IKKγ Facilitates the Activation of NF-κB by Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV core protein has been shown to modulate various cellular signaling pathways including the nuclear factor κB (NF-κB) pathway which is associated with inflammation, cell proliferation and apoptosis. However, there have been conflicting reports about the effect of HCV core protein on NF-κB pathway, and the mechanism by which the core protein affects NF-κB activity remains nuclear. In this study, the functional interaction of HCV core protein and IκB kinase γ (IKKγ) was investigated using the expression plasmids of core and the components of IKK complex. The data revealed that HCV core protein activates NF-κB. Also, HCV core protein up-regulated the phosphorylation and degradation of IκBα. The activating effect of HCV core protein on NF-κB was synergistically elevated by IKKγ. It was noticed that the N-terminal IKKβ binding site, C-terminal leucine zipper, and zinc finger domains of IKKγ are not necessary for its synergistic effect. HCV core protein and IKKγ appeared to activate NF-κB by up-regulating the IKKβ activity resulting in the degradation of IκBα. As expected, HCV core protein induced the expression of NF-κB-targeted pro-inflammatory genes such as iNOS, IL-1β and IL-6 in the transcription level. These results suggest that HCV core protein induces NF-κB through the interaction with IKKγ and may play a critical role in the development of inflammation and related liver diseases.

Evaluation of the role of TAX, HBZ, and HTLV-1 proviral load on the survival of ATLL patients

BACKGROUND: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive malignancy with very poor prognosis and short survival, caused by the human T-lymphotropic virus type-1 (HTLV-1). The HTLV-1 biomarkers trans-activator x (TAX) and HTLV-1 basic leucine zipper factor (HBZ) are main oncogenes and life-threatening elements. This study aimed to assess the role of the TAX and HBZ genes and HTLV-1 proviral load (PVL) in the survival of patients with ATLL. METHODS: Forty-three HTLV-1-infected individuals, including 18 asymptomatic carriers (AC) and 25 ATLL patients (ATLL), were evaluated between 2011 and 2015. The mRNA expression of TAX and HBZ and the HTLV-1 PVL were measured by quantitative PCR. RESULTS: Significant differences in the mean expression levels of TAX and HBZ were observed between the two study groups (ATLL and AC, P=0.014 and P=0.000, respectively). In addition, the ATLL group showed a significantly higher PVL than AC (P=0.000). There was a significant negative relationship between PVL and survival among all study groups (P=0.047). CONCLUSION: The HTLV-1 PVL and expression of TAX and HBZ were higher in the ATLL group than in the AC group. Moreover, a higher PVL was associated with shorter survival time among all ATLL subjects. Therefore, measurement of PVL, TAX, and HBZ may be beneficial for monitoring and predicting HTLV-1-infection outcomes, and PVL may be useful for prognosis assessment of ATLL patients. This research demonstrates the possible correlation between these virological markers and survival in ATLL patients.

Bovine adenovirus type 3 virions cannot be rescued in vivo after full-length viral genome transfection in the absence of detectable polypeptide IX

Bovine adenovirus type 3 (BAdV3) is being used in the development of potential vehicles for gene therapy and vectored vaccine. To that end, a more comprehensive description of BAdV3 biology is essential. In this study, we focused on the role of pIX in BAdV3 virion rescue after full-length BAdV3 genome transfection. Initially, pIX deletion or initiation codon mutation abolished the production of progeny virions, which suggested that pIX was essential for the rescue of BAdV3 containing a full-length genome. Moreover, through transfection of a panel of pIX mutant BAdV3 genomes, we observed that the conserved N-terminus and the putative leucine zipper element (PLZP) were essential for virion rescue, whereas the C-terminus following the coiled-coil domain was non-essential. In addition, swap of the PLZP element and its following region of BAdV3 pIX to corresponding domains of human adenovirus type 5 (HAdV5) did not affect virion production, whereas swap of the entire pIX abolished production of progeny virions. We suggest that failure of the full-length BAdV3 pIX swap might be due to species specificity of its N-terminus region before the PLZP element.

