Xenopus laevis skin Arg-Xaa-Val-Arg-Gly-endoprotease. A highly specific protease cleaving after a single arginine of a consensus sequence of peptide hormone precursors

Comparison of the precursor sequence for several peptide hormones of Xenopus laevis skin revealed a consensus sequence around a single arginine cleavage site which is 100% conserved on four residues Arg-Xaa-Val-Arg-Gly (RXVRG). A tetradecapeptide substrate (Asp-Val-Asp-Glu-Arg-Asp-Val-Arg-Gly-Phe-Ala-Ser-Phe-Leu-NH2) was used as a probe to purify and characterize the putative processing endoprotease. A hydrophobic enzyme was purified at least 9000-fold from Xenopus skin exudate by a four-step procedure. This highly specific activity cleaves the Arg-Gly bond and has no effect on the Arg-Xaa bond. It was strongly inhibited by divalent ion chelators, moderately by phenylmethylsulfonyl fluoride, aprotinin, and 1-tosylamide-2-phenylethyl chloromethyl ketone, but was insensitive to soybean trypsin inhibitor. Tetradecapeptide derivatives selectively modified on each of the amino acids of the consensus sequence demonstrated the relevance of this conserved pattern to endoprotease action. This enzyme, which we refer to as RXVRG-endoprotease, is proposed to be involved in the post-translational processing of pro-caerulein, promagainin, pro-xenopsin, pro-glycyl-leucine amide, and pro-levitide of X. laevis skin secretory granules.     

Feeding preferences of <Emphasis Type="Italic">Gomphotherium subtapiroideum</Emphasis> (Proboscidea,Mammalia) from the Miocene of Sandelzhausen (Northern Alpine Foreland Basin,southern Germany) through life and geological time: evidence from dental microwear analysis

The objective of this study is to estimate changes in feeding preferences of the proboscidean species Gomphotherium subtapiroideum (Schlesinger 1917) by means of dental microwear analyses. The dietary changes are first evaluated through the ontogeny of this species, between juveniles and adults, and are then studied through geological time, from early Middle Miocene (MN5) to middle Late Miocene (MN8–9) localities of the German Molasse Basin. The microwear patterns of juvenile and adult individuals of G. subtapiroideum from Sandelzhausen (MN5) differ merely by the variable “length of scratches”, emphasizing longer jaw movements during mastication in adults. The microwear signatures of G. subtapiroideum do not vary significantly between the two geological time periods studied, but reflect mixed feeding preferences in both cases. These results imply that, despite an important environmental change at that time (drying and opening), the ecology of G. subtapiroideum and, especially, its feeding habits were not affected. Its dental microwear pattern is then compared with those of other species of Proboscidea from the Middle-Late Miocene of Germany, namely Deinotherium giganteum and Gomphotherium steinheimense.     

Transfer-NMR and docking studies identify the binding of the peptide derived from activating transcription factor 4 to protein ubiquitin ligase beta-TrCP. Competition STD-NMR with beta-catenin

ATF4 plays a crucial role in the cellular response to stress. The E3 ubiquitin ligase, SCF beta-TrCP protein responsible for ATF4 degradation by the proteasome, binds to ATF4 through a DpSGXXXpS phosphorylation motif, which is similar but not identical to the DpSGXXpS motif found in most other substrates of beta-TrCP. NMR studies were performed on the free and bound forms of a peptide derived from this ATF4 motif that enabled the elucidation of the conformation of the ligand complexed to the beta-TrCP protein and its binding mode. Saturation transfer difference (STD) NMR allowed the study of competition for binding to beta-TrCP, between the phosphorylation motifs of ATF4 and beta-catenin, to characterize the ATF4 binding epitope. Docking protocols were performed using the crystal structure of the beta-catenin-beta-TrCP complex as a template and NMR results of the ATF4-beta-TrCP complex. In agreement with the STD results, in order to bind to beta-TrCP, the ATF4 DpSGIXXpSXE motif required the association of two negatively charged areas, in addition to the hydrophobic interaction in the beta-TrCP central channel. Docking studies showed that the ATF4 DpSGIXXpSXE motif fits the binding pocket of beta-TrCP through an S-turning conformation. The distance between the two phosphate groups is 17.8 A, which matched the corresponding distance 17.1 A for the other extended DpSGXXpS motif in the beta-TrCP receptor model. This study identifies the residues of the beta-TrCP receptor involved in ligand recognition. Using a new concept of STD competition experiment, we show that ATF4 competes and inhibits binding of beta-catenin to beta-TrCP.     

