[Show abstract][Hide abstract] ABSTRACT: The human papillomavirus (HPV) E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa) substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells.
CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA) showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2.
These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.
[Show abstract][Hide abstract] ABSTRACT: Octarepeats region sequence is one of the most important characteristics of PrP topology. To explore the mechanism of deleted and inserted octarepeats mutants PrP-caused apoptosis, wild-type PrP (PrP-PG5), and PrP with deleted octarepeats (PrP-PG0) and with four (PrP-PG9) and seven (PrP-PG12) extra octarepeats were transiently induced into SH-SY5Y cell. The results indicated PrP-PG9 and PrP-PG12 mainly retained in fraction of cytoplasm, while PrP-PG5 and PrP-PG0 presented both in cell membrane and cytoplasm. Cells expressing PrP-PG9 and PrP-PG12 were sensitive to endoplasmic reticulum (ER) stimuli, tunicamycin, and brefeldin A. ER-stress-related proteins, Grp94, XBP1, TRAF2, and CHOP, were significantly increased in cells expressing PrP-PG9 and PrP-PG12, while Grp78 increased markedly 12 h and pro-caspase-12 decreased sharply 20 h post-transfection. It indicates that expressions of PrP mutants with inserted octarepeats cause ER stress and lead to cell apoptosis lately. Meanwhile, cellular Cytochrome C increased and Bcl-2 decreased obviously in cells expressing PrP-PG0, indicating triggering a mitochondrial-related apoptosis. These data highlight that PrP mutants in region of octarepeats may undergo different pathways to trigger cell apoptosis, in which PrPs with inserted octarepeats via ER stress and PrP mutant without octarepeats via mitochondrial-related pathway.
[Show abstract][Hide abstract] ABSTRACT: Prion protein (PrP) is able to bind with tubulin and to interfere with the formation of microtubule. To investigate the influence of accumulation of cytosolic PrP in cytoplasm on microtubule, plasmid pcDNA3.1-PrP23-230 expressing human PrP23-230 was introduced into HeLa cells. Immunoprecipitation assays identified the molecular interaction between cytosolic PrP and cellular tubulin. Confocal microscopy showed the co-localization of the expressed cytosolic PrP with tubulin in cytoplasm. Immunofluorescent assays of tubulin illustrated remarkable disruption of microtubular structures in the cells accumulated with cytosolic PrP. Meanwhile, the expressed cytosolic PrP significantly reduced cell viability and induced cell apoptosis. The amounts of microtubule protein in the cells expressing cytosolic PrP were decreased. Moreover, the levels of endogenous tubulin in the brain tissues of scrapie-infected hamsters were significantly lower than that of normal one. It highlights a close linkage between disruption of microtubule framework and cell death caused by abnormal presence of cellular PrP in cytoplasm. The association of apoptosis with microtubule-disrupting activity caused by cytosolic PrP may further provide insight into the unresolved biological function of PrP in the neurons.
[Show abstract][Hide abstract] ABSTRACT: Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrP(C)) to the pathogenic one (PrP(Sc)). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease.
To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and caspase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed.
The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants within the transmembrane region undergo an ER-stress mediated cell apoptosis.
PLoS ONE 01/2011; 6(1):e14602. DOI:10.1371/journal.pone.0014602 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HPV-2 is a very common type of HPV which causes common warts. The E2 protein of virus can repress the activity of the viral early promoter through binding to the specific binding sites in viral LCR. Previously we reported that the repression of a mutated E2 protein of HPV-2 isolated from a patient with huge common wart on the viral early promoter was obviously decreased, and A338V mutation located at the C terminal DNA binding region of E2 protein. In this study, we expressed and purified the recombinant mutated and prototype E2 fusion proteins, both in the contexts of the C terminal and the full length, by prokaryotic expression system. The electrophoretic mobility shift assay showed E2 protein could bind to double-stranded DNA oligos labeled with biotin that covered two E2 binding sites. The DNA binding abilities of both C terminal and full-length mutated E2 proteins were stronger than the prototype analogs. This result indicates that the enhancement of the mutated E2 DNA binding ability may be the molecular mechanism for its impact on the activity of viral promoter, which correlates with the phenotype of extensive common wart.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 05/2010; 26(3):223-7.
[Show abstract][Hide abstract] ABSTRACT: Manganese may play some roles in the pathogenesis of prion diseases. In this study, recombinant human wild-type (WT) PrP and PrP mutants with deleted or inserted octarepeats were exposed to manganese, and their biochemical and biophysical characteristics were evaluated by proteinase K (PK) digestion, sedimentation experiments, transmission electron microscopy and circular dichroism. It demonstrated that incubation of manganese remarkably increased PK-resistances, protein aggregations and beta-sheet contents of the PrPs. Moreover, the PrP mutants of inserted or deleted octarepeats were much vulnerable to the influence of manganese, which showed obviously more aggregation and higher beta-sheet content than that of WT-PrP. It highlights that the effect of manganese on the PrP seems to lie on the incorrectness of the octarepeats numbers. The association of the octarepeats number of PrP with manganese may further provide insight into the unresolved biological function of PrP in the neurons.
