Identification of residues that determine the absence of a Ca(2+)/myristoyl switch in neuronal calcium sensor-1

The neuronal calcium sensor (NCS) family of Ca(2+)-binding proteins regulates a number of different processes in neurons and photoreceptor cells. The first of these proteins to be characterized, recoverin, was shown to exhibit a Ca(2+)/myristoyl switch whereby its N-terminal myristoyl group is sequestered in the Ca(2+)-free form and is exposed on Ca(2+) binding to allow the protein to become membrane-associated. It has subsequently been shown that certain other family members also exhibit this mechanism in living cells. In contrast, NCS-1 does not show the Ca(2+)/myristoyl switch and is membrane-associated even at low Ca(2+) concentrations. We have used sequence comparison combined with information from structural analyses to attempt to identify candidate residues within the NCS proteins that determine whether or not the Ca(2+)/myristoyl switch operates in cells and have tested their functional significance by mutagenesis. The results show that NCS-1 possesses residues within its N terminus that lock the myristoyl group in an exposed conformation. In addition, other structural aspects within the C-terminal domains are required to allow the switch to operate. We have determined a key role for residues within the motif EELTRK in NCS-1 in keeping the myristoyl group exposed and allowing the protein to be constitutively membrane-associated.     

N-terminal myristoylation alters the calcium binding pathways in neuronal calcium sensor-1

Neuronal calcium sensor-1 (NCS-1) interacts with many membranes and cytosolic proteins, both in a Ca²⁺-dependent and in a Ca₂₊-independent manner, and its physiological role is governed by its N-terminal myristoylation. To understand the role of myristoylation in altering Ca²⁺ response and other basic biophysical properties, we have characterized the Ca²⁺ filling pathways in both myristoylated (myr) and non-myristoylated (non-myr) forms of NCS-1. We have observed that Ca²⁺ binds simultaneously to all three active EF-hands in non-myr NCS-1, whereas in the case of myr NCS-1, the process is sequential, where the second EF-hand is filled first, followed by the third and fourth EF-hands. In the case of myr NCS-1, the observed sequential Ca²⁺ binding process becomes more prominent in the presence of Mg²⁺. Besides, the analysis of ¹⁵N-relaxation data reveals that non-myr NCS-1 is more dynamic than myr NCS-1. The overall molecular tumbling correlation time increases by approximately 20% upon myristoylation. Comparing the apo forms of non-myr NCS-1 and myr NCS-1, we found the possibility of existence of some substates, which are structurally closer to the holo form of the protein. There are more such substates in the case of non-myr NCS-1 than in the case of the myr NCS-1, suggesting that the former accesses larger volumes of conformational substates compared with the latter. Further, the study reveals that the possibility of Ca²⁺ binding simultaneously to different parts of the protein is more favourable in non-myr NCS-1 than in myr NCS-1.     

Genetic control of amino acid permeability in Neurospora crassa

Lester, Gabriel (Reed College, Portland, Ore.). Genetic control of amino acid permeability in Neurospora crassa. J. Bacteriol. 91:677-684. 1966.-Strains of Neurospora crassa resistant to 4-methyltryptophan (4-MT) were isolated from populations of conidia exposed to ultraviolet light. In genetic crosses, 4-MT resistance behaved as a single-gene difference. Resistance to 4-MT could not be attributed to a relaxation of control of the formation or the activity of the enzymes of tryptophan biosynthesis. Growth studies involving tryptophan auxotrophs carrying the aberrant mt gene and uptake studies with normal and 4-MT-resistant strains showed that 4-MT resistance could be attributed to an inability of 4-MT-resistant strains to take up tryptophan and its methyl analogues. The mt gene is not specific for tryptophan; strains resistant to 4-MT are also resistant to ethionine, and they have a markedly reduced ability to take up serine, leucine, and alpha-aminoisobutyric acid. No difference was observed between strains carrying either mt allele in their ability to take up glucose; also, the uptake of anthranilic acid or of indole was not sufficiently impaired by the aberrant mt gene to prevent these tryptophan precursors from satisfying the nutritional requirement of certain tryptophan auxotrophs. The role of the mt gene in determining the permeability of N. crassa to amino acids is discussed.     

Developmental regulation of amino acid transport in Neurospora crassa

Conidia of Neurospora crassa exhibit an ability to transport various amino acids against a concentration gradient. The conidial transport system has previously been characterized in terms of kinetics, competitions, and genetic control. This study describes the development of a new and highly active transport capability which is elaborated during the early stages of development but prior to evident germination. It has been named “postconidial” transport activity and represents as much as 20-fold greater initial rates as compared to those observed with conidia. Development of the postconidial transport activity requires protein synthesis and can be partially repressed when the substrate amino acid is present during the developmental preincubation period. A mutant has been utilized which exhibits normal conidial but fails to develop normal postconidial transport activity for any amino acid examined. Although temperature optimum and pH dependence are similar in conidial and postconidial systems, there is evidence that the new activity is not a simple amplification of an existing capability. This is reflected as a change in competition patterns between particular amino acids as development proceeds.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.     

