Effects of Ultraviolet Light on Melanocyte Differentiation: Studies with Mouse Neural Crest Cells and Neural Crest‐derived Cell Lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L ‐3,4‐dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT‐ or DOPA‐positive cells between the UV‐irradiated cultures and the non‐irradiated cultures. We then examined the effects of UV light on KIT‐positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase‐positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with α‐melanocyte‐stimulating hormone (α‐MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase‐negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal‐regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as α‐MSH and/or endothelin‐1.     

Evaluation of mutation effects on UGT1A1 activity toward 17beta-estradiol using liquid chromatography-tandem mass spectrometry

Mutations in the gene encoding UDP-glucuronosyltransferase 1A1 (UGT1A1) may reduce the glucuronidation of estradiol, bilirubin, etc. In the present study, we used a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to assay the activities of recombinant mutated UGT1A1 toward 17beta-estradiol (E2), by determining its glucuronide (E2G) content. Direct evidence for glucuronide formation was provided by E2G-specific ion peaks. The UGT1A1 activities of G71R (exon 1), F83L (exon 1), I322V (exon 2) and G493R (exon 5) mutants were 24, 30, 18 and 0.6% of the normal UGT1A1 activity, respectively. In conclusion, our study showed that LC/MS/MS enabled accurate evaluation of the effects of mutations on recombinant UGT1A1 activity towards E2.     

Glutamate:glyoxylate aminotransferase modulates amino acid content during photorespiration

In photorespiration, peroxisomal glutamate:glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to produce 2-oxoglutarate and glycine. Previous studies demonstrated that alanine aminotransferase-like protein functions as a photorespiratory GGAT. Photorespiratory transamination to glyoxylate, which is mediated by GGAT and serine glyoxylate aminotransferase (SGAT), is believed to play an important role in the biosynthesis and metabolism of major amino acids. To better understand its role in the regulation of amino acid levels, we produced 42 GGAT1 overexpression lines that express different levels of GGAT1 mRNA. The levels of free serine, glycine, and citrulline increased markedly in GGAT1 overexpression lines compared with levels in the wild type, and levels of these amino acids were strongly correlated with levels of GGAT1 mRNA and GGAT activity in the leaves. This accumulation began soon after exposure to light and was repressed under high levels of CO(2). Light and nutrient conditions both affected the amino acid profiles; supplementation with NH(4)NO(3) increased the levels of some amino acids compared with the controls. The results suggest that the photorespiratory aminotransferase reactions catalyzed by GGAT and SGAT are both important regulators of amino acid content.     

A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.     

The Erf4 Subunit of the Yeast Ras Palmitoyl Acyltransferase Is Required for Stability of the Acyl-Erf2 Intermediate and Palmitoyl Transfer to a Ras2 Substrate

Protein S-palmitoylation is a posttranslational modification in which a palmitoyl group is added to a protein via a thioester linkage on cysteine. Palmitoylation is a reversible modification involved in protein membrane targeting, receptor trafficking and signaling, vesicular biogenesis and trafficking, protein aggregation, and protein degradation. An example of the dynamic nature of this modification is the palmitoylation-depalmitoylation cycle that regulates the subcellular trafficking of Ras family GTPases. The Ras protein acyltransferase (PAT) consists of a complex of Erf2-Erf4 and DHHC9-GCP16 in yeast and mammalian cells, respectively. Both subunits are required for PAT activity, but the function of the Erf4 and Gcp16 subunits has not been established. This study elucidates the function of Erf4 and shows that one role of Erf4 is to regulate Erf2 stability through an ubiquitin-mediated pathway. In addition, Erf4 is required for the stable formation of the palmitoyl-Erf2 intermediate, the first step of palmitoyl transfer to protein substrates. In the absence of Erf4, the rate of hydrolysis of the active site palmitoyl thioester intermediate is increased, resulting in reduced palmitoyl transfer to a Ras2 substrate. This is the first demonstration of regulation of a DHHC PAT enzyme by an associated protein.     