Effect of MITF-M and MITF-A Overexpression on the Dendrtic Formation in Melanocytes / 체질인류학회지

The microphthalmia-associated transcription factor (MITF), has been described as the master regulator of the basic helix-loop-helix leucine zipper family, involves melanogenesis in melanocytes. MITF consists of at least six isoforms, called MITF-M, MITF-A, MITF-B, MITF-C, MITF-H, and MITF-J. Previously, we found that not only MITF-M is expressed in the human hair follicle, but also MITF-A, MITF-C, MITF-H, and MITF-J isoforms are expressed in the skin. The aim of this study was to conform the MITF isoforms expressed in human skin, and investigate novel role of MITF isoforms in the melanocytes. Expression of MITF-M and MITF-A was found in primary melanoctyes and the melanoma cell lines. Interestingly, when MITF-M and MITF-A were overexpressed in the SK-MEL-24 melanoma cells by adenoviral transfection, length of the dendrites, serves as the principal conduit for melanosomes transfer, was significantly increased in the MITF-M overexpressed cells compared with the control group, and number of the dendtrites was significantly increased in the MITF-A overexpressed cells. A signal molecule involve in actin polymerization during dendrite formation, Rac1, was increased in the SK-MEL-24 melanoma cells treated with adenoviral MITF-M and MITF-A vectors. These results suggest that MITF-M and MITF-A induce dendrite formation via Rac1 signaling in the melanocytes.

SoxD Transcription Factors: Multifaceted Players of Neural Development

SoxD transcription factor subfamily includes three members, Sox5, Sox6, and Sox13. Like other Sox genes, they contain the High-Mobility-Group (HMG) box as the DNA binding domain but in addition feature the subgroup-specific leucine zipper motif. SoxD genes are expressed in diverse cell types in multiple organs during embryogenesis and in adulthood. Among the cells expressing them are those present in the developing nervous system including neural stem (or progenitor) cells as well as differentiating neurons and oligodendrocytes. SoxD transcription factors do not contain distinct activator or repressor domain, and they are believed to function in modulation of other transcription factors in promoter- specific manners. This brief review article will attempt to summarize the latest studies on the function of SoxD genes in embryogenesis with a particular emphasis on the regulation of neural development.

Engagement of CD99 Reduces AP-1 Activity by Inducing BATF in the Human Multiple Myeloma Cell Line RPMI8226

CD99 signaling is crucial to a diverse range of biological functions including survival and proliferation. CD99 engagement is reported to augment activator protein-1 (AP-1) activity through mitogen-activated protein (MAP) kinase pathways in a T-lymphoblastic lymphoma cell line Jurkat and in breast cancer cell lines. In this study, we report that CD99 differentially regulated AP-1 activity in the human myeloma cell line RPMI8226. CD99 was highly expressed and the CD99 engagement led to activation of the MAP kinases, but suppressed AP-1 activity by inducing the expression of basic leucine zipper transcription factor, ATF-like (BATF), a negative regulator of AP-1 in RPMI8226 cells. By contrast, engagement of CD99 enhanced AP-1 activity and did not change the BATF expression in Jurkat cells. CD99 engagement reduced the proliferation of RPMI8226 cells and expression of cyclin 1 and 3. Overall, these results suggest novel CD99 functions in RPMI8226 cells.

Association Study of FOS-Like Antigen-2 Promoter Polymorphisms With Papillary Thyroid Cancer in Korean Population

