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Sample records for nf-kappab p65 subunit

  1. Short communication: molecular characterization of dog and cat p65 subunits of NF-kappaB.

    PubMed

    Ishikawa, Shingo; Takemitsu, Hiroshi; Li, Gebin; Mori, Nobuko; Yamamoto, Ichiro; Arai, Toshiro

    2015-04-01

    Nuclear factor kappa B (NF-κB) plays an important role in the immune system. The p65 subunit is an important part of NF-κB unit, and studies of dog and cat p65 subunits of NF-κB (dp65 and cp65) are important in understanding their immune function. In this study, we described the molecular characterization of dp65 and cp65. The dp65 and cp65 complementary DNA encoded 542 and 555 amino acids, respectively, showing a high sequence homology with the mammalian p65 subunit (>87.5%). Quantitative polymerase chain reaction revealed that the p65 messenger RNA is highly expressed in the dog stomach and cat heart and adipose tissue. Functional NF-κB promoter-luciferase reporter vectors revealed that our isolated dp65 and cp65 cDNA encodes a functionally active protein. Transiently expressed dp65 and cp65 up-regulated pro-inflammatory cytokine expression levels in dog and cat, respectively. These findings suggest that dp65 and cp65 play important roles in regulating immune function.

  2. The DEAD-Box RNA Helicase DDX3 Interacts with NF-κB Subunit p65 and Suppresses p65-Mediated Transcription

    PubMed Central

    Gao, Yu; Song, Feifei; Guo, Liang; Ma, Li; Sun, Guihong; Liu, Dan; Guo, Deyin

    2016-01-01

    RNA helicase family members exhibit diverse cellular functions, including in transcription, pre-mRNA processing, RNA decay, ribosome biogenesis, RNA export and translation. The RNA helicase DEAD-box family member DDX3 has been characterized as a tumour-associated factor and a transcriptional co-activator/regulator. Here, we demonstrate that DDX3 interacts with the nuclear factor (NF)-κB subunit p65 and suppresses NF-κB (p65/p50)-mediated transcriptional activity. The downregulation of DDX3 by RNA interference induces the upregulation of NF-κB (p65/p50)-mediated transcription. The regulation of NF-κB (p65/p50)-mediated transcriptional activity was further confirmed by the expression levels of its downstream cytokines, such as IL-6 and IL-8. Moreover, the binding of the ATP-dependent RNA helicase domain of DDX3 to the N-terminal Rel homology domain (RHD) of p65 is involved in the inhibition of NF-κB-regulated gene transcription. In summary, the results suggest that DDX3 functions to suppress the transcriptional activity of the NF-κB subunit p65. PMID:27736973

  3. Regulation of RelA (p65) Function by the Large Subunit of Replication Factor C

    PubMed Central

    Anderson, Lisa A.; Perkins, Neil D.

    2003-01-01

    The RelA (p65) subunit of NF-κB is an important regulator of inflammation, proliferation, and apoptosis. We have discovered that the large subunit, p140, of replication factor C (RFC) can function as a regulator of RelA. RFC is a clamp loader, facilitating the addition and removal of proliferating-cell nuclear antigen from DNA during replication and repair but can also interact directly with the retinoblastoma tumor suppressor protein and the transcription factor C/EBPα. We find that RFC (p140) interacts with RelA both in vitro and in vivo and stimulates RelA transactivation. In contrast, coexpression of fragments of RFC (p140) that mediate the interaction with RelA results in transcriptional inhibition. The significance of this regulation was confirmed by using short interfering RNA oligonucleotides targeted to RFC (p140). Down regulation of endogenous RFC (p140) inhibits expression from a chromosomally integrated reporter plasmid induced by endogenous, TNF-α-activated NF-κB. Dominant negative fragments of RFC (p140) also cooperate with overexpressed RelA to induce cell death. Interestingly, RFC (p140) also interacts with the tumor suppressor p53. Taken together, these observations suggest that, in addition to its previously described function in DNA replication and repair, RFC (p140) has an important role as a regulator of transcription and NF-κB activity. PMID:12509469

  4. Piperlongumine selectively suppresses ABC-DLBCL through inhibition of NF-κB p65 subunit nuclear import

    SciTech Connect

    Niu, Mingshan; Shen, Yangling; Xu, Xiaoyu; Yao, Yao; Fu, Chunling; Yan, Zhiling; Wu, Qingyun; Cao, Jiang; Sang, Wei; Zeng, Lingyu; Li, Zhenyu; Liu, Xuejiao; and others