Medical Microbiology and Immunology 08/2009; 198(4):239-45. DOI:10.1007/s00430-009-0120-y · 3.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The molecular interaction between PrP and 14-3-3 beta and the possible interactional domain between two proteins were studied by co-immunoprecipitation, pull down and FRET assays. The results showed that PrP protein could interact with 14-3-3 beta in vitro and in vivo. The domain which responded for the interaction was located at C-terminal of PrP (amino acid residues 106 to 126). This study of the interaction between PrP and 14-3-3 protein further provided the insight into the potential role of 14-3-3 in the biological function of PrP and the pathogenesis of prion disease.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 06/2009; 25(3):208-12.
[Show abstract][Hide abstract] ABSTRACT: In human prion diseases, phosphorylated-tau deposition has been described in a rare genetic form, Gerstmann-Straussler-Scheinker disease, but is not considered part of the neuropathological picture of Creutzfeldt-Jakob disease. To investigate the possible changes of tau and phosphorylated tau (Ser396/Ser404) in transmissible spongiform encephalopathies (TSEs), the expressions and transcriptions of above biological factors in the brain tissues of 263K- and 139A-infected hamsters were evaluated by Western blots and Real Time PCR, respectively, followed by quantitative analyses of immunoblot images and relative transcriptional levels compared with normal animals. The contents of total tau increased, but phosphorylated tau at Ser396 and Ser404 decreased, regardless of the types of scrapie agents and clinical incubations. Transcriptions of two tau isoforms were also markedly increased. These findings suggested that dephosphorylation of tau at Ser396/Ser404 was a illness-correlative phenomenon in TSEs. Alterations of tau and phosphorylated tau (Ser396/Ser404) were either intermediate or consequent events in TSE pathogenesis and proposed the potential linkage of these bioactive proteins with the pathogenesis of prion diseases.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 06/2009; 25(3):202-7.
[Show abstract][Hide abstract] ABSTRACT: Nerve growth factor (NGF), a member of the neurotrophin family, is essential for the development and maintenance of sensory neurons and for the formation of central pain circuitry. The current study was designed to evaluate the expression of NGF in the brain of rats with spared nerve injury (SNI), using immunohistochemical technique. The results showed that the level of NGF in the Red nucleus (RN) of SNI rats was apparently higher than that of sham-operated rats. To further study the effect of NGF in the development of neuropathic pain, different doses of anti-NGF antibody (20, 2.0 and 0.2 microg/ml) were microinjected into the RN contralateral to the nerve injury side of SNI rats. The data suggested that the higher doses of anti-NGF antibody (20 and 2.0 microg/ml) significantly attenuated the mechanical allodynia of neuropathic rats, while the 0.2 microg/ml antibody showed no analgesic effect. These results suggest that the NGF of RN is involved in the development of neuropathic allodynia in SNI rats.
Neurochemical Research 04/2009; 34(9):1612-8. DOI:10.1007/s11064-009-9950-7 · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gliosis of glial fibrillary acidic protein (GFAP) associated astrocytes is considered to be one of the hallmarks of transmissible spongiform encephalopathies (TSEs). In the present study, remarkable GFAP-PrP(Sc) or GFAP-PrP(C) complexes were separately detected in the brain homogenates of 263 K (Scrapie)-infected or normal hamsters by co-immunoprecipitation assay. To get more exact molecular evidences for interaction between prion protein (PrP) and GFAP, various recombinant PrP or GFAP proteins were expressed using prokaryotic-expressing and in vitro translation system. Using pull down and co-immunoprecipitation assays, reliable molecular interaction between PrP and GFAP was observed, and proteinase K (PK)-digested PrP(Sc) molecules were confirmed to be able to bind the recombinant GFAP specifically as well. The region within PrP that was responsible for interaction with GFAP was narrowed to PK-resistant core of PrP (i.e. aa 91-230). The study of the association of PrP with GFAP supplies the molecular evidence for the observation of co-localization of PrP(Sc) and GFAP in the brains of TSEs and may further provide insight into a potential role of GFAP in the biological function of PrP and the pathogenesis of prion diseases.
Medical Microbiology and Immunology 12/2008; 197(4):361-8. DOI:10.1007/s00430-007-0071-0 · 3.04 Impact Factor