OBJECTIVES: FOS-like antigen-2 (FOSL-2), a member of the FOS gene family, encode leucine zipper proteins that can heterodimerize with proteins of Jun family. Thus, activating protein (AP)-1 transcription factor is formed, has a crucial role in proliferation, differentiation and apoptosis of normal tissue as well as oncogenic transformation and progression. We performed an association study of single nucleotide polymorphisms (SNPs) in the FOSL-2 with papillary thyroid cancer (PTC). We also estimated the relationships between the SNPs and the clinicopathologic characteristics of PTC. METHODS: One promoter SNPs (rs925255) of FOSL-2 gene were genotyped with direct sequencing method in 94 PTC and 213 controls. PTC patients were dichotomized and compared with respect to clinical parameters of PTC. Genetic data were analyzed using Helixtree, SNPAnalyzer, SNPStats. Multivariate logistic regression analysis was fulfilled to evaluate the genetic effect with adjustment for age and sex. RESULTS: SNP (rs925255) in FOSL-2 showed a significant association (codominant 1 model [G/G vs. A/G]: odds ratio [OR], 0.531, 95% confidence interval [CI], 0.293 to 0.96, P=0.036; dominant model: OR, 0.50, 95% CI, 0.28 to 0.89, P=0.015) with PTC. The frequency of allele G in rs925255 was also significantly associated with PTC (OR, 0.59; 95% CI, 0.34 to 0.91; P=0.02). But we fail to prove significant association between this polymorphism (rs925255) and clinico-pathological parameters. CONCLUSION: Our findings suggest that the rs925255 SNP and its allele G show significant association with the PTC in Korean population.

Leucine zippers improves protein splicing-mediated coagulation factor VIII gene delivery by dual-vector system / 药学学报

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.

Progress of TSC-22 gene research / 中南大学学报(医学版)

Transformation growth factor β -inducible gene 22 (TSC-22) is a putative negative growth regulation and tumor suppressor gene. It has the ability to combine with other transcription factors to regulate the cell growth and apoptosis. TSC-22 is lowly expressed in many types of tumors,which may be related to the tumorgenesis and development.

Expression of a Functional zipFv Antibody Fragment and Its Fusions with Alkaline Phosphatase in the Cytoplasm of an Escherichia coli

BACKGROUND: Expression of recombinant antibodies and their derivatives fused with other functional molecules such as alkaline phosphatase in Escherichia coli is important in the development of molecular diagnostic reagents for biomedical research. METHODS: We investigated the possibility of applying a well-known Fos-Jun zipper to dimerize V(H) and V(L) fragments originated from the Fab clone (SP 112) that recognizes pyruvate dehydrogenase complex-E2 (PDC-E2), and demonstrated that the functional zipFv-112 and its alkaline phosphatase fusion molecules (zipFv-AP) can be produced in the cytoplasm of Origami(DE3) trxB gor mutant E. coli strain. RESULTS: The zipFv-AP fusion molecules exhibited higher antigen-binding signals than the zipFv up to a 10-fold under the same experimental conditions. However, conformation of the zipFv-AP seemed to be influenced by the location of an AP domain at the C-terminus of V(H) or V(L) domain [zipFv-112(H-AP) or zipFv-112(L-AP)], and inclusion of an AraC DNA binding domain at the C-terminus of V(H) of the zipFv-112(L-AP), termed zipFv-112(H-AD/L-AP), was also beneficial. Cytoplasmic co-expression of disulfide-binding isomerase C (DsbC) helped proper folding of the zipFv-112(H-AD/L-AP) but not significantly. CONCLUSION: We believe that our zipFv constructs may serve as an excellent antibody format bi-functional antibody fragments that can be produced stably in the cytoplasm of E. coli.

The centrosomal localization of KM-HN-1 (MGC33607) depends on the leucine zipper motif and the C-terminal coiled-coil domain

KM-HN-1 is a C-terminal coiled-coil domain containing protein previously referred to as image clone MGC33607. This protein has been previously identified as a cancer/testis antigen and reported as nuclear and chromatin localizing protein. We raised polyclonal antisera with the GST fusion protein and identified them as a 105 kDa protein. Motif analysis showed that this protein harbors the leucine zipper motif in internal 1/3 region and the coiled-coil domain in the C-terminal region. Using the full length and various deletion mutants, we determined the motif that governs the subcellular localization of KM-HN-1. Immunofluorescence staining of the endogenous KM-HN-1 and various kinds of GFP-tagged KM-HN-1 revealed that KM-HN-1 localizes to the centrosomes as well as nucleus. The centrosomal localization-determining region of this protein is C-terminal coiled-coil domain in which the leucine zipper motif and the nuclear export signal (NES) harbor.