    2015-07-10

    Constitutive NF-κB activation is required for survival of activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL). However, current NF-κB targeting strategies lack cancer cell specificity. Here, we identified a novel inhibitor, piperlongumine, features direct binding to NF-κB p65 subunit and suppression of p65 nuclear import. This was accompanied by NF-κB reporter activity suppression and NF-κB target gene downregulation. Moreover, mutation of Cys{sup 38} to Ser in p65 abolished this effect of piperlongumine on inhibition of p65 nuclear import. Furthermore, we show that piperlongumine selectively inhibited proliferation and induced apoptosis of ABC-DLBCL cells. Most notably, it has been reported that piperlongumine did not affect normal cells even at high doses and was nontoxic to animals. Hence, our current study provides new insight into piperlongumine's mechanism of action and novel approach to ABC-DLBCL target therapy. - Highlights: • Current NF-κB targeting strategies lack cancer cell specificity. • Piperlongumine inhibits NF-κB p65 subunit nuclear import via directly binding to p65. • Piperlongumine selectively inhibits proliferation of ABC-DLBCL cells. • This study provides a novel approach to ABC-DLBCL target therapy.

  5. Exposure to coplanar PCBs induces endothelial cell inflammation through epigenetic regulation of NF-κB subunit p65

    PubMed Central

    Liu, Dandan; Perkins, Jordan T.; Petriello, Michael C.; Hennig, Bernhard

    2015-01-01

    Epigenetic modifications of DNA and histones alter cellular phenotypes without changing genetic codes. Alterations of epigenetic marks can be induced by exposure to environmental pollutants and may contribute to associated disease risks. Here we test the hypothesis that endothelial cell dysfunction induced by exposure to polychlorinated biphenyls (PCBs) is mediated in part though histone modifications. In this study, human vascular endothelial cells were exposed to physiologically relevant concentrations of several PCBs congeners (e.g., PCBs 77, 118, 126 and 153) followed by quantification of inflammatory gene expression and changes of histone methylation. Only exposure to coplanar PCBs 77 and 126 induced the expression of histone H3K9 trimethyl demethylase jumonji domain-containing protein 2B (JMJD2B) and nuclear factor-kappa B (NF-κB) subunit p65, activated NF-κB signaling as evidenced by nuclear translocation of p65, and up-regulated p65 target inflammatory genes, such as interleukin (IL)-6, C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and IL-1α/β. The increased accumulation of JMJD2B in the p65 promoter led to a depletion of H3K9me3 repression mark, which accounts for the observed up-regulation of p65 and associated inflammatory genes. JMJD2B gene knockdown confirmed a critical role for this histone demethylase in mediating PCB-induced inflammation of the vascular endothelium. Finally, it was determined, via chemical inhibition, that PCB-induced up-regulation of JMJD2B was estrogen receptor-alpha (ER-α) dependent. These data suggest that coplanar PCBs may exert endothelial cell toxicity through changes in histone modifications. PMID:26519613

  6. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-{kappa}B p65 subunit and cytotoxicity in renal proximal tubule cells

    SciTech Connect

    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Lee, Sang Yong; Han, Myung Kwan; Kim, Duk Hoon; Kim, Won

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Cisplatin increases acetylation of NF-{kappa}B p65 subunit in HK2 cells. Black-Right-Pointing-Pointer SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. Black-Right-Pointing-Pointer Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-{kappa}B (NF-{kappa}B) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD{sup +})-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-{kappa}B p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-{kappa}B during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-{kappa}B p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-{kappa}B through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

  7. MKRN2 is a novel ubiquitin E3 ligase for the p65 subunit of NF-κB and negatively regulates inflammatory responses

    PubMed Central

    Shin, Chanyoung; Ito, Yuma; Ichikawa, Shota; Tokunaga, Makio; Sakata-Sogawa, Kumiko; Tanaka, Takashi

