E. coli expression of TIMP-4 and comparative kinetic studies with TIMP-1 and TIMP-2: insights into the interactions of TIMPs and matrix metalloproteinase 2 (gelatinase A)

The inhibitory properties of TIMP-4 for matrix metalloproteinases (MMPs) were compared to those of TIMP-1 and TIMP-2. Full-length human TIMP-4 was expressed in E. coli, folded from inclusion bodies, and the active component was purified by MMP-1 affinity chromatography. Progress curve analysis of MMP inhibition by TIMP-4 indicated that association rate constants (k(on)) and inhibition constants (K(i)) were similar to those for other TIMPs ( approximately 10(5) M(-)(1) s(-)(1) and 10(-)(9)-10(-)(12) M, respectively). Dissociation rate constants (k(off)) for MMP-1 and MMP-3 determined using alpha(2)-macroglobulin to capture MMP dissociating from MMP-TIMP complexes were in good agreement with values deduced from progress curves ( approximately 10(-)(4) s(-)(1)). K(i) and k(on) for the interactions of TIMP-1, -2, and -4 with MMP-1 and -3 were shown to be pH dependent. TIMP-4 retained higher reactivity with MMPs at more acidic conditions than either TIMP-1 or TIMP-2. Molecular interactions of TIMPs and MMPs investigated by IAsys biosensor analysis highlighted different modes of interaction between proMMP-2-TIMP-2 (or TIMP-4) and active MMP-2-TIMP-2 (or TIMP-4) complexes. The observation that both active MMP-2 and inactive MMP-2 (with the active site blocked either by the propeptide or a hydroxamate inhibitor) have essentially identical affinities for TIMP-2 suggests that there are two TIMP binding sites on the hemopexin domain of MMP-2: one with high affinity that is involved in proMMP-2 or hydroxamate-inhibited MMP-2; and the other with low affinity involved in formation of the complex of active MMP-2 and TIMP-2. Similar models of interaction may apply to TIMP-4. The latter low-affinity site functions in conjunction with the active site of MMP-2 to generate a tight enzyme-inhibitor complex.     

Comparison of the expression of Bacillus thuringiensis full-length and N-terminally truncated vip3A gene in Escherichia coli

AIMS: Studies were performed to demonstrate the function of the putative signal peptide of Vip3A proteins in Escherichia coli. METHODS AND RESULTS: The full-length vip3A-S184 gene was isolated from a soil-isolated Bacillus thuringiensis, and the vip3AdeltaN was constructed by deleting 81 nucleotides at the 5'-terminus of vip3A-S184. Both were transformed and expressed in E. coli. About 19.2% of Vip3A-S184 proteins secreted soluble proteins and others formed inclusion bodies in the periplasmic space. In contrast, the Vip3AdeltaN was insoluble and formed inclusion bodies in the cytoplasm. Bioassay indicated that Vip3A-S184 showed different toxicity against Spodoptera exigua, Helicoverpa armigera and S. litura, but Vip3AdeltaN showed no toxicity to either of them because of the deletion of the first 27 amino acids at the N-terminus. CONCLUSIONS: The results suggest that the deleted N-terminal sequences were essential for the secretion of Vip3A-S184 protein in E. coli and might be required for toxicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The function of the putative signal peptide of Vip3A protein in E. coli was investigated. These would be helpful to make clear the unknown secretion pathway of Vip3A protein in B. thuringiensis and determine the receptor-binding domain or toxic fragment of Vip3A-S184 protein.     

肽转运载体的分子特征及其分布

动物体内的肽转运载体目前发现的至少有五种,其中研究最为广泛的是：PepT1和PepT2。PepT1和PepT2都是依质子的寡肽转运载体(POT)家族的成员。PepT1是低亲和力／高容量的肽载体,PepT2高亲和力／低容量的肽载体。PepT1主要在消化道中表达,在肾脏中也有微弱的表达;PepT2主要在肾脏中表达。这些肽载体的分子结构特征主要有：(1)有12个假想的穿膜区,在9区和10区之间有一大的胞外环,且所有穿膜区内的序列都高度保守,胞外环上的序列保守的很少;(2)被编码的蛋白上有多个N-糖基化和蛋白激酶的识别位点,它们可能参与肽转运的调控;(3)PepT1上的His-57和PepT2上的His-87是最关键的组氨酸残基,它们可能是转运蛋白发挥吸收功能时最关键的结合位点;(4)不同动物肽转运蛋白的氨基酸范围在707到729之间,且不同动物相同器官肽转运载体的同源性高(大约80％),同种动物不同器官肽转运载体的同源性低(大约50％)。了解肽载体的分子特征和组织分布,可以更好地理解肽吸收的分子机制并有利于肽类药物的研发。     

Oligonucleotide-based microarray for DNA methylation analysis: principles and applications

