Raymond A. Grant

Characterization and cloning of a receptor for BMP-2 and BMP-4 from NIH 3T3 cells.

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor beta (TGF-beta)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57- to 62-kDa) and type II (75- to 82-kDa) receptor components for TGF-beta and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-beta and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP-4 binding and the appearance of a major affinity-labeled product of approximately 64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs. Images

Dandruff-associated Malassezia genomes reveal convergent and divergent virulence traits shared with plant and human fungal pathogens

Fungi in the genus Malassezia are ubiquitous skin residents of humans and other warm-blooded animals. Malassezia are involved in disorders including dandruff and seborrheic dermatitis, which together affect >50% of humans. Despite the importance of Malassezia in common skin diseases, remarkably little is known at the molecular level. We describe the genome, secretory proteome, and expression of selected genes of Malassezia globosa. Further, we report a comparative survey of the genome and secretory proteome of Malassezia restricta, a close relative implicated in similar skin disorders. Adaptation to the skin environment and associated pathogenicity may be due to unique metabolic limitations and capabilities. For example, the lipid dependence of M. globosa can be explained by the apparent absence of a fatty acid synthase gene. The inability to synthesize fatty acids may be complemented by the presence of multiple secreted lipases to aid in harvesting host lipids. In addition, an abundance of genes encoding secreted hydrolases (e.g., lipases, phospholipases, aspartyl proteases, and acid sphingomyelinases) was found in the M. globosa genome. In contrast, the phylogenetically closely related plant pathogen Ustilago maydis encodes a different arsenal of extracellular hydrolases with more copies of glycosyl hydrolase genes. M. globosa shares a similar arsenal of extracellular hydrolases with the phylogenetically distant human pathogen, Candida albicans, which occupies a similar niche, indicating the importance of host-specific adaptation. The M. globosa genome sequence also revealed the presence of mating-type genes, providing an indication that Malassezia may be capable of sex.

Regional protein alterations in rat kidneys induced by lead exposure.

Lead is a potent neuro‐ and nephrotoxin in humans and a renal carcinogen in rats. Previous studies have detected lead‐induced increases in the activities of specific detoxification enzymes in distinct kidney cell types preceding irreversible renal damage. While preferential susceptibility of the highly vascularized cortex to the effects of lead is clear, lead effects on the medullary region have remained unexplored. The present study was undertaken to investigate the extent to which regional renal protein expression differs and to determine which, if any, regionally distinct protein markers indicative of leads renotoxic mechanism might be detected in kidney cortical and medullary cytosols. We examined protein expression in these two functionally and anatomically distinct regions, and identified several proteins that are differentially expressed in those regions and were significantly altered by lead. Kidney cytosols from rats injected with lead acetate (114 mg/kg, three consecutive daily injections) were separated by two‐dimensional electrophoresis. Lead exposure significantly (P<0.001) altered the abundance (either ↑ or ↓) of 76 proteins in the cortex and only 13 in the medulla. Eleven of the proteins altered in the protein patterns were conclusively identified either by matrix‐assisted laser desorption/ionization mass spectrometry / electrospray ionization‐mass spectrometry (MALDI‐MS/ESI‐MS) analysis of peptide digests, immunological methods, or by gel matching. Several of the cortical proteins altered by lead were unchanged in the medulla while others underwent similar but lesser alterations. These observations reflect the complexity of leads nephrotoxicity and endorse the application of proteomics in mechanistic studies as well as biomarker development in a variety of toxicologic paradigms.

Zinc Pyrithione Inhibits Yeast Growth through Copper Influx and Inactivation of Iron-Sulfur Proteins

ABSTRACT Zinc pyrithione (ZPT) is an antimicrobial material with widespread use in antidandruff shampoos and antifouling paints. Despite decades of commercial use, there is little understanding of its antimicrobial mechanism of action. We used a combination of genome-wide approaches (yeast deletion mutants and microarrays) and traditional methods (gene constructs and atomic emission) to characterize the activity of ZPT against a model yeast, Saccharomyces cerevisiae. ZPT acts through an increase in cellular copper levels that leads to loss of activity of iron-sulfur cluster-containing proteins. ZPT was also found to mediate growth inhibition through an increase in copper in the scalp fungus Malassezia globosa. A model is presented in which pyrithione acts as a copper ionophore, enabling copper to enter cells and distribute across intracellular membranes. This is the first report of a metal-ligand complex that inhibits fungal growth by increasing the cellular level of a different metal.