    2017-01-01

    Activation of NF-κB transcription factor is strictly regulated to prevent excessive inflammatory responses leading to immunopathology. However, it still remains unclear how NF-κB activation is negatively controlled. The PDZ-LIM domain-containing protein PDLIM2 is a nuclear ubiquitin E3 ligase targeting the p65 subunit of NF-κB for degradation, thus terminating NF-κB-mediated inflammation. Using yeast two-hybrid screening, we sought to isolate PDLIM2-interacting proteins that are critical for suppressing NF-κB signaling. Here we identified MKRN2, a RING finger domain-containing protein that belongs to the makorin ring finger protein gene family, as a novel p65 ubiquitin E3 ligase. MKRN2 bound to p65 and promoted the polyubiquitination and proteasome-dependent degradation of p65 through the MKRN2 RING finger domain, thereby suppressing p65-mediated NF-κB transactivation. Notably, MKRN2 and PDLIM2 synergistically promote polyubiquitination and degradation of p65. Consistently, MKRN2 knockdown in dendritic cells resulted in larger amounts of nuclear p65 and augmented production of proinflammatory cytokines in responses to innate stimuli. These results delineate a novel role of MKRN2 in negatively regulating NF-κB-mediated inflammatory responses, cooperatively with PDLIM2. PMID:28378844

  8. Cyclophilin A (CypA) Interacts with NF-κB Subunit, p65/RelA, and Contributes to NF-κB Activation Signaling

    PubMed Central

    Zhang, James Beiji; Ha, Albert; Yokoyama, Kazunari K.; Chiu, Robert H.

    2014-01-01

    Background Peptidyl-prolyl isomerase cyclophilin A (CypA) plays important roles in signaling, protein translocation, inflammation, and cancer formation. However, little is known about the mechanisms by which CypA exerts its effects. C57BL/6 Ppia (encoding CypA)-deficient embryonic fibroblasts show reduced activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), the p65/RelA subunit, suggesting that CypA may mediate modulation of NF-κB activity to exert its biological effects. Methodology Western blotting and qRT-PCR analyses were used to evaluate the association of CypA deficiency with reduced activation of NF-κB/p65 at the protein level. GST pull-down and co-immunoprecipitation were used to examine interactions between CypA and p65/RelA. Truncation mutants and site-directed mutagenesis were used to determine the sequences of p65/RelA required for interactions with CypA. Enhancement of p65/RelA nuclear translocation by CypA was assessed by co-transfection and immunofluorescent imaging. Treatment of cells with cycloheximide that were harvested at various time points for Western blot analyses was carried out to evaluate p65/RelA protein stability. The functional activity of NF-κB was assessed by electrophoretic mobility-shift assays (EMSA), luciferase assays, and changes in expression levels of target genes. Results GST pull-down assays in vitro and co-immunoprecipitation analyses in vivo provided evidence for protein-protein interactions. These interactions were further supported by identification of a CypA-binding consensus-like sequence within NF-κB subunit p65 at the N-terminal 170–176 amino acid residues. Significantly, CypA provided stability for NF-κB p65 and promoted NF-κB p65 nuclear translocation, resulting in increased nuclear accumulation and enhanced NF-κB activity. Conclusions Our findings revealed important mechanisms that regulate NF-κB activation, and offer new insights into the role of CypA in aberrant

  9. Inhibition of BMP2-induced bone formation by the p65 subunit of NF-κB via an interaction with Smad4.

    PubMed

    Hirata-Tsuchiya, Shizu; Fukushima, Hidefumi; Katagiri, Takenobu; Ohte, Satoshi; Shin, Masashi; Nagano, Kenichi; Aoki, Kazuhiro; Morotomi, Takahiko; Sugiyama, Goro; Nakatomi, Chihiro; Kokabu, Shoichiro; Doi, Takahiro; Takeuchi, Hiroshi; Ohya, Keiichi; Terashita, Masamichi; Hirata, Masato; Kitamura, Chiaki; Jimi, Eijiro