Nitrogen regulation of amino acid utilization by Neurospora crassa.

The production of an extracellular deaminase activity involved with the utilization of amino acids as sole sources of nitrogen is under the control of the nit-2 locus of Neurospora crassa. This locus is the sole major nitrogen regulatory locus described for N. crassa and is believed to encode a positive effector required for induction of activities involved with the utilization of alternate nitrogen sources. Production of deaminase activity requires the lifting of nitrogen metabolite repression, the presence of a functional nit-2 gene product, and specific induction by amino acids. Additional parameters of enzyme production are described.     

Calcium and chlorpromazine binding to the EF-hand peptides of neuronal calcium sensor-1

Neuronal calcium sensor-1, a protein of calcium sensor family, is known to have four structural EF-hands. We have synthesised peptides corresponding to all the four EF-hands and studied their conformation and calcium-binding. Our data confirm that the first putative site, a non-canonical one (EF1), does not bind calcium. We have investigated if this lack of binding is due to the presence of non-favoured residues (particularly at +x and -z co-ordinating positions) of the loop. We have mutated these residues and found that after modification the peptides bound calcium. However, these mutated peptides (EF1 and its functional mutants) do not show any Ca(2+) induced changes in far-UV CD. EF2, EF3, and EF4 peptides bind Ca(2+), EF3 being the strongest binder, followed by EF4. Our data of Ca(2+)-binding to individual EF peptides show that there are three active Ca(2+)-binding sites in NCS-1. We have also studied the binding of a neuroleptic drug, chlorpromazine, with the protein as well as with its EF-hands. CPZ binds myristoylated as well as non-myristoylated NCS-1 in Ca(2+)-dependent manner, with dynamic interaction to myristoylated protein. CPZ does not bind to EF1, but binds to functional EF-hand peptides and induces changes in far-UV CD. Our results suggest that NCS-1 could be a target of such antipsychotic and neuroleptic drugs.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3 ^? null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

Intestinal inflammation modulates expression of the synaptic vesicle protein neuronal calcium sensor-1

The calcium-binding protein neuronal calcium sensor 1 (NCS-1) is involved in modulation of neurotransmitter release in the peripheral and central nervous systems. Since intestinal inflammation impairs neurotransmitter release, we evaluated the expression of NCS-1 in the normal rat colon and in dinitrobenzene sulfonic acid (DNBS)-induced colitis. Immunocytochemistry and Western blots showed high levels of NCS-1 in the myenteric plexus and in axons in the smooth muscle layers; 23 +/- 2% of myenteric neurons were NCS-1 positive, with staining restricted to the largest neurons. NCS-1-positive axons decreased to 13.3 +/- 0.4% of total axons by day 2 and dropped further to 7.0 +/- 0.1% by day 4, returning to control levels by day 16. Dual-label Western blot analysis showed that the expression of NCS-1 relative to PGP 9.5 decreased by 50% on day 4 but returned to control by day 16. The selective loss of NCS-1 during colitis may underlie the altered neural function seen in the inflamed intestine.     

Identification of amino acid residues in BK virus VP1 that are critical for viability and growth

BK virus (BKV) is a ubiquitous pathogen that establishes a persistent infection in the urinary tract of 80% of the human population. Like other polyomaviruses, the major capsid protein of BKV, virion protein 1 (VP1), is critical for host cell receptor recognition and for proper virion assembly. BKV uses a carbohydrate complex containing alpha(2,3)-linked sialic acid attached to glycoprotein and glycolipid motifs as a cellular receptor. To determine the amino acids important for BKV binding to the sialic acid portion of the complex, we generated a series of 17 point mutations in VP1 and scored them for viral growth. The first set of mutants behaved identically to wild-type virus, suggesting that these amino acids were not critical for virus propagation. Another group of VP1 mutants rendered the virus nonviable. These mutations failed to protect viral DNA from DNase I digestion, indicating a role for these domains in capsid assembly and/or packaging of DNA. A third group of VP1 mutations packaged DNA similarly to the wild type but failed to propagate. The initial burst size of these mutations was similar to that of the wild type, indicating that there is no defect in the lytic release of the mutated virions. Binding experiments revealed that a subset of the BKV mutants were unable to attach to their host cells. These motifs are likely important for sialic acid recognition. We next mapped these mutations onto a model of BKV VP1 to provide atomic insight into the role of these sites in the binding of sialic acid to VP1.     

