Expansion of amino acid homo-sequences in proteins: insights into the role of amino acid homo-polymers and of the protein context in aggregation.

Expansion of amino acid homo-sequences, such as polyglutamines or polyalanines, in proteins has been directly implicated in various degenerative diseases through a mechanism of protein misfolding and aggregation. However, it is still unclear how the nature of the expansion and the protein context influence the tendency of a protein to aggregate. Here, we have addressed these questions using spinocerebellar ataxia type-3 (ATX3) protein, the best characterised of the polyglutamine proteins, chosen as a model system. Using a transfected mammalian cell line, we demonstrate that ATX3 aggregation is noticeably reduced by deletion or replacement of regions other than the polyglutamine tract. The nature of the amino acid homo-sequences also has a strong influence on aggregation. From our studies, we draw general conclusions on the effect of the protein architecture and of the amino acid homo-sequence on pathology.

Role of pHyde novel gene product as an intrinsic factor for apoptotic pathway in prostate cancer.

Induction of apoptotic cell death mechanism can be regulated by internal factor(s), such as by gene product(s) that directly upregulate the apoptosis pathway or indirectly by down-regulating the anti-apoptosis gene. This homeostasis is a normal phenomenon in a biological system disturbed by cancer. It is thus important to find any gene functioning as an upregulator for the apoptosis pathway that may have a potential application in the context of cancer gene therapy. We have cloned a novel rat gene, denoted as pHyde, that fulfilled this objective. Internally, this pHyde gene product renders the stable transfectant of rat prostatic cancer cell lines more susceptible to apoptosis even without any external inducer. By using an external agent, such as 5-fluoro-2'-deoxyuridine (FdUr), apoptotic responses of the stable transfectants are even higher, suggesting that both intrinsic and extrinsic factors work synergistically. The pHyde gene product was termed an intrinsic factor, whereas FdUr was considered an extrinsic factor for the apoptosis in rat prostate cancer model.

Interaction of a novel cysteine and histidine-rich cytoplasmic protein with galectin-3 in a carbohydrate-independent manner.

We have used the yeast two-hybrid system to search for cytoplasmic proteins that might assist in the intracellular trafficking of the soluble beta-galactoside-binding protein, galectin-3. We utilised as bait murine full-length galectin-3 to screen a murine 3T3 cDNA library. Several interacting clones were found to encode a partial open reading frame and a full-length clone was obtained by rapid amplification of cDNA ends methodology. In various assays in vitro the novel protein was shown to bind galectin-3 in a carbohydrate-independent manner. The novel protein contains an unusually high content of cysteine and histidine residues and shows significant sequence homologies with several metal ion-binding motifs present in known proteins. Confocal immunofluorescence microscopy of permeabilised 3T3 cells shows a prominent perinuclear, as well as cytoplasmic, localisation of the novel protein.

Determinants in the N-terminal domains of galectin-3 for secretion by a novel pathway circumventing the endoplasmic reticulum-Golgi complex.

Galectin-3 is a beta-galactoside-binding protein that is secreted from many cells although the protein lacks a signal sequence for transfer into the endoplasmic reticulum and Golgi compartments and entry into classical secretory pathways. Previously it was shown that attachment of the first 120 amino acid residues of the N-terminal sequence of hamster galectin-3 to the cytoplasmic protein chloramphenicol acetyltransferase (CAT) supported the rapid secretion of the fusion protein from transiently transfected Cos cells under conditions in which CAT protein was not secreted. Here we report that progressive N-terminal truncation gradually reduced secretion of the fusion proteins, eventually to very low levels compared with the starting product, but did not totally eliminate secretion until a significant majority of the sequence was removed. Mutant CAT fusion proteins containing internal deletions in residues 97-120 of the galectin-3 N-terminal sequence were also secreted to a similar extent to the starting product, but further deletion of residues 89-96 abolished detectable secretion. Proline to alanine mutagenesis of the sequence YP(90)SAP(93)GAY in two secretion-competent CAT fusion proteins greatly reduced or abolished their secretion, whereas similar mutagenesis of proline pairings present elsewhere in the galectin-3 N-terminal segments of these proteins had no effect. The results indicate that this sequence is one essential determinant for secretion of galectin-3-CAT fusion proteins and by inference galectin-3, at least from transfected Cos cells. However, the short sequence of residues 89-96 by itself is insufficient to direct secretion of CAT fusion proteins and appears to be active only in the context of a larger portion of the galectin-3 N-terminal sequence.

Inflammatory cytokine (interleukins 1, 6 and 8 and tumor necrosis factor-alpha) release from cultured human fetal membranes in response to endotoxic lipopolysaccharide mirrors amniotic fluid concentrations.