    2014-09-01

    Bone morphogenic proteins (BMPs) stimulate bone formation in vivo and osteoblast differentiation in vitro via a Smad signaling pathway. Recent findings revealed that the activation of nuclear factor-κB (NF-κB) inhibits BMP-induced osteoblast differentiation. Here, we show that NF-κB inhibits BMP signaling by directly targeting the Smad pathway. A selective inhibitor of the classic NF-κB pathway, BAY11-770682, enhanced BMP2-induced ectopic bone formation in vivo. In mouse embryonic fibroblasts (MEFs) prepared from mice deficient in p65, the main subunit of NF-κB, BMP2, induced osteoblastic differentiation via the Smad complex to a greater extent than that in wild-type MEFs. In p65(-/-) MEFs, the BMP2-activated Smad complex bound much more stably to the target element than that in wild-type MEFs without affecting the phosphorylation levels of Smad1/5/8. Overexpression of p65 inhibited BMP2 activity by decreasing the DNA binding of the Smad complex. The C-terminal region, including the TA2 domain, of p65 was essential for inhibiting the BMP-Smad pathway. The C-terminal TA2 domain of p65 associated with the MH1 domain of Smad4 but not Smad1. Taken together, our results suggest that p65 inhibits BMP signaling by blocking the DNA binding of the Smad complex via an interaction with Smad4. Our study also suggests that targeting the association between p65 and Smad4 may help to promote bone regeneration in the treatment of bone diseases.

  10. Light induces translocation of NF-κB p65 to the mitochondria and suppresses expression of cytochrome c oxidase subunit III (COX III) in the rat retina.

    PubMed

    Tomita, Hiroshi; Tabata, Kitako; Takahashi, Maki; Nishiyama, Fumiaki; Sugano, Eriko

    2016-05-13

    The transcription factor nuclear factor kappaB (NF-κB) plays various roles in cell survival, apoptosis, and inflammation. In the rat retina, NF-κB activity increases after exposure to damaging light, resulting in degeneration of photoreceptors. Here, we report that in dark-adapted rats exposed for 6 h to bright white light, the p65 subunit of retinal NF-κB translocates to the mitochondria, an event associated with a decrease in expression of cytochrome c oxidase subunit III (COX III). However, sustained exposure for 12 h depleted p65 from the mitochondria, and enhanced COX III expression. Treatment with the protective antioxidant PBN prior to light exposure prevents p65 depletion in the mitochondria and COX III upregulation during prolonged exposure, and apoptosis in photoreceptor cells. These results indicate that COX III expression is sensitive to the abundance of NF-κB p65 in the mitochondria, which, in turn, is affected by exposure to damaging light.

  11. Id-1 and the p65 subunit of NF-κB promote migration of nasopharyngeal carcinoma cells and are correlated with poor prognosis.

    PubMed

    Sun, Wei; Guo, Ming-Ming; Han, Ping; Lin, Ji-Zhen; Liang, Fa-Ya; Tan, Guang-Mou; Li, Hua-Bin; Zeng, Musheng; Huang, Xiao-Ming

    2012-04-01

    Inhibitor of differentiation (Id)-1 and nuclear factor-kappa B (NF-κB) have been detected in many malignant tumors, and their presence has been correlated with the metastatic potential of these tumors. This study was undertaken to investigate the prognostic significance of the expression of Id-1 and the p65 subunit of NF-κB (NF-κB/p65) and the proteins' roles in the invasion process of nasopharyngeal carcinoma (NPC) cells. The messenger RNA (mRNA) and protein levels of Id-1 and NF-κB/p65 in normal nasopharyngeal epithelial cells and NPC cell lines were examined using reverse transcription-PCR and western blot analysis, whereas the mRNA and protein levels of Id-1 and NF-κB/p65 in clinical NPC specimens were determined by reverse transcription-PCR and immunohistochemistry. Short hairpin RNA (shRNA) was used to silence Id-1 and NF-κB/p65 to allow for the examination of matrix metalloproteinase (MMP)-9 expression and migratory capacity changes in CNE-2 cells. Multivariate Cox analysis revealed that elevated Id-1 expression was a significant independent predictor of the 5 year overall survival rate (hazards ratio = 16.720, P = 0.005). Furthermore, elevated expression of both Id-1 and NF-κB/p65 was associated with poor clinical survival (P = 0.049). Targeting Id-1 and NF-κB/p65 mRNA with shRNA in CNE-2 cells inhibited MMP-9 expression and decreased the migratory capacity of CNE-2 cells. In conclusion, Id-1 expression is a novel independent prognostic marker molecule that helps identify NPC patients with a poor prognosis. Additionally, combined analysis of Id-1 and NF-κB/p65 can be useful for identifying patients at risk for unfavorable clinical outcomes. Id-1 or/and NF-κB/p65 enhanced tumor cell migration, which is associated with the secretion of MMP-9.