Hypoxia-induced metastasis model in embryonic zebrafish

Hypoxia facilitates tumor invasion and metastasis by promoting neovascularization and co-option of tumor cells in the peritumoral vasculature, leading to dissemination of tumor cells into the circulation. However, until recently, animal models and imaging technology did not enable monitoring of the early events of tumor cell invasion and dissemination in living animals. We recently developed a zebrafish metastasis model to dissect the detailed events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent DiI-labeled human or mouse tumor cells are implanted into the perivitelline cavity of 48-h-old zebrafish embryos, which are subsequently placed in hypoxic water for 3 d. Tumor cell invasion, metastasis and pathological angiogenesis are detected under fluorescent microscopy in the living fish. The average experimental time for this model is 7 d. Our protocol offers a remarkable opportunity to study molecular mechanisms of hypoxia-induced cancer metastasis.     

Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B

Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.     

Myristate exposure in the human immunodeficiency virus type 1 matrix protein is modulated by pH

Human immunodeficiency virus type 1 (HIV-1) encodes a polypeptide called Gag that is capable of forming virus-like particles (VLPs) in vitro in the absence of other cellular or viral constituents. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. A combination of in vivo, in vitro, and structural studies have shown that Gag targeting and assembly on the PM are mediated by specific interactions between the myristoylated matrix [myr(+)MA] domain of Gag and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Exposure of the MA myristyl (myr) group is triggered by PI(4,5)P2 binding and is enhanced by factors that promote protein self-association. In the studies reported here, we demonstrate that myr exposure in MA is modulated by pH. Our data show that deprotonation of the His89 imidazole ring in myr(+)MA destabilizes the salt bridge formed between His89(Hδ2) and Glu12(COO-), leading to tight sequestration of the myr group and a shift in the equilibrium from trimer to monomer. Furthermore, we show that oligomerization of a Gag-like construct containing matrix-capsid is also pH-dependent. Disruption of the His?Glu salt bridge by single-amino acid substitutions greatly altered the myr-sequestered?myr-exposed equilibrium. In vivo intracellular localization data revealed that the H89G mutation retargets Gag to intracellular compartments and severely inhibits virus production. Our findings reveal that the MA domain acts as a “pH sensor” in vitro, suggesting that the effect of pH on HIV-1 Gag targeting and binding to the PM warrants investigation.     

Potent and selective inhibition of angiotensin AT1 receptor signaling by RGS2: roles of its N-terminal domain

Emerging evidence indicates that R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in functional regulation in the cardiovascular system. In this study, we compared effects of three R4/B subfamily proteins, RGS2, RGS4 and RGS5 on angiotensin AT1 receptor signaling, and investigated roles of the N-terminus of RGS2. In HEK293T cells expressing AT1 receptor stably, intracellular Ca²⁺ responses induced by angiotensin II were much more strongly attenuated by RGS2 than by RGS4 and RGS5. N-terminally deleted RGS2 proteins lost this potent inhibitory effect. Replacement of the N-terminal residues 1-71 of RGS2 with the corresponding residues (1-51) of RGS5 decreased significantly the inhibitory effect. On the other hand, replacement of the residues 1-51 of RGS5 with the residues 1-71 of RGS2 increased the inhibitory effect dramatically. Furthermore, we investigated functional contribution of N-terminal subdomains of RGS2, namely, an N-terminal region (residues 16-55) with an amphipathic α helix domain (the subdomain N1), a probable non-specific membrane-targeting subdomain, and another region (residues 56-71) between the α helix and the RGS box (the subdomain N2), a probable GPCR-recognizing subdomain. RGS2 chimera proteins with the residues 1-33 or 34-52 of RGS5 showed weak inhibitory activity, and either of RGS5 chimera proteins with residues 1-55 or 56-71 of RGS2 showed strong inhibitory effects on AT1 receptor signaling. The present study indicates the essential roles of both N-terminal subdomains for the potent inhibitory activity of RGS2 on AT1 receptor signaling.