility of sperm and enhanced fertilization in vitro. These actions were inhibited by a PAF receptor antagonist (UR-12519) and by extracellular recombinant PAF:acetylhydrolase (an enzyme that degrades PAF to a biologically inert form). Seminal plasma contained an acid-labile PAF:acetylhydrolase, whereas capacitation was inhibited by an acid-labile factor within seminal plasma, implicating this factor as a potential decapacitation factor within seminal plasma. Sperm from a PAF receptor knock-out mouse strain failed to express the receptor and displayed a significantly (p < 0.01) reduced rate of capacitation, as assessed by the spontaneous onset of the acrosome reaction in vitro. When used for in vitro fertilization, sperm from PAF receptor knock-out mice gave a significantly lower rate of fertilization (21.5%) than did wild-type sperm (66.7%). The study shows for the first time the operation of an autocrine loop that induces capacitation in sperm in vitro and shows that this loop acts in concert with other mediators of capacitation to promote efficient fertilization.     

Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers

The Na+/H+ exchangers (NHEs) comprise a family of transporters that catalyze cell functions such as regulation of the pH and volume of a cell and epithelial absorption of Na+ and bicarbonate. Ubiquitous calcineurin B homologous protein (CHP or p22) is co-localized and co-immunoprecipitated with expressed NHE1, NHE2, or NHE3 independently of its myristoylation and Ca2+ binding, and its binding site was identified as the juxtamembrane region within the carboxyl-terminal cytoplasmic domain of exchangers. CHP binding-defective mutations of NHE1-3 or CHP depletion by injection of the competitive CHP-binding region of NHE1 into Xenopus oocytes resulted in a dramatic reduction (>90%) in the Na+/H+ exchange activity. The data suggest that CHP serves as an essential cofactor, which supports the physiological activity of NHE family members.     

Identification of Ipaf, a human caspase-1-activating protein related to Apaf-1

Procaspase-9 contains an NH2-terminal caspase-associated recruitment domain (CARD), which is essential for direct association with Apaf-1 and activation. Procaspase-1 also contains an NH2-terminal CARD domain, suggesting that its mechanism of activation, like that of procaspase-9, involves association with an Apaf-1-related molecule. Here we describe the identification of a human Apaf-1-related protein, named Ipaf that contains an NH2-terminal CARD domain, a central nucleotide-binding domain, and a COOH-terminal regulatory leucine-rich repeat domain (LRR). Ipaf associates directly and specifically with the CARD domain of procaspase-1 through CARD-CARD interaction. A constitutively active Ipaf lacking its COOH-terminal LRR domain can induce autocatalytic processing and activation of procaspase-1 and caspase-1-dependent apoptosis in transfected cells. Our results suggest that Ipaf is a specific and direct activator of procaspase-1 and could be involved in activation of caspase-1 in response to pro-inflammatory and apoptotic stimuli.     

The covalent structure of the small subunit from Pseudomonas putida amine dehydrogenase reveals the presence of three novel types of internal cross-linkages, all involving cysteine in a thioether bond

Pseudomonas putida contains an amine dehydrogenase that is called a quinohemoprotein as it contains a quinone and two hemes c as redox active groups. Amino acid sequence analysis of the smallest (8.5 kDa), quinone-cofactor-bearing subunit of this heterotrimeric enzyme encountered difficulties in the interpretation of the results at several sites of the polypeptide chain. As this suggested posttranslational modifications of the subunit, the structural genes for this enzyme were determined and mass spectrometric de novo sequencing was applied to several peptides obtained by chemical or enzymatic cleavage. In agreement with the interpretation of the X-ray electronic densities in the diffraction data for the holoenzyme, our results show that the polypeptide of the small subunit contains four intrachain cross-linkages in which the sulfur atom of a cysteine residue is involved. Two of these cross-linkages occur with the beta-carbon atom of an aspartic acid, one with the gamma-carbon atom of a glutamic acid and the fourth with a tryptophanquinone residue, this adduct constituting the enzyme's quinone cofactor, CTQ. The thioether type bond in all four of these adducts has never been found in other proteins. CTQ is a novel cofactor in the series of the recently discovered quinone cofactors.     

Cytokine-independent Jak3 activation upon T cell receptor (TCR) stimulation through direct association of Jak3 and the TCR complex

Jak3 is responsible for growth signals by various cytokines such as interleukin (IL)-2, IL-4, and IL-7 through association with the common gamma chain (gammac) in lymphocytes. We found that T cells from Jak3-deficient mice exhibit impairment of not only cytokine signaling but also early activation signals and that Jak3 is phosphorylated upon T cell receptor (TCR) stimulation. TCR-mediated phosphorylation of Jak3 is independent of IL-2 receptor/gammac but is dependent on Lck and ZAP-70. Jak3 was found to be assembled with the TCR complex, particularly through direct association with CD3zeta via its JH4 region, which is a different region from that for gammac association. These results suggest that Jak3 plays a role not only in cell growth but also in T cell activation and represents cross-talk of a signaling molecule between TCR and growth signals.     