  12. Identification of Genes, including the Gene Encoding p27Kip1, Regulated by Serine 276 Phosphorylation of the p65 Subunit of NF-κB

    PubMed Central

    Prasad, Ratna Chakraborty; Wang, Xiaohui L.; Law, Brian K.; Davis, Bradley; Green, Gail; Boone, Braden; Sims, Lauren; Law, Mary

    2009-01-01

    Phosphorylation of the p65 subunit of NF-κB is required for its transcriptional activity. Recent reports show that phosphorylation of p65 at serine 276 regulates only a subset of genes, such as those encoding IL-6, IL-8, Gro-β, and ICAM-1. In order to identify additional genes regulated by serine 276 phosphorylation, HepG2 hepatoma cells were infected with adenoviruses encoding either wild-type p65 or the S276A mutant of p65, followed by DNA microarray analysis. The results show that mutation of serine 276 affected the expression of several genes that encode proteins involved in cell cycle regulation, signal transduction, transcription, and metabolism. Notably, expression of S276A increased the mRNA and protein level of p27, a cell cycle inhibitory protein, which led to an increased association of p27 with cdk2, and inhibition of cdk2 activity. Furthermore, while wild-type NF-κB is known to increase cell proliferation in a number of different cancer cell lines, our data show that S276A inhibits cell proliferation. Evidence is mounting that NF-κB plays a pivotal role in oncogenesis. Therapeutic agents that regulate the phosphorylation of serine 276 and p27 gene expression, therefore, may be useful as anti-cancer agents in the future. PMID:19038492

  13. Neural progenitor number is regulated by nuclear factor-kappaB p65 and p50 subunit-dependent proliferation rather than cell survival.

    PubMed

    Young, Kaylene M; Bartlett, Perry F; Coulson, Elizabeth J

    2006-01-01

    The number of cells generated by a proliferating stem or precursor cell can be influenced both by proliferation and by the degree of cell death/survival of the progeny generated. In this study, the extent to which cell survival controls progenitor number was examined by comparing the growth characteristics of neurosphere cultures derived from mice lacking genes for the death-inducing Bcl-2 homologue Hara Kiri (Hrk), apoptosis-associated protein 1 (Apaf1), or the prosurvival nuclear factor-kappaB (NFkappaB) subunits p65, p50, or c-rel. We found no evidence that Hrk or Apaf1, and by inference the mitochondrial cell death pathway, are involved in regulating the number of neurosphere-derived progeny. However, we identified the p65p50 NFkappaB dimer as being required for the normal growth and expansion of neurosphere cultures. Genetic loss of both p65 and p50 NFkappaB subunits resulted in a reduced number of progeny but an increased proportion of neurons. No effect on cell survival was observed. This suggests that the number and fate of neural progenitor cells are more strongly regulated by cell cycle control than survival.

  14. Isoliensinine induces dephosphorylation of NF-κB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction

    PubMed Central

    Shu, Guangwen; Zhang, Lang; Jiang, Shanqing; Cheng, Zhuo; Wang, Guan; Huang, Xu; Yang, Xinzhou

    2016-01-01

    Our previous study discovered that isoliensinine (isolie) triggers hepatocellular carcinoma (HCC) cell apoptosis via inducing p65 dephosphorylation at Ser536 and inhibition of NF-κB. Here, we showed that isolie promoted p65/PP2A interaction in vitro and in vivo. Repression of PP2A activity or knockdown of the expression of PP2A-C (the catalytic subunit of PP2A) abrogated isolie-provoked p65 dephosphorylation. I2PP2A is an endogenous PP2A inhibitor. Isolie directly impaired PP2A/I2PP2A interaction. Knockdown of I2PP2A boosted p65/PP2A association and p65 dephosphorylation. Overexpression of I2PP2A restrained isolie-induced p65 dephosphorylation. Untransformed hepatocytes were insensitive to isolie-induced NF-κB inhibition and cell apoptosis. In these cells, basal levels of I2PP2A and p65 phosphorylation at Ser536 were lower than in HCC cells. These findings collectively indicated that isolie suppresses NF-κB in HCC cells through impairing PP2A/I2PP2A interaction and stimulating PP2A-dependent p65 dephosphorylation at Ser536. PMID:27244888

  15. Isoliensinine induces dephosphorylation of NF-kB p65 subunit at Ser536 via a PP2A-dependent mechanism in hepatocellular carcinoma cells: roles of impairing PP2A/I2PP2A interaction.

