The solution structure of the disulphide-linked homodimer of the human trefoil protein TFF1

The trefoil factor family protein, TFF1, forms a homodimer, via a disulphide linkage, that has greater activity in wound healing assays than the monomer. Having previously determined a high-resolution solution structure of a monomeric analogue of TFF1, we now investigate the structure of the homodimer formed by the native sequence. The two putative receptor/ligand recognition domains are found to be well separated, at opposite ends of a flexible linker. This contrasts sharply with the known fixed and compact arrangement of the two trefoil domains of the closely related TFF2, and has significant implications for the mechanism of action and functional specificity of the TFF of proteins.     

The RNA interacting domain but not the protein interacting domain is highly conserved in ribosomal protein P0

Protein P0 interacts with proteins P1alpha, P1beta, P2alpha, and P2beta, and forms the Saccharomyces cerevisiae ribosomal stalk. The capacity of RPP0 genes from Aspergillus fumigatus, Dictyostelium discoideum, Rattus norvegicus, Homo sapiens, and Leishmania infantum to complement the absence of the homologous gene has been tested. In S. cerevisiae W303dGP0, a strain containing standard amounts of the four P1/P2 protein types, all heterologous genes were functional except the one from L. infantum, some of them inducing an osmosensitive phenotype at 37 degrees C. The polymerizing activity and the elongation factor-dependent functions but not the peptide bond formation capacity is affected in the heterologous P0 containing ribosomes. The heterologous P0 proteins bind to the yeast ribosomes but the composition of the ribosomal stalk is altered. Only proteins P1alpha and P2beta are found in ribosomes carrying the A. fumigatus, R. norvegicus, and H. sapiens proteins. When the heterologous genes are expressed in a conditional null-P0 mutant whose ribosomes are totally deprived of P1/P2 proteins, none of the heterologous P0 proteins complemented the conditional phenotype. In contrast, chimeric P0 proteins made of different amino-terminal fragments from mammalian origin and the complementary carboxyl-terminal fragments from yeast allow W303dGP0 and D67dGP0 growth at restrictive conditions. These results indicate that while the P0 protein RNA-binding domain is functionally conserved in eukaryotes, the regions involved in protein-protein interactions with either the other stalk proteins or the elongation factors have notably evolved.     

Human Fas-associated factor 1, interacting with ubiquitinated proteins and valosin-containing protein, is involved in the ubiquitin-proteasome pathway

Human Fas-associated factor 1 (hFAF1) is a novel protein having multiubiquitin-related domains. We investigated the cellular functions of hFAF1 and found that valosin-containing protein (VCP), the multiubiquitin chain-targeting factor in the degradation of the ubiquitin-proteasome pathway, is a binding partner of hFAF1. hFAF1 is associated with the ubiquitinated proteins via the newly identified N-terminal UBA domain and with VCP via the C-terminal UBX domain. The overexpression of hFAF1 and a truncated UBA domain inhibited the degradation of ubiquitinated proteins and increased cell death. These results suggest that hFAF1 binding to ubiquitinated protein and VCP is involved in the ubiquitin-proteasome pathway. We hypothesize that hFAF1 may serve as a scaffolding protein that regulates protein degradation in the ubiquitin-proteasome pathway.     

A protein of the Z class of liver cytosolic proteins in the rat that preferentially binds heme

A low molecular weight protein purified from rat liver cytosol was observed to bind heme with an affinity higher than that for other organic anions. Purification was achieved by two procedures, one employing affinity chromatography on oleic acid-agarose, and the other using sequential ion-exchange and gel filtration chromatography after initial removal of aprotinin-sensitive proteases. Removal rather than inhibition of proteases improved the yield four times. Both procedures produced a stable protein. The purified protein binds heme with a higher affinity (Kd 0.15 microM) than any other organic anion tested including other (metallo)porphyrins, bilirubin, and oleic acid. Based on its molecular weight, amino acid composition, immunological properties, and the increase of its tissue levels in response to the administration of hypolipidemic agents, the protein was identified as being related to proteins of the Z class, whose members include fatty acid binding protein and sterol carrier protein. Like other Z proteins, our protein exhibits several forms on electrofocusing, but differs from fatty acid-binding protein and sterol carrier protein in that its major form exhibits a pI of 7.4. In view of its distinct isoelectric focusing pattern, its higher affinity for heme than for oleic acid, and its apparent inability to bind cholesterol and steroids, we cannot identify this protein as any of the above-mentioned proteins of the Z class. Consequently we have provisionally designated it heme-binding protein.     

