Structural properties of the chloroplast stromal processing peptidase required for its function in transit peptide removal

The stromal processing peptidase (SPP) catalyzes removal of transit peptides from a diversity of precursor proteins imported into chloroplasts. SPP contains an HXXEH zinc-binding motif characteristic of members of the metallopeptidase family M16. We previously found that the three steps of precursor processing by SPP (i.e. transit peptide binding, removal, and conversion to a degradable subfragment) are mediated by features that reside in the C-terminal 10-15 residues of the transit peptide. In this study, we performed a mutational analysis of SPP to identify structural elements that determine its function. SPP loses the ability to proteolytically remove the transit peptide when residues of the HXXEH motif, found in an N-terminal region, are mutated. Deletion of 240 amino acids from its C terminus also abolishes activity. Interestingly, however, SPP can still carry out the initial binding step, recognizing the C-terminal residues of the transit peptide. Hence, transit peptide binding and removal are two separable steps of the overall processing reaction. Transit peptide conversion to a subfragment also depends on the HXXEH motif. The precursor of SPP, containing an unusually long transit peptide itself, is not proteolytically active. Thus, the SPP precursor is synthesized as a latent form of the metallopeptidase.     

Functionalized self-assembled monolayer on gold for detection of human mitochondrial tRNA gene mutations

We developed a rapid and simple method to identify single-nucleotide polymorphisms (SNPs) in the human mitochondrial tRNA genes. This method is based on a universal, functionalized, self-assembled monolayer, XNA on Gold chip platform. A set of probes sharing a given allele-specific sequence with a single base substitution near the middle of the sequence was immobilized on chips and the chips were then hybridized with fluorescence-labeled reference targets produced by asymmetric polymerase chain reaction from patient DNA. The ratio of the hybridization signals from the reference and test targets with each probe was then calculated. A ratio of above 3 indicates the presence of a wild-type sequence and a ratio of below 0.3 indicates a mutant sequence. We tested the sensitivity of the chip for known mutations in tRNA(Leu(UUR)) and tRNA(Lys) genes and found that it can also be used to discriminate multiple mutations and heteroplasmy, two typical features of human mitochondrial DNA. The XNA on Gold biochip method is a simple and rapid microarray method that can be used to test rapidly and reliably any SNP in the mitochondrial genome or elsewhere. It will be particularly useful for detecting SNPs associated with human diseases.     

Regulation of PCNA and CAF-1 expression by the two tuberous sclerosis gene products

Tuberous sclerosis is an autosomal dominant tumor suppressor gene syndrome affecting about 1 in 6000 individuals. Two genes have been shown to be responsible for this disease: TSC1, encoding hamartin and TSC, encoding tuberin. A variety of tumors characteristically occur in different organs of tuberous sclerosis patients and are believed to result from defects in cell cycle/cell size control. In this study, we performed two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after overexpression of TSC1 or TSC2. We found expression of PCNA and the p48 subunit of CAF-1 to be regulated by two tuberous sclerosis gene products. CAF-1 and PCNA interact as major regulators of chromatin assembly during DNA repair. We suggest that deregulation of the control of chromatin assembly might contribute to development of tumors in tuberous sclerosis patients and provide important new insights into the molecular development, especially since deregulation of chromatin assembly and DNA repair results in genomic instability, a hallmark of tumor development.     

Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.     

Determinants for removal and degradation of transit peptides of chloroplast precursor proteins

The stromal processing peptidase (SPP) cleaves a large diversity of chloroplast precursor proteins, removing an N-terminal transit peptide. We predicted previously that this key step of the import pathway is mediated by features of the transit peptide that determine precursor binding and cleavage followed by transit peptide conversion to a degradable substrate. Here we performed competition experiments using synthesized oligopeptides of the transit peptide of ferredoxin precursor to investigate the mechanism of these processes. We found that binding and processing of ferredoxin precursor depend on specific interactions of SPP with the region consisting of the C-terminal 12 residues of the transit peptide. Analysis of four other precursors suggests that processing depends on the same region, although their transit peptides are highly divergent in primary sequence and length. Upon processing, SPP terminates its interaction with the transit peptide by a second cleavage, converting it to a subfragment form. From the competition experiments we deduce that SPP releases a subfragment consisting of the transit peptide without its original C terminus. Interestingly, examination of the ATP-dependent metallopeptidase activity responsible for degradation of transit peptide subfragments suggests that it may recognize other unrelated peptides and, hence, act separately from SPP as a novel stromal oligopeptidase.     

Synthesis and biological activity of selective pipecolic acid-based TNF-alpha converting enzyme (TACE) inhibitors

A series of novel, selective TNF-alpha converting enzyme inhibitors based on 4-hydroxy and 5-hydroxy pipecolate hydroxamic acid scaffolds is described. The potency and selectivity of TACE inhibition is dramatically influenced by the nature of the sulfonamide group which interacts with the S1' site of the enzyme. Substituted 4-benzyloxybenzenesulfonamides exhibit excellent TACE potency with >100x selectivity over inhibition of matrix metalloprotease-1 (MMP-1). Alkyl substituents on the ortho position of the benzyl ether moiety give the most potent inhibition of TNF-alpha release in LPS-treated human whole blood.     