Identification, cloning and functional characterization of novel beta-defensins in the rat (Rattus norvegicus)

Background  

beta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation.     

Neurospora crassa conidial germination: role of endogenous amino acid pools.

The levels of the endogenous amino acid pools in conidia, germinating conidia, and mycelia of wild-type Neurospora crassa were measured. Three different chromatographic procedures employing the amino acid analyzer were used to identify and quantitatively measure 28 different ninhydrin-positive compounds. All of the common amino acids were detected in conidial extracts except proline, methionine, and cystine. The levels of these three amino acid pools were also very low in mycelia. During the first hour of germination in minimal medium, the levels of most of the free amino acid pools decreased. The pool of glutamic acid, the predominant free amino acid in conidia, decreased 70% during the first hour. Very little glutamic acid or any other amino acid was excreted into the medium. During the first 20 min of germination, the decrease in the glutamic acid pool was nearly equivalent to the increase in the aspartic acid pool. The aspartic acid and lambda-aminobutyric acid pools were the only amino acid pools that increased to maximum levels within the first 20 min of germination and then decreased. It is proposed that an important metabolic event that occurs during the early stages of conidial germination is the production of reduced pyridine nucleotides. The degradation of the large glutamic acid pool existing in the conidia (2.5% of the conidial dry weight) could produce these reduced coenzymes.     

Identification of amino acid residues critical for biological activity in human interleukin-18

Interleukin-18 (IL-18) is a pro-inflammatory cytokine, and IL-18-binding protein (IL-18BP) is a naturally occurring protein that binds IL-18 and neutralizes its biological activities. Computer modeling of human IL-18 identified two charged residues, Glu-42 and Lys-89, which interact with oppositely charged amino acid residues buried in a large hydrophobic pocket of IL-18BP. The cell surface IL-18 receptor alpha chain competes with IL-18BP for IL-18 binding, although the IL-18 receptor alpha chain does not share significant homology to IL-18BP. In the present study, Glu-42 was mutated to Lys and Lys-89 to Glu; Glu-42 and Lys-89 were also deleted separately. The deletion mutants (E42X and K89X) were devoid of biological activity, and the K89E mutant lost 95% of its activity. In contrast, compared with wild-type (WT) IL-18, the E42K mutant exhibited a 2-fold increase in biological activity and required a 4-fold greater concentration of IL-18BP for neutralization. The binding of WT IL-18 and its various mutants to human natural killer cells was evaluated by competition assays. The mutant E42K was more effective than WT IL-18 in inhibiting the binding of (125)I-IL-18 to natural killer cells, whereas the three inactive mutants E42X, K89E, and K89X were unable to compete with (125)I-IL-18 for binding. Similarly, WT IL-18 and the E42K mutant induced degradation of Ikappa-Balpha, whereas the three biologically inactive mutants did not induce degradation. The present study reveals that Glu-42 and Lys-89 are critical amino acid residues for the integrity of IL-18 structure and are important for binding to cell surface receptors, for signal transduction, and for neutralization by IL-18BP.     

Participation of an extracellular deaminase in amino acid utilization by Neurospora crassa

A strain of Neurospora crassa defective in amino acid transport can utilize a variety of amino acids for growth when readily metabolizable nitrogen is limiting. Growth is accompanied by the production of an extracellular deaminase that converts the amino acid to its respective keto acid plus equimolar quantities of utilizable nitrogen in the ammonium ion form. Production of the deaminase is subject to ammonium repression. The relationship between the ability of an amino acid to trigger deaminase production and the presence of particular amino acid permease deficiencies is complex. Four classes of amino acids have been defined with respect to this relationship. The existence of multiple extracellular deaminases is discussed.     

The amino acid sequence of ribonuclease N1, a guanine-specific ribonuclease from the fungus Neurospora crassa

The complete amino acid sequence of ribonuclease N1 (RNase N1), a guanine-specific ribonuclease from a fungus, Neurospora crassa, was determined by conventional protein sequencing, using peptide fragments obtained by tryptic digestion of cyanogen bromide-treated RNase N1 and by Staphylococcus aureus V8 protease digestion of heat-denatured RNase N1. The results showed that the protein is composed of a single polypeptide chain of 104 amino acid residues cross-linked by two disulfide bonds and has a molecular weight of 11,174: (sequence; see text) (Disulfide bonds: C2-C10, C6-C103) The amino acid sequence was homologous with those of RNase T1 (65% identity) and related microbial RNases.