Pharmacoproteomics in drug development

ABSTRACTThe field of proteomics is taking on increased significance as the relevance of investigating and understanding protein expression in disease and drug development is appreciated. Recent advances in proteomics have been driven by the availability of numerous annotated whole-genome sequences and a broad range of technological and bioinformatic developments that underscore the complexity of the proteome. This review briefly addresses some of the various technologies that comprise Expression Proteomics and Functional Proteomics, citing examples where these emerging approaches have been applied to pharmacology, toxicology, and the development of drugs.

Tandem mass spectrometry methods for definitive protein identification in proteomics research.

Optimized procedures have been developed for the addition of sulfonic acid groups to the N‐termini of low‐level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two‐dimensional (2‐D) gel electrophoresis. The derivatized peptides were sequenced using matrix‐assisted laser desorption/ionization (MALDI) post‐source decay (PSD) and electrospray ionization‐tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub‐picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization‐tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine‐terminated peptides. The spectra are easily interpreted de novo, and they facilitate error‐tolerant identification of proteins whose sequences have been entered into databases.

Proteomic analysis of simulated occupational jet fuel exposure in the lung.

We analyzed protein expression in the cytosolic fraction prepared from whole lung tissue in male Swiss‐Webster mice exposed 1 h/day for seven days to aerosolized JP‐8 jet fuel at concentrations of 1000 and 2500 mg/m3, simulating military occupational exposure. Lung cytosol samples were solubilized and separated via large scale, high resolution two‐dimensional electrophoresis (2‐DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant quantitative and qualitative changes in tissue cytosol proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting, confirmed by sequence tag analysis, and related to impaired protein synthetic machinery, toxic/metabolic stress and detoxification systems, ultrastructural damage, and functional responses to CO2 handling, acid‐base homeostasis and fluid secretion. These results demonstrate a significant but comparatively moderate JP‐8 effect on protein expression and corroborate previous morphological and biochemical evidence. Further molecular marker development and mechanistic inferences from these observations await proteomic analysis of whole tissue homogenates and other cell compartment, i.e., mitochondria, microsomes, and nuclei of lung and other targets.

Proteomic analysis of the renal effects of simulated occupational jet fuel exposure

We analyzed protein expression in the cytosolic fraction prepared from whole kidneys in male Swiss‐Webster mice exposed 1 h/day for five days to aerosolized JP‐8 jet fuel at a concentration of 1000 mg/m3, simulating military occupational exposure. Kidney cytosol samples were solubilized and separated via large‐scale, high‐resolution two‐dimensional electrophoresis (2‐DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant changes in soluble kidney proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting and related to ultrastructural abnormalities, altered protein processing, metabolic effects, and paradoxical stress protein/detoxification system responses. These results demonstrate a significant but comparatively moderate JP‐8 effect on protein expression in the kidney and provide novel molecular evidence of JP‐8 nephrotoxicity. Human risk is suggested by these data but conclusive assessment awaits a noninvasive search for biomarkers in JP‐8 exposed humans.

Subunit 6 of the Fo-ATP synthase complex from cytoplasmic male-sterile radish: RNA editing and NH2-terminal protein sequencing

RNA editing and NH2-terminal processing of subunit 6 (atp6) of the mitochondrial Fo-ATPase complex has been investigated for the normal (fertile) and Ogura (male-sterile) radish cytoplasms to determine if previously identified differences between the Ogura atp6 locus and its normal radish counterpart are associated with cytoplasmic male sterility. Analysis of cDNA clones from five different sterile and fertile radish lines identified one C-to-U transition, which results in the replacement of a proline with a serine, in several of the lines. No editing of atp6 transcripts was observed in two lines, Scarlet Knight (normal radish) and sterile CrGC15 (Ogura radish). This is the first example of a naturally occurring plant mitochondrial gene that is not edited. The Ogura atp6 polypeptide is synthesized with a predicted NH2-terminal extension of 174 amino acids in contrast to the nine amino acid extension found in normal radish. In spite of the lack of similarity between the two extensions, NH2-terminal sequence analysis indicates that both polypeptides are processed to yield identical core proteins with a serine as the NH2-terminal residue. These results indicate that ATPase subunit 6 is synthesized normally in Ogura radish, and that it is unlikely that the atp6 locus is associated with Ogura cytoplasmic male sterility.