Synergistic effects of systemic trefoil factor family 1 (TFF1) peptide and epidermal growth factor in a rat model of colitis

Novel therapies for the treatment of colitis are required. We therefore examined the potential value of the trefoil factor family 1 (TFF1) peptide and epidermal growth factor (EGF) alone and in combination. Effects of TFF1- Cys58 +/- EGF on an in vitro HT29 cell wounding model of restitution showed synergistic activity when used in combination. In addition, animals had colitis induced by adding 4% dextran sulphate sodium (DSS) to the drinking water for 7 days and they also received twice daily subcutaneous injections of test peptides. Treatment with TFF1-Cys58 alone (100 microg/kg) reduced histological colitis score by 22%, but the TFF1-Ser58 variant was ineffective. In a second study, TFF1-Cys58 reduced histological colitis score by 15%, EGF (600 microg/kg) by 26%, and an additive response (42% reduction) was demonstrated when used together (P < 0.01 versus either peptide given alone). Similar results were found using tissue myeloperoxidase (MPO) activity as a marker of inflammation. Where clinical risk/benefit seems justified, these initial studies suggest that combination therapy of systemic EGF and TFF peptides may prove useful for treatment of colitis in patients with disease extending beyond the reach of topical (enema) therapy.     

Polymorphism of estrogen response element in TFF1 gene promoter is associated with an increased susceptibility to gastric cancer

TFF1 is a cysteine-rich protein that forms a characteristic trefoil domain through disulfide bonds, which render it resistant to vigorous conditions and it involves in maintaining the integrity of the gastric mucosa. Decreased expression of TFF1 gene plays a role in the development of gastric cancer. We examined the association between the promoter polymorphisms of the TFF1 gene and the risk of development of gastric cancer, in a case-control study including 199 controls and 141 patients with gastric cancer. Assessment of single nucleotide polymorphisms in the promoter region of the TFF1 gene was performed by sequencing and polymerase chain reaction-based restriction fragment length polymorphism. We found a statistically significant increased risk of gastric cancer associated with − 394 TT genotypes (OR = 8.78, CI = 2.85-27.05, p < 0.001) and CT (OR = 1.64, CI = 1.04-2.60, p = 0.033). This single nucleotide polymorphism occurs naturally in an estrogen response element. According to induction of the TFF1 gene by estrogen, it is possible that the substitution of C to T results in a decreased estrogen receptor binding affinity to the estrogen response element and in turn it decreases the expression of the TFF1 gene that may be involved in development of gastric cancer over a lifetime.     

Gastrointestinal tract specific gene GDDR inhibits the progression of gastric cancer in a TFF1 dependent manner

The novel gene GDDR, also named trefoil factor interactions 1, is abundantly expressed in human gastric epithelial cells but significantly down-regulated in gastric cancer. In this study, we first demonstrated that GDDR was specifically expressed in the gastrointestinal tract epithelium, while suppressed in gastric cancer cells. Forced expression of GDDR suppressed the tumor growth as shown by MTT and xenograft assay. siRNA mediated TFF1 inhibition nearly blocked the growth inhibitory role of GDDR. In summary, our study suggested that gastrointestinal tract specific gene GDDR might inhibit gastric cancer growth in a TFF1 dependent manner.     

Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7

Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-β (IFN-β) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.     

TFF2在胃癌中的表达及与幽门螺杆菌感染关系的研究

目的探讨三叶因子Ⅱ(Trefoil factors2,TFF2)在胃癌和癌前病变中的表达及与幽门螺杆菌感染(Helicobacter pylori,H.pylori)的关系。方法选取经病理证实的慢性浅表性胃炎、胃溃疡、慢性萎缩性胃炎和胃癌4种不同胃黏膜病变的标本140例,用免疫组化法检测标本中TFF2的表达及H.pylori的感染情况,并分析TFF2的表达与H.pylori的感染的关系。结果在慢性浅表性胃炎、胃溃疡、慢性萎缩性胃炎和胃癌中,TFF2和H.pylori的表达率依序呈逐渐增加的趋势,但TFF2在胃癌组织中表达降低。H.pylori阳性组TFF2的表达率低于阴性组,TFF2的阳性率与H.pylori感染率之间呈负相关(r=-0.335,P<0.05)。结论 TFF2的表达和H.pylori的感染与肿瘤的发生密切相关,检测该指标可为胃癌诊断、判断预后和指导治疗提供理论依据。     