OBJECTIVE: This study was conducted to quantitate and compare the amount of cytokines released from human fetal membranes in response to treatment with bacterial lipopolysaccharide and to compare this with amniotic fluid levels. STUDY DESIGN: Amniochorionic membranes were collected from women undergoing elective repeat cesarean section and showing no signs of infection- or pregnancy-related complications. Membranes were maintained in an organ explant system and stimulated with bacterial lipopolysaccharide for 24 hours. Media samples were collected and stored at -20 degrees C until cytokine levels were assayed by enzyme-linked immunosorbent assay. RESULTS: Enzyme-linked immunosorbent assay results demonstrated that lipopolysaccharide stimulated production of interleukins 1, 6 and 8 and tumor necrosis factor-alpha by the fetal membranes in comparison with the control cultures. A greater release of interleukin-6 and interleukin-8 compared with interleukin-1 and tumor necrosis factor-alpha was noticed. The relationships between cytokine concentrations observed in culture mirror those seen in amniotic fluid. CONCLUSION: Amniochorionic membranes can respond to an infectious process with increased secretion of interleukins 1, 6 and 8 and tumor necrosis factor-alpha. Cytokines produced from both amnion and chorion (interleukin-6 and interleukin-8) are released in greater quantities than those cytokines produced from chorion or amnion alone (interleukin-1 and tumor necrosis factor-alpha). These studies support a major role for amnion in infection-induced preterm labor.

Primary sequence and evolutionary conservation of ribosomal protein genes from the domestic cat.

Ribosomal proteins complex with ribosomal RNA to form the subunits of the ribosome, and as such, serve essential functions in protein synthesis. Accumulating evidence suggests that ribosomal proteins are bifunctional molecules with roles in diverse cellular processes in addition to protein synthesis. Previous studies have demonstrated that expression of four ribosomal proteins. L41, S3a, S4, and S17, is elevated in malignant lymphomas of the domestic cat. Reported here is the nucleotide sequence of cDNA clones encoding feline ribosomal proteins L41, S3a, S4, and S17.

Oxidant stress causes alteration in the attachment of mononuclear cells to collagen.

The effect of oxidant stress on the attachment of blood mononuclear cells to collagen was investigated. Study of the kinetics of attachment of mononuclear cells pretreated with 0.2% H2O2 for 10' showed significantly lower attachment to collagen I substrata when compared to untreated controls. Reduced glutathione at 2.5mM concentration partially reversed the H2O2 induced alteration in attachment. Pretreatment with H2O2 caused a reduction in the number of free thiol groups both at the cell surface and intracellular sites. Changes in cell surface free thiol groups could be reversed by reduced glutathione. These results point to the importance of a stable redox status in the cellular environment for normal interaction of mononuclear cells with extracellular matrix components.

Enhanced adhesion of human mononuclear cells to nonenzymatically glycosylated collagen I.

Nonenzymatic glycosylation of extracellular matrix components may contribute to altered interaction of cells with the matrix. We have examined the interaction of mononuclear cells with early glycosylated collagen I. Significantly more cells attached to glycosylated collagen compared to normal collagen. Radioiodinated glycosylated collagen I specifically bound to mononuclear cells in a time and concentration dependent manner with a Kd of 2.45 x 10(9) M. Maximum binding was observed in the presence of Mn++ ions. The iodinated ligand bound to mononuclear cell membrane immobilized on nitrocellulose disks and the interaction was found to be saturable. These results suggested an alteration in the interaction of human blood mononuclear cells with collagen I, when it gets glycosylated non enzymatically and also indicate that early glycosylated collagen interacts with mononuclear cells through specific, high affinity cell surface molecules.

Binding of collagen causes intracellular mobilization of calcium in human mononuclear cells.

The possibility that extracellular matrix-cell surface interaction might lead to signalling was examined in human mononuclear cells. In mononuclear cells loaded with Indo-1 acetoxymethylester, collagen binding elicited a 2-3 fold increase in cytosolic free calcium concentration within seconds. Plasma fibronectin, serum albumin and heparin did not cause any significant change in intracellular calcium levels. Collagen I, collagen IV and glucosylated collagen I evoked an increase in intracellular Ca++ both in the presence and absence of extracellular calcium. However, in the presence of 2mM EDTA, the increase in cytosolic free calcium concentration caused by collagen I and collagen IV was partly abolished, suggesting the requirement of a cation-dependent interaction of collagen with mononuclear cells. Glucosylated collagen induced intracellular calcium mobilization even in the presence of EDTA suggesting a cation-independent interaction. These results indicate that collagen binding induces rapid mobilization of calcium in human mononuclear cells, apparently from intracellular sources.

Isolation of a type IV collagen binding protein from human platelets.

In order to study the molecular basis of platelet interaction with collagen IV of the basement membrane separating the arterial endothelium from the underlying subendothelial connective tissue, the possibility of presence of platelet membrane protein with affinity to type IV collagen was examined by subjecting the platelet membrane extract to affinity chromatography on collagen IV-sepharose. Urea (4 M) eluate was found to contain a protein with an apparent mol. wt of 68 kDa. The radioiodinated protein was isolated and used to test its specificity. By dot blot assay on nitrocellulose disks and solid-phase assays, the 68 kDa protein was found to bind with high affinity to collagen IV. Lack of significant binding to fibronectin and laminin when compared to albumin control indicated its high specificity for collagen. The radioiodinated protein was inserted into egg yolk lecithin liposomes. While these liposomes attached to microtitre plates coated with collagen IV, there was no significant binding to fibronectin or laminin coated wells, suggesting the membrane associated character of the protein as well as its specificity for collagen. These results indicate that presence of a 68 kDa protein in platelet membrane which interacts with very high specificity to collagen IV.