Identification of essential residues in the type II Hsp40 Sis1 that function in polypeptide binding

Sis1 is an essential yeast Type II Hsp40 protein that assists cytosolic Hsp70 Ssa1 in the facilitation of processes that include translation initiation, the prevention of protein aggregation, and proteasomal protein degradation. An essential function of Sis1 and other Hsp40 proteins is the binding and delivery of non-native polypeptides to Hsp70. How Hsp40s function as molecular chaperones is unknown. The crystal structure of a Sis1 fragment that retains peptide-binding activity suggests that Type II Hsp40s utilize hydrophobic residues located in a solvent-exposed patch on carboxyl-terminal domain I to bind non-native polypeptides. To test this model, amino acid residues Val-184, Leu-186, Lys-199, Phe-201, Ile-203, and Phe-251, which form a depression in carboxyl-terminal domain I, were mutated, and the ability of Sis1 mutants to support cell viability and function as molecular chaperones was examined. We report that Lys-199, Phe-201, and Phe-251 are essential for cell viability and required for Sis1 polypeptide binding activity. Sis1 I203T could support normal cell growth, but when purified it exhibited severe defects in chaperone function. These data identify essential residues in Sis1 that function in polypeptide binding and help define the nature of the polypeptide-binding site in Type II Hsp40 proteins.     

Analysis on additive effects and additive-by-additive epistatic effects of QTLs for yield traits in a recombinant inbred line population of rice

A linkage map consisting of 158 DNA markers were constructed by using a recombinant inbred line (RIL) population derived from the indica-indica rice cross Zhenshan 97B 2 Milyang 46. Quantitative trait loci (QTLs) conditioning grain yield and five yield component traits were determined at the one-locus and two-locus levels, and genotype-by-environment (GE) interactions were analyzed. Thirty-one QTLs were detected to have significant additive effects for yield traits, of which 12 also exhibited significant epistatic effects. Sixteen significant additive-by-additive (AA) interactions were detected, of which nine occurred between QTLs with own additive effects (M_epQTLs), four occurred between QTLs showing epistatic effects only (epQTLs), and three occurred between M_epQTLs and epQTLs. Significant GE interactions were found for six QTLs with additive effects and one AA interaction. Generally, the contributions to the phenotypic variation were higher due to QTL main effects than to epistatic effects. The detection of additive effects and AA effects of a QTL interfered with each other, indicating that the detection of QTLs with main effects, as well as the magnitude and directions of the additive effects, might vary depending on their interactions with other loci.     

Effect of alpha2M on earthworm fibrinolytic enzyme III-1 from Lumbricus rubellus

Though it is known that human alpha2-macroglobulin (alpha2M) inhibits most proteases, the effect of alpha2M has not been investigated on earthworm fibrinolytic enzymes (EFEs) from Lumbricus rubellus, which could be transported from intestine epithelium into blood as an intact molecule (Fan et al., Biochim. Biophys. Acta 1526 (2001) 286). The activity of earthworm fibrinolytic III-1 (EFE-III-1) decreased to 65% when incubated with alpha2M, while it decreased to 30% in plasma under the same conditions. The first order rate of the inactivation of EFE-III-1 with alpha2M was similar to that of fast phase with plasma, indicating that alpha2M may be the inhibitor initially binding to the enzyme in blood. SDS-PAGE showed that incubation of EFE-III-1 with alpha2M a released fragment ( approximately 90 kDa), followed by formation of a high molecular weight complex (approximately 700 kDa). There was a linear relationship between the apparent inhibition rate constant (k1) and [alpha2M], by double reciprocal plot. It was suggested, as described by Tsou (Acta Biochem. Biophys. Sinica 5 (1965) 398) and Tian (Biochem. J. 21 (1982) 1028), that the mechanism of alpha2M/EFE-III-1 interaction could be coincided with a complexing irreversible inhibition. Experiments in both the inactivation and the intrinsic fluorescence showed that alpha2M bound to the enzyme mole by mole equivalently. The intrinsic fluorescence of alpha2M was enhanced with an observable blue shift in emission maxima, suggesting that alpha2M was one of the important inhibitors to EFEs when it absorbed into blood.     

Visualization of the uptake of high-density lipoprotein by rat aortic endothelial cells and smooth muscle cells in vitro

High-density lipoproteins (HDL) were conjugated to Fluorescein 1,1-dioctadecyl 3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) or colloidal gold for the investigation of ultrastructural aspects of binding and uptake of HDL by cholesterol-loaded cultured endothelial and smooth muscle cells from rat aorta. When cells were incubated for 2h at 4°C, HDL–DiI and HDL–gold conjugates were seen only on the cell surface. When cells were returned to incubation at 37°C for 5min, HDL–DiI appeared in the cytoplasm and colocalized with the fluorescent cholesteryl ester tag BODIPY-FL-C₁₂. HDL–gold conjugates appeared in the plasmalemmal invaginations and plasmalemmal vesicles. After incubation for 15min, most of the HDL–gold conjugates reappeared on the cell surface. After incubation for 30min, only a few conjugates were observed and they localized in lysosomal-like bodies. Quantitative data indicated that when the cholesterol-loaded cells were incubated at 4°C for 2h, the numbers of HDL–gold associated in clusters on the endothelial cell surface was 1.18 clusters/m. When cells were returned to incubation at 37°C for 5min, this value decreased to 0.7, increased again to 1.13 at 15min, and decreased to 0.29 at 30min. The numbers of clusters in the plasmalemmal invaginations were 0.06 clusters/m at 4°C for 2h, increased to 0.34 at 37°C for 5min and decreased gradually to 0.19 and 0.04 at 15 and 30min, respectively. The incidence of clusters in the plasmalemmal vesicles per non-nuclear cytoplasm was 0.01 clusters/m² at 4°C for 2h, increased significantly to 1.08 at 37°C for 5min, and decreased to 0.43 and 0.14 at 15 and 30min, respectively. This work supports that the plasmalemmal invaginations and plasmalemmal vesicles are linked to the HDL uptake in cholesterol-loaded aortic endothelial cells and smooth muscle cells.     