GSTM1 null allele is a risk factor for gastric cancer development in Asians

IL-1 strikingly enhances antigen-driven responses of CD4 and CD8 T cells. It is substantially more effective than LPS and when added to a priming regime of antigen plus LPS, it strikingly enhances cell expansion. The effect is mediated by direct action on CD4 and CD8 T cells; the response occurs when OT-I or OT-II cells are transferred to B6 IL-1R1-/- recipients and only cells that express IL-1 receptors can respond. The major mechanism through which IL-1 enhances responses is by increasing survival of responding cells. IL-1 enhances the proportion of responding CD4 T cells that differentiate into Th17 cells and increases the proportion of responding CD8 cells that express granzyme B. Of a wide range of cytokines tested, only IL-1α and IL-1β mediate this function. The potency of IL-1 as an enhancer of T cell responses suggests that it could act to enhance responses to weak vaccines and that the pathway utilized by IL-1 might be considered in the design of new generations of adjuvants.     

EndoPDI,a novel protein-disulfide isomerase-like protein that is preferentially expressed in endothelial cells acts as a stress survival factor

We have identified a novel protein-disulfide isomerase and named it endothelial protein-disulfide isomerase (EndoPDI) because of its high expression in endothelial cells. Isolation of the full-length cDNA showed EndoPDI to be a 48 kDa protein that has three APWCGHC thioredoxin motifs in contrast to the two present in archetypal PDI. Ribonuclease protection and Western analysis has shown that hypoxia induces EndoPDI mRNA and protein expression. In situ hybridization analysis showed that EndoPDI expression is rare in normal tissues, except for keratinocytes of the hair bulb and syncytiotrophoblasts of the placenta, but was present in the endothelium of tumors and in other hypoxic lesions such as atherosclerotic plaques. We have compared the function of EndoPDI to that of PDI in endothelial cells using specific siRNA. PDI was shown to have a protective effect on endothelial cells under both normoxia and hypoxia. In contrast, EndoPDI has a protective effect only in endothelial cells exposed to hypoxia. The loss of EndoPDI expression under hypoxia caused a significant decrease in the secretion of adrenomedullin, endothelin-1, and CD105; molecules that protect endothelial cells from hypoxia-initiated apoptosis. The identification of an endothelial PDI further extends this increasing multigene family and EndoPDI, unlike archetypal PDI, may be a molecule with which to target tumor endothelium.     

Effects of MCRS1 on proliferation,migration, invasion,and epithelial mesenchymal transition of gastric cancer cells by interacting with Pkmyt1 protein kinase

Microspherule protein 1(MCRS1) is known to be an oncogene in several tumors. However, recent studies have shown that MCRS1 inhibits lymphatic metastasis in gastric cancer (GC) patients by inhibiting telomerase activity. Protein kinase, membrane associated tyrosine/threonine 1(Pkmyt1), a member of the WEE1 family, has been found to interact with MCRS1 by yeast two-hybrid assay; however, how these two proteins interact in GC is still unclear. Hence, this study aimed to investigate the effect of MCRS1 interaction with Pkmyt1 on GC cell proliferation, migration, and invasion. Initially, we observed increased expression of MCRS1 in GC SGC-7901 cells and decreased expression in GC BGC-823 cells. Hence, we down-regulated MCRS1 expression in SGC-7901 cells and up-regulated it in BGC-823 cells. Our results showed that overexpression of MCRS1 inhibits the growth, invasion and migration of GC cells, while downregulation of MCRS1 promotes the growth, invasion and migration of GC cells. When MK1775, an inhibitor of WEE1 kinase, was added after downregulation of MCRS1, phenotypic recovery effects were observed. Overexpression of MCRS1 also inhibited the expression of Pkmyt1 and vice versa. This indicated that there might be a possible interaction between MCRS1 and Pkmyt1. Furthermore, immunoprecipitation assay revealed the interaction between MCRS1 and Pkmyt1 in virto, and immunofluorescence experiments showed that the two proteins were co-localized in the cytoplasm. In conclusion, our study confirmed the specific tumor suppressive activity of MCRS1 in GC proliferation, invasion and migration and suggested that it might inhibit the progression of GC through its interaction with Pkmyt1.