Gene silencing via promoter CpG island hypermethylation offers tumor cells growth advantages. This epigenetic event is pharmacologically reversible, and uncovering a unique set of methylation-silenced genes in tumor cells can bring a new avenue to cancer treatment. However, high-throughput tools capable of surveying the methylation status of multiple gene promoters are needed for this discovery process. Herein we describe an oligonucleotide-based microarray technique that is both versatile and sensitive in revealing hypermethylation in defined regions of the genome. DNA samples are bisulfite-treated and PCR-amplified to distinguish CpG dinucleotides that are methylated from those that are not. Fluorescently labeled PCR products are hybridized to arrayed oligonucleotides that can discriminate between methylated and unmethylated alleles in regions of interest. Using this technique, two clinical subtypes of non-Hodgkin's lymphomas, mantle cell lymphoma, and grades I/II follicular lymphoma, were further separated based on the differential methylation profiles of several gene promoters. Work is underway in our laboratory to extend the interrogation power of this microarray system in multiple candidate genes. This novel tool, therefore, holds promise to monitor the outcome of various epigenetic therapies on cancer patients.     

DcR3/TR6 modulates immune cell interactions

DcR3/TR6, a secreted protein, is a member of TNF receptor family. Its ligands include FasL, LIGHT, and TL1A, all TNF family members. TR6 can interfere with FasL- or LTbetaR-mediated apoptosis; it can also inhibit T-cell costimulation by blocking the two-way signaling between TR2 and LIGHT, and the one-way signaling from TL1A to DR3. In this study, we discovered that TR6 was secreted by peripheral blood mononuclear cells (PBMC) stimulated by T-cell mitogens. It inhibited actin polymerization of T cells upon mitogen stimulation, and repress T-cell pseudopodium formation, which is known to be important for cell-cell interaction. As a consequence, T-cell aggregation stimulated by alloantigens, anti-CD3 or PHA was suppressed by either soluble or solid phase TR6-Fc. This result suggests that TR6 might regulate T-cell interaction with other cells such as antigen-presenting cells (APC) or their fellow T cells by preventing them from forming inseparable cell clusters, which are undesirable for the progression of immune responses.     

Adaptive diversification of bitter taste receptor genes in Mammalian evolution

The diversity and evolution of bitter taste perception in mammals is not well understood. Recent discoveries of bitter taste receptor (T2R) genes provide an opportunity for a genetic approach to this question. We here report the identification of 10 and 30 putative T2R genes from the draft human and mouse genome sequences, respectively, in addition to the 23 and 6 previously known T2R genes from the two species. A phylogenetic analysis of the T2R genes suggests that they can be classified into three main groups, which are designated A, B, and C. Interestingly, while the one-to-one gene orthology between the human and mouse is common to group B and C genes, group A genes show a pattern of species- or lineage-specific duplication. It is possible that group B and C genes are necessary for detecting bitter tastants common to both humans and mice, whereas group A genes are used for species-specific bitter tastants. The analysis also reveals that phylogenetically closely related T2R genes are close in their chromosomal locations, demonstrating tandem gene duplication as the primary source of new T2Rs. For closely related paralogous genes, a rate of nonsynonymous nucleotide substitution significantly higher than the rate of synonymous substitution was observed in the extracellular regions of T2Rs, which are presumably involved in tastant-binding. This suggests the role of positive selection in the diversification of newly duplicated T2R genes. Because many natural poisonous substances are bitter, we conjecture that the mammalian T2R genes are under diversifying selection for the ability to recognize a diverse array of poisons that the organisms may encounter in exploring new habitats and diets.     

A recombinant anti-erbB2, scFv-Fc-IL-2 fusion protein retains antigen specificity and cytokine function

Elevated erbB2 expression is detected in many in situ and invasive human ductal carcinomas. Anti-erbB2 antibody directed at the extracellular domain of erbB2 can result in an antitumor response in some patients with tumors overexpressing erbB2 oncoprotein. By combining interleukin 2 (IL-2) activities with a tumor specific antibody, immunotherapy of tumors might be more effective in the future. In this study, a fusion protein consisting of erbB2 single chain antibody (scFv), Fc fragment of human IgG1 and IL-2 was constructed. The molecular weight of fusion proteins is 66 kDa, only one third of whole antibody-IL-2 fusion protein or 44% whole Ig molecule. The fusion proteins retained the activities of both antigen binding and IL-2. The scFv-Fc-IL-2 fusion protein may have advantages over whole antibody-IL-2 fusion proteins, such as smaller molecule, better activity of penetration, more favorable pharmacokinetic properties.     

New perspectives in arsenic-induced cell signal transduction

Although the carcinogenicity of arsenic has been well established, the underlying molecular mechanisms have not yet been fully identified. Accumulating evidence indicates that the alteration of cellular signal transduction is directly related to the carcinogenesis of arsenic. This review focuses on recent advances in arsenic-induced signal transduction, including reactive oxygen species (ROS) production, tyrosine phosphorylation, MAPK signaling, NF-kappaB activation, cell cycle arrest, and apoptosis.