Preparation and identification of soluble trimeric CD40-isoleucine zipper fusion protein / 生物工程学报

The interaction of CD40 with its cognate ligand, CD40L (CD154), plays important roles in immune responses. Blockade of CD40-CD40L signal pathway can protect the progression of antibody- and cell-mediated autoimmune diseases, and reduce allograft rejection thus prolonging graft survival, even engendering long-lived antigen-specific tolerance. The present study aims to enhance the binding activity of CD40 by incorporating an isoleucine zipper (IZ) trimeric motif into CD40 ectodomain to promote the formation of soluble CD40 trimers, which would be useful for blocking CD40-CD40L interaction. A prokaryotic expression vector for soluble human CD40 ectodomain fused with an IZ motif and a hexa-histidine (His6) tag at its carboxyl terminus (sCD40IZ) was constructed by multiple round PCR using cloned CD40 cDNA as a template. The recombinant sCD40IZ protein was expressed highly in Escherichia coli (E. coli) with a molecular weight of 27kD, which is consistent with its theoretical value. It mainly existed in inclusion bodies. After refolding from inclusion bodies, soluble sCD40IZ protein was purified by gel filtration. Its molecular weight in solution was about 91kD when determined by gel filtration, suggesting that it most probably existed in the form of trimers. Moreover, this protein could bind to CD40L expressed on Jurkat T cells and its binding activity was significantly higher than that of soluble CD40 without an IZ motif. These results suggest that incorporation of an IZ motif at the carboxyl terminus of soluble CD40 can facilitate the formation of trimers and enhance its binding activity with CD40L. Thus, the trimeric CD40 protein may be used to block CD40-CD40L signal pathway, suggesting that it may have potential application in preventing autoimmune diseases and transplantation rejection.

Leucine Zipper, fyve-finger protein interacts with activating transcription factor 2 [ATF-2] and regulates its transcriptional activity

This study identified two novel proteins, 52-Zn and 67-LZ, that interact with ATF-2 in yeast two-hybrid assay. The study showed that their mRNA expression is restricted to the embryonic stage of mouse development. 52-Zn contains a putative zinc finger domain and interacts only with full-length ATF-2 in vivo. In contrast, 67-LZ contains a leucine zipper-like domain followed by a phosphatidylinositol 3-phosphate [PI3P]-binding FYVE-finger domain, is able to bind to the DNA-binding/leucine zipper domain of ATF-2 in vivo and in vitro and markedly stimulates in vitro DNA-binding by ATF-2. The ability of 67-LZ to functionally interact with ATF-2 in HeLa cell transfection assay is dependent on MEKK-1 activity, suggesting that ATF-2 phosphorylation by p38 kinase and/or Jun kinase is required for in vivo interaction between ATF-2 and this protein

Detection of point mutation at C-terminal region of phagosomal coat protein (TACO) in patients with leprosy / 나학회지

Mycobacteria, which are highly successful pathogen, resist delivary to lysosomes and instead survive within a specialized vacuole, the mycobacterial phagosome. The bacteria survive intracellularly because they are able to actively recruit and retain TACO ( tryptophane aspartate-containing coat protein ) at the mycobacterial phagosome, where it prevents lysosomal delivary in a cholesterol-dependent manner. In this study, we investigated the difference of TACO expression is whether related to mutant in coro1a gene in patients with leprosy and normal volunteer. First, we screened for detection of a mutant in the leucine zipper motif within the exon 11, and then in the exon 9 to 10, and finally in the coiled-coil region. Interestingly, single base substitutions ( point mutation ) presents at assembly site of U1 snRNP, around of 5' splice site in the intron 9, there are a C to T and G to A transition are at 9 bp and 14 bp downstream of 5' splice site, respectively, and both of it. Among the 3 types of polymorphism, frequency of a G to A transition is markedly increased in patients of lepromatous type, which are new cases or relapsed. Both a C to T and G to A transitions are found in 1 case of tuberculoid type and 2 cases in lepromatoue type, but not found in control group. The silent mutation in leucine zipper motif within the exon 11 is located at codon at 454 ( CTG-->CTA), which is 1st leucine from C-terminal among four leucine zipper. In coiled-coil region, no mutation is found in genomic DNA of patients with leprosy. Further, we will do functional study about the identified point mutation and will screen any possible mutation in the region of promotor and WD repeat.