Protein Repair l-Isoaspartyl Methyltransferase1 Is Involved in Both Seed Longevity and Germination Vigor in Arabidopsis

The formation of abnormal amino acid residues is a major source of spontaneous age-related protein damage in cells. The protein l-isoaspartyl methyltransferase (PIMT) combats protein misfolding resulting from l-isoaspartyl formation by catalyzing the conversion of abnormal l-isoaspartyl residues to their normal l-aspartyl forms. In this way, the PIMT repair enzyme system contributes to longevity and survival in bacterial and animal kingdoms. Despite the discovery of PIMT activity in plants two decades ago, the role of this enzyme during plant stress adaptation and in seed longevity remains undefined. In this work, we have isolated Arabidopsis thaliana lines exhibiting altered expression of PIMT1, one of the two genes encoding the PIMT enzyme in Arabidopsis. PIMT1 overaccumulation reduced the accumulation of l-isoaspartyl residues in seed proteins and increased both seed longevity and germination vigor. Conversely, reduced PIMT1 accumulation was associated with an increase in the accumulation of l-isoaspartyl residues in the proteome of freshly harvested dry mature seeds, thus leading to heightened sensitivity to aging treatments and loss of seed vigor under stressful germination conditions. These data implicate PIMT1 as a major endogenous factor that limits abnormal l-isoaspartyl accumulation in seed proteins, thereby improving seed traits such as longevity and vigor. The PIMT repair pathway likely works in concert with other anti-aging pathways to actively eliminate deleterious protein products, thus enabling successful seedling establishment and strengthening plant proliferation in natural environments.     

The key-role of tyrosine 155 in the mechanism of prion transconformation as highlighted by a study of sheep mutant peptides

Prion protein is a strongly conserved and ubiquitous glycoprotein. The conformational conversion of the non-pathogenic cellular prion isoform (PrP(C)) into a pathogenic scrapie isoform (PrP(Sc)) is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this conversion, helix H1 and its two flanking loops are known to undergo a conformational transition into a beta-like structure. In order to understand mechanisms which trigger this transconformation, sheep prion protein synthetic peptides spanning helix 1 and beta-strand 2 (residues 142-166) were studied: (1) the N3 peptide, studied earlier, is known to fold into beta-hairpin-like conformation in phosphate buffer at neutral pH and to adopt a helix H1 conformation when dissolved in trifluoroethanol/phosphate buffer mixture, (2) The R156A mutant (peptide R15) and (3) the Y155A mutant (peptide Y14) of the N3 peptide are studied by circular dichroism and NMR spectroscopy in this article. Structural characterization of these peptides highlights the key role of tyrosine 155 in the stabilization of the beta-hairpin-like conformation of the sheep peptide in phosphate buffer. We propose a model where tyrosine 155 could stabilize the beta-hairpin structure by creating a hydrophobic core in phosphate buffer, necessary to initiate the beta-type structure formation. In the turn, the side chain ionic interaction, E152-R156 described before, seems to play a minor role relative to the hydrophobic packing, as observed with the R156A mutation (peptide R15). Interestingly, homology at amino acid residue 155 could be responsible for the species barrier in TSE.     

Seed aging and survival mechanisms

Aging and death are universal to living systems. In temperate climate latitudes the mature seeds of higher plants are exposed to aging and have developed resistance mechanisms allowing survival and plant propagation. In addition to the physicochemical properties of the seed that confer stress resistance, the protein metabolism contributes importantly to longevity mechanisms. Recently, genetic studies have demonstrated the occurrence of the Protein L-isoaspartyl methyltransferase repair enzyme in controlling age-related protein damages and seed survival. These protective mechanisms by protein repair are widespread in all kingdoms, so that the use of seeds as models to study these controlling processes offers the prospect of understanding longevity mechanisms better.     

Visible minorities and ‘White’–‘non-White’ conjugal unions in Canadian large cities

The study investigates assortative mating patterns with respect to race (visible minority status) in Canada. Using the 2001 Census data, the article analyses the occurrence of White/non-White unions in Montreal, Toronto and Vancouver. Log-linear models indicate that the relative levels of interracial relationships vary across racial groups, immigration status, and place of residence. First, the highest odds of cohabiting or marrying a White person are found among Blacks. Whereas the high level of racial exogamy of Blacks is observed in all metropolitan areas under study, the relative position of other groups varies. Second, the highest levels of racial exogamy are found among couples composed of an immigrant and a non-immigrant but this effect varies across racial groups. Third, our hypothesis that residents of Montreal (Quebec) will inter-partner less was confirmed only for unions between two native-born Canadians. Finally, we found that French Canadians are not more inclusive of their linguistic counterparts than Anglophones.     

The S4-S5 linker of KCNQ1 channels forms a structural scaffold with the S6 segment controlling gate closure

In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac potassium current (I(Ks)). Structurally, they share their topology with other Kv channels and consist out of six transmembrane helices (S1-S6) with the S1-S4 segments forming the voltage-sensing domain (VSD). The opening or closure of the intracellular channel gate, which localizes at the bottom of the S6 segment, is directly controlled by the movement of the VSD via an electromechanical coupling. In other Kv channels, this electromechanical coupling is realized by an interaction between the S4-S5 linker (S4S5(L)) and the C-terminal end of S6 (S6(T)). Previously we reported that substitutions for Leu(353) in S6(T) resulted in channels that failed to close completely. Closure could be incomplete because Leu(353) itself is the pore-occluding residue of the channel gate or because of a distorted electromechanical coupling. To resolve this and to address the role of S4S5(L) in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5(L). The residues with a "high impact" on channel gating (when mutated) clustered on one side of the S4S5(L) α-helix. Hence, this side of S4S5(L) most likely contributes to the electromechanical coupling and finds its residue counterparts in S6(T). Accordingly, substitutions for Val(254) resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu(353) in S6(T). Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.