NADPH oxidase is involved in prostaglandin F2alpha-induced hypertrophy of vascular smooth muscle cells: induction of NOX1 by PGF2alpha

Prostaglandin (PG) F(2alpha), one of the primary prostanoids generated in vascular tissue, is known to cause hypertrophy in vascular smooth muscle cells. To clarify the molecular mechanisms underlying PGF(2alpha)-induced hypertrophy, the involvement of reactive oxygen species was examined in a rat vascular smooth muscle cell line, A7r5. PGF(2alpha) and (+)-fluprostenol, a selective agonist of the PGF receptor, significantly increased intracellular O(2)(-) in A7r5. The PGF(2alpha)-induced O(2)(-) increase was suppressed by diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase that has been reported to be the major source of O(2)(-) in vascular cells. The augmented synthesis of the protein induced by PGF(2alpha) or (+)-fluprostenol was suppressed in the presence of DPI. In PGF(2alpha) or (+)-fluprostenol-treated cells, a dose-dependent increase in the expression of NOX1, a homolog of the catalytic subunit of the phagocyte NADPH oxidase gp91(phox), was demonstrated by Northern blot analysis. Finally, depletion of NOX1 mRNA in the cells transfected with ribozymes targeted for three independent cleavage sites on the mRNA sequence significantly reduced the PGF(2alpha)-induced increase in protein synthesis. Taken together, these results suggest that hypertrophy of vascular smooth muscle cells caused by PGF(2alpha) is mediated by NOX1 induction and the resultant overproduction of O(2)(-) by NADPH oxidase.     

Exploring the stereochemistry of CXCR4-peptide recognition and inhibiting HIV-1 entry with D-peptides derived from chemokines

Chemokine receptor CXCR4 plays an important role in the immune system and the cellular entry of human immunodeficiency virus type 1 (HIV-1). To probe the stereospecificity of the CXCR4-ligand interface, d-amino acid peptides derived from natural chemokines, viral macrophage inflammatory protein II (vMIP-II) and stromal cell-derived factor-1alpha (SDF-1alpha), were synthesized and found to compete with (125)I-SDF-1alpha and monoclonal antibody 12G5 binding to CXCR4 with potency and selectivity comparable with or higher than their l-peptide counterparts. This was surprising because of the profoundly different side chain topologies between d- and l-enantiomers, which circular dichroism spectroscopy showed adopt mirror image conformations. Further direct binding experiments using d-peptide labeled with fluorescein (designated as FAM-DV1) demonstrated that d- and l-peptides shared similar or at least overlapping binding site(s) on the CXCR4 receptor. Structure-activity analyses of related peptide analogs of mixed chiralities or containing alanine replacements revealed specific residues at the N-terminal half of the peptides as key binding determinants. Acting as CXCR4 antagonists and with much higher biological stability than l-counterparts, the d-peptides showed significant activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. These results show the remarkable stereochemical flexibility of the CXCR4-peptide interface. Further studies to understand the mechanism of this unusual feature of the CXCR4 binding surface might aid the development of novel CXCR4-binding molecules like the d-peptides that have high affinity and stability.     

Models and tests of linkage and association studies of quantitative trait locus for multi-allele marker Loci

In this paper, we explore models and tests for association and linkage studies of a quantitative trait locus (QTL) linked to a multi-allele marker locus. Based on the difference between an offspring's conditional trait means of receiving and not receiving an allele from a parent at marker locus, we propose three statistics T(m), T(m,row) and T(m,col) to test association or linkage disequilibrium between the marker locus and the QTL. These tests are composite tests, and use the offspring marginal sample means including offspring data of both homozygous and heterozygous parents. For the linkage study, we calculate the offspring's conditional trait mean given the allele transmission status of a heterozygous parent at the marker locus. Based on the difference between the conditional means of a transmitted and a nontransmitted allele from a heterozygous parent, we propose statistics T(parsi), T(satur), T(gen) and T(m,het) to perform composite tests of linkage between the marker locus and the quantitative trait locus in the presence of association. These tests only use the offspring data that are related to the heterozygous parents at the marker locus. T(parsi) is a parsimonious or allele-wise statistic, T(satur) and T(gen )are satured or genotype-wise statistics, and T(m,het) compares the row and column sample means for offspring data of heterozygous parents. After comparing the powers and the sample sizes, we conclude that T(parsi) has higher power than those of the bi-allele tests, T(satur), T(gen), and T(m,het). If there is tight linkage between the marker and the trait locus, T(parsi) is powerful in detecting linkage between the marker and the trait locus in the presence of association. By investigating the goodness-of-fit of T(parsi), we find that T(satur) does not gain much power compared to that of T(parsi). Moreover, T(parsi) takes into account the pattern of the data that is consistent with linkage and linkage disequilibrium. As the number of alleles at the marker locus increases, T(parsi) is very conservative, and can be useful even for sparse data. To illustrate the usefulness and the power of the methods proposed in this paper, we analyze the chromosome 6 data of the Oxford asthma data, Genetic Analysis Workshop 12.