Seroprevalence of Trypanosoma cruzi in raccoons from South Carolina and Georgia

Trypanosoma cruzi, the causative agent of American trypanosomiasis or Chagas' disease, is of both medical and veterinary importance as is evidenced by chronic phase myocarditis in humans and dogs. Further, T. cruzi has been reported from over 20 species of wildlife reservoir hosts in the USA, with raccoons (Procyon lotor) and opossums (Didelphis virginiana) being the most common. Whereas previous studies on T. cruzi in raccoons have included only culture and direct examination of blood, the indirect immunofluorescent antibody test (IFAT) was used in the current study to detect anti-T. cruzi antibodies in the serum of raccoons. Of 221 raccoons trapped at 13 sites representing the five physiographic regions of South Carolina plus five sites in the Piedmont region of Georgia (from April 1997 to February 2000), 104 (47%) were seropositive. A higher seroprevalence in raccoons was observed in the coastal regions, with seroprevalence in the Lower Coastal Plain South (61%) being significantly higher than that in the Foothills (37%), Piedmont (42%), and Upper Coastal Plain (40%) regions. However, at a seroprevalence of 52%, the Lower Coastal Plain North was not significantly different from any other region. Although more female raccoons were infected than males, no statistical difference in prevalence was observed between sexes. The high seroprevalence of T. cruzi in raccoons, together with a few reports of wildlife isolates being infective for other wildlife species and domestic/laboratory animals, suggests that risk of T. cruzi infection may be higher than previously suspected.     

Translocation of an intracellular antigen to the surface of medullary breast cancer cells early in apoptosis allows for an antigen-driven antibody response elicited by tumor-infiltrating B cells

Tumor-infiltrating lymphoplasmacytic cells are a key feature of medullary carcinoma of the breast (MCB), a distinct subtype of human breast cancer that, despite cytologically anaplastic characteristics, has a more favorable prognosis than other types of breast cancer. Since it has been proposed that the improved clinical outcome is due at least in part to the presence of a prominent lymphoplasmacytic cell infiltrate in the tumor stroma, we recently examined the tumor-infiltrating B cell response in MCB and showed that it is oligoclonal and directed against an intracellular protein translocated to the cell surface upon MCB cell apoptosis. Human Abs cloned from MCB lymphoplasmacytic infiltrate-derived phage display libraries and reflecting the dominant part of the response were used to identify the target Ag as actin. Here, we have characterized in detail the cloned human IgG Abs and the translocation process of actin to the cell surface of apoptotic MCB cells. Our analysis shows that the cloned Abs bind specifically and with high affinity to actin, as determined by ELISA and surface plasmon resonance. Sequence analysis revealed that the Abs are highly somatically mutated, with high replacement to silent ratios, indicative of an Ag-driven, affinity-matured response. Interestingly, the tumor-infiltrating B cells in half the MCB patients mainly exhibited an IgG2 response, while IgG1 dominated in the others. To gain insight to the molecular events that may elicit such an Ab response, we examined the translocation of actin to the cell surface of apoptotic MCB cells using flow cytometry and laser scanning cytometry. Our results show that actin becomes exposed on the cell surface of a large proportion of apoptotic MCB cells as an early apoptotic event. We propose that the Ab response against actin produced by tumor-infiltrating B lymphoplasmacytic cells is Ag-driven, affinity-matured, and elicited due to the increased rate of apoptosis occurring within the MCB tumor that facilitates the translocation and proteolytic fragmentation of intracellular proteins.     

Brca1 and differentiation

Breast cancer is one of the most frequent malignancies affecting women. The human breast cancer gene 1 (BRCA1) gene is mutated in a distinct proportion of hereditary breast and ovarian cancers. Tumourigenesis in individuals with germline BRCA1 mutations requires somatic inactivation of the remaining wild-type allelle. Although, this evidence supports a role for BRCA1 as a tumour suppressor, the mechanisms through which its loss leads to tumourigenesis remain to be determined. Neither the expression pattern nor the described functions of human BRCA1 and murine breast cancer gene 1 (Brca1) can explain the specific association of mutations in this gene with the development of breast and ovarian cancer. Investigation of the role of Brca1 in normal cell differentiation processes might provide the basis to understand the tissue-restricted properties.     

Hexose phosphorylation and the putative calcium channel component Mid1p are required for the hexose-induced transient elevation of cytosolic calcium response in Saccharomyces cerevisiae

Saccharomyces cerevisiae responds to environ-mental stimuli such as an exposure to pheromone or to hexoses after carbon source limitation with a transient elevation of cytosolic calcium (TECC) response. In this study, we examined whether hexose transport and phosphorylation are necessary for the TECC response. We found that a mutant strain lacking most of the known hexose transporters was unable to carry out the TECC response when exposed to glucose. A mutant strain that lacked the ability to phosphorylate glucose was unable to respond to glucose addition, but displayed a normal TECC response after the addition of galactose. These results indicate that hexose uptake and phosphorylation are required to trigger the hexose-induced TECC response. We also found that the TECC response was significantly smaller than normal when the level of environmental calcium was reduced, and was abolished in a mid1 mutant that lacked a subunit of the high-affinity calcium channel of the yeast plasma membrane. These results indicate that most or all of the TECC response is mediated by an influx of calcium from the extracellular space. Our results indicate that this transient increase in plasma membrane calcium permeability may be linked to the accumulation of Glc-1-P (or a related glucose metabolite) in yeast.