Brownian dynamics simulation of helix-capping motifs

Helix-capping motifs are believed to play an important role in stabilizing alpha-helices and defining helix start and stop signals. We performed microsecond scale Brownian dynamics simulations to study ten XAAD sequences, with X = (A,E,I,L,N,Q,S,T,V,Y), to examine their propensity to form helix capping motifs and correlate these results with those obtained from analyzing a structural database of proteins. For the widely studied capping box motif S**D, where the asterisk can be any amino acid residue, the simulations suggested that one of the two hydrogen bonds proposed earlier as a stabilizing factor might not be as important. On the other hand, side-chain interactions between the capping residue and the third residue downstream on the polypeptide chain might also play a role in stabilizing this motif. These results are consistent with explicit-solvent molecular dynamics simulations of two capping box motifs found in the proteins BPTI and alpha-dendrotoxin. Principal component analysis of the SAAD trajectory showed that the first three principal components, after those corresponding to translational-rotational motion were removed, accounted for more than half of the conformational fluctuations. The first component separated the conformational space into two parts with the all-helical conformation and the capping box motif lying largely in one part. The second component, on the other hand, could be used to describe conformational transitions between the all-helical form and the capping box motif.     

The establishment of Caenorhabditis elegans germline pattern is controlled by overlapping proximal and distal somatic gonad signals

We investigated the control of proliferation and differentiation in the larval Caenorhabditis elegans hermaphrodite germ line through analysis of glp-1 and lag-2 mutants, cell ablations, and ultrastructural data. After the first several rounds of germ cell division, GLP-1, a receptor of the LIN-12/Notch family, governs germline proliferation. We analyzed the proximal proliferation (Pro) phenotype in glp-1(ar202) and found that initial meiosis was delayed and spatially mispositioned. This is due, at least in part, to a heightened response of the mutant GLP-1 receptor to multiple sources of the somatic ligand LAG-2, including the proximal somatic gonad. We investigated whether proximal LAG-2 affects germline proliferation in the wild type. Our results indicate that (1) LAG-2 is necessary for GLP-1-mediated germline proliferation and prevention of early meiosis, and (2) several distinct anatomical sources of LAG-2 in the larval somatic gonad functionally overlap to promote proliferation and prevent early meiosis. Ultrastructural studies suggest that mitosis is not restricted to areas of direct DTC-germ line contact and that the germ line shares a common cytoplasm in larval stages. We propose that downregulation of the GLP-1 signaling pathway in the proximal germ line at the time of meiotic onset is under tight temporal and spatial control.     

gp38k (CHI3L1) is a novel adhesion and migration factor for vascular cells

gp38k (CHI3L1) is a secreted heparin-binding glycoprotein whose expression, in vitro, is associated with vascular smooth muscle cell (VSMC) migration and invasion into the underlying gelatinous matrix. gp38k is expressed at high levels in postconfluent "nodular" VSMC cultures and at low levels in subconfluent proliferating cultures. In vivo, expression of gp38k homologs is high in regions of tissue remodeling and now has been detected in atherosclerotic plaques and in the developing heart. We tested the hypothesis that gp38k functions to modulate VSMC adhesion and migration. By use of modified Boyden chambers, gp38k at a concentration as low as 1 ng/ml has profound effects on VSMC migration but little or no effect on fibroblast migration. In addition, gp38k adsorbed to polystyrene surfaces directly promotes VSMC attachment and spreading. Attachment is inhibited in the presence of affinity-purified anti-gp38k or 10 mM EDTA. These results establish that gp38k is a new vascular cell adhesion and migration factor that may have a role in processes leading to vascular occlusion and heart development. gp38k may interact with VSMC via an EDTA-sensitive mechanism consistent with integrin mediated cell-matrix interaction.     

Membrane protein folding: beyond the two stage model

The folding of alpha-helical membrane proteins has previously been described using the two stage model, in which the membrane insertion of independently stable alpha-helices is followed by their mutual interactions within the membrane to give higher order folding and oligomerization. Given recent advances in our understanding of membrane protein structure it has become apparent that in some cases the model may not fully represent the folding process. Here we present a three stage model which gives considerations to ligand binding, folding of extramembranous loops, insertion of peripheral domains and the formation of quaternary structure.     

A tomato xylem sap protein represents a new family of small cysteine-rich proteins with structural similarity to lipid transfer proteins

The coding sequence of a major xylem sap protein of tomato was identified with the aid of mass spectrometry. The protein, XSP10, represents a novel family of extracellular plant proteins with structural similarity to plant lipid transfer proteins. The XSP10 gene is constitutively expressed in roots and lower stems. The decline of XSP10 protein levels in tomato infected with a fungal vascular pathogen may reflect breakdown or modification by the pathogen.     

