No abstract

No abstract

Vesicular transport between secretory compartments requires specific recognition molecules called SNAREs. Here we report the identification of three putative SNAREs, p14 (Sft1p), p28 (Gos1p), and a detailed characterization of p26 (Ykt6p). All three were originally isolated as interacting partners of the cis Golgi target membrane-associated SNARE Sed5p, when Sec18p (yeast NSF) was inactivated. YKT6 is an essential gene that codes for a novel vesicle-associated SNARE functioning at the endoplasmic reticulum-Golgi transport step in the yeast secretory pathway. Depletion of Ykt6p results in the accumulation of the p1 precursor (endoplasmic reticulum form) of the vacuolar enzyme carboxypeptidase Y and morphological abnormalities consistent with a defect in secretion. Membrane localization of Ykt6p is essential for protein function and is normally mediated by isoprenylation. However, replacement of the isoprenylation motif with a bona fide transmembrane anchor results in a functional protein confirming that membrane localization, but not isoprenylation per se, is required for function. Ykt6p and its homologues are highly conserved from yeast to human as demonstrated by the functional complementation of the loss of Ykt6p by its human counterpart. This is the first example of a human SNARE protein functionally replacing a yeast SNARE. This observation implies that the specific details of the vesicle targeting code, like the genetic code, are conserved in evolution.

ST2/ST2L, a member of the IL-1R gene family, is expressed by fibroblasts, mast cells, and Th2, but not Th1, cells. It exists in both membrane-bound (ST2L) and soluble forms (ST2). Although ST2L has immunoregulatory properties, its ligand, cellular targets, and mode of action remain unclear. Using a soluble ST2-human IgG fusion protein, we demonstrated that ST2 bound to primary bone marrow-derived macrophages (BMM) and that this binding was enhanced by treatment with LPS. The sST2 treatment of BMMs inhibited production of the LPS-induced proinflammatory cytokines IL-6, IL-12, and TNF-α but did not alter IL-10 or NO production. Treatment of BMMs with sST2 down-regulated expression of Toll-like receptors-4 and -1 but induced nuclear translocation of NF-κB. Administration of sST2 in vivo after LPS challenge significantly reduced LPS-mediated mortality and serum levels of IL-6, IL-12, and TNF-α. Conversely, blockade of endogenous ST2 through administration of anti-ST2 Ab exacerbated the toxic effects of LPS. Thus, ST2 has anti-inflammatory properties that act directly on macrophages. We demonstrate here a novel regulatory pathway for LPS-induced shock via the ST2-Toll-like receptor 4 route. This may be of considerable therapeutic potential for reducing the severity and pathology of inflammatory diseases.

a-Amylases belong to the alp-barrel protein family in which the active site is created by residues located at the C-terminus of the p strands and in the helix-connecting loops extending from these ends. In the a-amylase family, a small separate domain B protrudes at the C-terminus of the third p strand of the @/a),-barrel framework. The 80% identical barley a-amylase isozymes 1 and 2 (AMYl and AMY2, respectively) differ in substrate affinity and turnover rate, CaC1, stimulation of activity, sensitivity to the endogenous 21-kDa a-amylaselsubtilisin inhibitor, and stability at low pH. To identify regions that confer these isozyme-specific variations, AMY1 -AMY2 hybrid cDNAs were generated by in vivo homologous recombination in yeast. The hybrids AMYl-(l-9O)-AMY2-(90-403) and AMY1-(1-161)-AMY2-(161-403) characterized in this study contain the 90-residue and 161 -residue N-terminal sequences, respectively, of AMY 1 and complementary C-terminal regions of AMY2. AMYl-(1-90)-AMY2-(90-403) comprises the 60-amino-acid domain B of AMY2 and resembles this isozyme in sensitivity to a-amylaselsubtilisin inhibitor and its low affinity for the substrates p-nitrophenyl a-D-maltoheptaoside, amylose and the inhibitor acarbose. Only AMY1-( 1-161)-AMY2-(161-403) and AMYl, which both share domain B, are stable at low pH. However, AMY2 and both hybrid AMY species, but not AMY1, show maximum enzyme activity on insoluble blue starch at approximately 10 mM CaC1,. Domain B thus determines several functional and stability properties that distinguish the barley a-amylase isozymes.a-Amylases hydrolyse internal a-l,4-glucosidic linkages of starch and related dextrins and are widely occurring in microorganisms, higher plants and animals. In cereals, aamylases represent a major starch-degrading activity important in seed germination (MacGregor, 1987). a-Amylases and related amylolytic enzymes are @/a),-barrel proteins (Matsuura et al