CD98-mediated links between amino acid transport and beta 1 integrin distribution in polarized columnar epithelia

In non-polarized cells, CD98 has been shown to both influence beta(1) integrins and heterodimerize with LAT-2, which confers amino acid transport capability on the LAT-2/CD98 heterodimer. Since LAT-2 is most heavily expressed in intestine and CD98 associates with the beta(1) integrin splice form selectively found in such epithelia, we investigated the relationship and polarity of these proteins using the intestinal epithelial model Caco2-BBE. CD98 was found to selectively coimmunoprecipitate with both LAT-2 and beta(1) integrin, and, logically, all three proteins were polarized to the same (basolateral) domain. Furthermore, expression of CD98 in polarized epithelia lacking human CD98 (MDCK cells) disrupted beta(1) integrin surface distribution and cytoskeletal architecture, suggesting that CD98 can influence integrin function. Expression of a CD98 mutant lacking the specific residues conferring LAT-2 binding similarly affected cells, confirming that the latter effect was not due to LAT-2 sequestration. Use of CD98 truncation mutants suggest that a 10-amino acid domain located at the putative cytoplasmic tail/transmembrane domain interface was necessary and sufficient to induce the phenotype change. We conclude that the CD98/LAT-2 amino acid transporter is polarized to the same domain on which beta(1) integrin resides. CD98 appears to associate with beta(1) integrin and, in doing so, may influence its function as revealed by disruption of the outside-in signaling that confers cytoskeletal organization. Furthermore, such findings suggest a link between classic transport events and a critical element of barrier function: integrin-mediated influences on cytoskeletal organization.     

Glial cell line-derived neurotrophic factor-stimulated phosphatidylinositol 3-kinase and Akt activities exert opposing effects on the ERK pathway: importance for the rescue of neuroectodermic cells

Glial cell line-derived neurotrophic factor (GDNF) plays a crucial role in rescuing neural crest cells from apoptosis during their migration in the foregut. This survival factor binds to the heterodimer GDNF family receptor alpha1/Ret, inducing the Ret tyrosine kinase activity. ret loss-of-function mutations result in Hirschsprung's disease, a frequent developmental defect of the enteric nervous system. Although critical to enteric nervous system development, the intracellular signaling cascades activated by GDNF and their importance in neuroectodermic cell survival still remain elusive. Using the neuroectodermic SK-N-MC cell line, we found that the Ret tyrosine kinase activity is essential for GDNF to induce phosphatidylinositol 3-kinase (PI3K)/Akt and ERK pathways as well as cell rescue. We demonstrate that activation of PI3K is mandatory for GDNF-induced cell survival. In addition, evidence is provided for a critical up-regulation of the ERK pathway by PI3K at the level of Raf-1. Conversely, Akt inhibits the ERK pathway. Thus, both PI3K and Akt act in concert to finely regulate the level of ERK. We found that Akt activation is indispensable for counteracting the apoptotic signal on mitochondria, whereas ERK is partially involved in precluding procaspase-3 cleavage. Altogether, these findings underscore the importance of the Ret/PI3K/Akt pathway in GDNF-induced neuroectodermic cell survival.     

Chicken avidin exhibits pseudo-catalytic properties. Biochemical, structural, and electrostatic consequences

Avidin and its bacterial analogue streptavidin exhibit similarly high affinities toward the vitamin biotin. The extremely high affinity of these two proteins has been utilized as a powerful tool in many biotechnological applications. Although avidin and streptavidin have similar tertiary and quaternary structures, they differ in many of their properties. Here we show that avidin enhances the alkaline hydrolysis of biotinyl p-nitrophenyl ester, whereas streptavidin protects this reaction even under extreme alkaline conditions (pH > 12). Unlike normal enzymatic catalysis, the hydrolysis reaction proceeds as a single cycle with no turnover because of the extremely high affinity of the protein for one of the reaction products (i.e. free biotin). The three-dimensional crystal structures of avidin (2 A) and streptavidin (2.4 A) complexed with the amide analogue, biotinyl p-nitroanilide, as a model for the p-nitrophenyl ester, revealed structural insights into the factors that enhance or protect the hydrolysis reaction. The data demonstrate that several molecular features of avidin are responsible for the enhanced hydrolysis of biotinyl p-nitrophenyl ester. These include the nature of a decisive flexible loop, the presence of an obtrusive arginine 114, and a newly formed critical interaction between lysine 111 and the nitro group of the substrate. The open conformation of the loop serves to expose the substrate to the solvent, and the arginine shifts the p-nitroanilide moiety toward the interacting lysine, which increases the electron withdrawing characteristics and consequent electrophilicity of the carbonyl group of the substrate. Streptavidin lacked such molecular properties, and analogous interactions with the substrate were consequently absent. The information derived from these structures may provide insight into the action of artificial protein catalysts and the evolution of catalytic sites in general.