Genetic analysis of Caenorhabditis elegans glp-1 mutants suggests receptor interaction or competition

glp-1 encodes a member of the highly conserved LIN-12/Notch family of receptors that mediates the mitosis/meiosis decision in the C. elegans germline. We have characterized three mutations that represent a new genetic and phenotypic class of glp-1 mutants, glp-1(Pro). The glp-1(Pro) mutants display gain-of-function germline pattern defects, most notably a proximal proliferation (Pro) phenotype. Each of three glp-1(Pro) alleles encodes a single amino acid change in the extracellular part of the receptor: two in the LIN-12/Notch repeats (LNRs) and one between the LNRs and the transmembrane domain. Unlike other previously described gain-of-function mutations that affect this region of LIN-12/Notch family receptors, the genetic behavior of glp-1(Pro) alleles is not consistent with simple hypermorphic activity. Instead, the mutant phenotype is suppressed by wild-type doses of glp-1. Moreover, a trans-heterozygous combination of two highly penetrant glp-1(Pro) mutations is mutually suppressing. These results lend support to a model for a higher-order receptor complex and/or competition among receptor proteins for limiting factors that are required for proper regulation of receptor activity. Double-mutant analysis with suppressors and enhancers of lin-12 and glp-1 further suggests that the functional defect in glp-1(Pro) mutants occurs prior to or at the level of ligand interaction.     

Endocrine correlates of intra-specific variation in the mating system of the St. Peter's fish (Sarotherodon galilaeus)

The Challenge Hypothesis postulates that androgen levels are a function of the social environment in which the individual is living. Thus, it is predicted that in polygynous males that engage in social interactions, androgen levels should be higher than in monogamous animals that engage in parental care. In this study, we tested this hypothesis at the intra-specific level using a teleost species, Sarotherodon galilaeus, which exhibits a wide variation in its mating system. Experimental groups of individually marked fish were formed in large ponds with different operational sex-ratios (OSR) to study the effects of partner availability on blood plasma levels of sex steroids [11-ketotestosterone (11-KT), testosterone (T), and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P)] and gonadosomatic index (GSI). Polygyny mostly occurred in the female biased OSR groups. 17,20beta-P and gonadosomatic index did not differ among OSR groups. However, 11-KT was high in male biased OSR and positively correlated with aggressive challenges, thereby supporting the central postulate of the Challenge Hypothesis. The results of T were the inverse of those of 11-KT, probably because 11-KT is metabolized from T. 11-KT levels of polygynous males did not differ neither from those of monogamous males, nor from those of males that participated in parental care. These results do not support the expected relationships between polygyny, parental care, and androgen levels. The differences from expectations for 11-KT may be related to the fact that in S. galilaeus, the mating and the parenting phase are not clearly separated and thus, males may still fight and court while they are brooding.     

Targeting presenilin-type aspartic protease signal peptide peptidase with gamma-secretase inhibitors

Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the gamma-secretase complex that catalyzes intramembrane cleavage of beta-amyloid precursor protein, the last step in the generation of amyloidogenic Abeta peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of gamma-secretase are considered potential therapeutics for the treatment of this disease because they prevent production of Abeta peptides. Recently, we discovered a family of presenilin-type aspartic proteases. The founding member, signal peptide peptidase, catalyzes intramembrane cleavage of distinct signal peptides in the endoplasmic reticulum membrane of animals. In humans, the protease plays a crucial role in the immune system. Moreover, it is exploited by the hepatitis C virus for the processing of the structural components of the virion and hence is an attractive target for anti-infective intervention. Signal peptide peptidase and presenilin share identical active site motifs and both catalyze intramembrane proteolysis. These common features let us speculate that gamma-secretase inhibitors directed against presenilin may also inhibit signal peptide peptidase. Here we demonstrate that some of the most potent known gamma-secretase inhibitors efficiently inhibit signal peptide peptidase. However, we found compounds that showed higher specificity for one or the other protease. Our findings highlight the possibility of developing selective inhibitors aimed at reducing Abeta generation without affecting other intramembrane-cleaving aspartic proteases.     

Eosinophil peroxidase-derived reactive brominating species target the vinyl ether bond of plasmalogens generating a novel chemoattractant,alpha-bromo fatty aldehyde

Plasmalogens are a subclass of glycerophospholipids that are enriched in the plasma membrane of many mammalian cells. The vinyl ether bond of plasmalogens renders them susceptible to oxidation. Accordingly, it was hypothesized that reactive brominating species, a unique oxidant formed at the sites of eosinophil activation, such as in asthma, might selectively target plasmalogens for oxidation. Here we show that reactive brominating species produced by the eosinophil peroxidase system of activated eosinophils attack the vinyl ether bond of plasmalogens. Reactive brominating species produced by eosinophil peroxidase target the vinyl ether bond of plasmalogens resulting in the production of a neutral lipid and lysophosphatidylcholine. Chromatographic and mass spectrometric analyses of this neutral lipid demonstrated that it was 2-bromohexadecanal (2-BrHDA). Reactive brominating species produced by eosinophil peroxidase attacked the plasmalogen vinyl ether bond at acidic pH. Bromide was the preferred substrate for eosinophil peroxidase, and chloride was not appreciably used even at a 1000-fold molar excess. Furthermore, 2-BrHDA production elicited by eosinophil peroxidase-derived reactive brominating species in the presence of 100 microM NaBr doubled with the addition of 100 mM NaCl. The potential physiological significance of this pathway was suggested by the demonstration that 2-BrHDA was produced by phorbol myristate acetate-stimulated eosinophils and by the demonstration that 2-BrHDA is a phagocyte chemoattractant. Taken together, the present studies demonstrate the targeting of the vinyl ether bond of plasmalogens by the reactive brominating species produced by eosinophil peroxidase and by activated eosinophils, resulting in the production of brominated fatty aldehydes.