    PubMed

    Shu, Guangwen; Zhang, Lang; Jiang, Shanqing; Cheng, Zhuo; Wang, Guan; Huang, Xu; Yang, Xinzhou

    2016-06-28

    Our previous study discovered that isoliensinine (isolie) triggers hepatocellular carcinoma (HCC) cell apoptosis via inducing p65 dephosphorylation at Ser536 and inhibition of NF-κB. Here, we showed that isolie promoted p65/PP2A interaction in vitro and in vivo. Repression of PP2A activity or knockdown of the expression of PP2A-C (the catalytic subunit of PP2A) abrogated isolie-provoked p65 dephosphorylation. I2PP2A is an endogenous PP2A inhibitor. Isolie directly impaired PP2A/I2PP2A interaction. Knockdown of I2PP2A boosted p65/PP2A association and p65 dephosphorylation. Overexpression of I2PP2A restrained isolie-induced p65 dephosphorylation. Untransformed hepatocytes were insensitive to isolie-induced NF-κB inhibition and cell apoptosis. In these cells, basal levels of I2PP2A and p65 phosphorylation at Ser536 were lower than in HCC cells. These findings collectively indicated that isolie suppresses NF-κB in HCC cells through impairing PP2A/I2PP2A interaction and stimulating PP2A-dependent p65 dephosphorylation at Ser536.

  16. A family of serine proteases expressed exclusively in myelo-monocytic cells specifically processes the nuclear factor-kappa B subunit p65 in vitro and may impair human immunodeficiency virus replication in these cells

    PubMed Central

    1994-01-01

    Two groups of U937 promonocytic cells were obtained by limiting dilution cloning which differed strikingly in their ability to support human immunodeficiency virus 1 (HIV-1) replication. "Plus" clones replicated the virus efficiently, whereas "minus" clones did not. We examined these clones for differences in nuclear factor (NF)-kappa B activity which might account for the observed phenomenon. Stimulation of plus clones liberated the classical p50-p65 complex from cytoplasmic pools, whereas minus clones produced an apparently novel, faster- migrating complex, as judged by electrophoretic mobility shift assays. It is surprising that the faster-migrating complex was composed also of p50 and p65. However, the p65 subunit was COOH-terminally truncated, as shown by immunoprecipitation. The truncation resulted from limited proteolysis of p65 during cellular extraction which released particular lysosomal serine proteases, such as elastase, cathepsin G, and proteinase 3. These specific proteases are coordinately expressed and were present exclusively in the minus U937 clones, but not in the plus clones, as demonstrated in the case of cathepsin G. In addition, these proteases were detected in certain subclones of THP-1 and HL-60 cells and in primary monocytes, in each case correlating with the truncated from of p65. We demonstrate in vitro cleavage of p65 by purified elastase and cathepsin G. It is possible that particular serine proteases may have inhibiting effects on the replication of HIV-1 in myelo-monocytic cells. The data also demonstrate that special precautions must be taken when making extracts from myelo-monocytic cells. PMID:7931077

  17. The response of L5178Y lymphoma sublines to oxidative stress: antioxidant defence, iron content and nuclear translocation of the p65 subunit of NF-kappaB.

    PubMed

    Bouzyk, E; Gradzka, I; Iwaneńko, T; Kruszewski, M; Sochanowicz, B; Szumiel, I

    2000-01-01

    We examined the response to hydrogen peroxide of two L5178Y (LY) sublines which are inversely cross-sensitive to hydrogen peroxide and X-rays: LY-R cells are radio-resistant and hydrogen peroxide-sensitive, whereas LY-S cells are radiosensitive and hydrogen peroxide-resistant. Higher initial DNA breaks and higher iron content (potentially active in the Fenton reaction) were found in the hydrogen peroxide sensitive LY-R cells than in the hydrogen peroxide resistant LY-S cells, whereas the antioxidant defence of LY-R cells was weaker. In particular, catalase activity is twofold higher in LY-S than in LY-R cells. The content of monobromobimane-reactive thiols is 54% higher in LY-S than in LY-R cells. In contrast, the activity of glutathione peroxidase (GPx) is about two times higher in LY-R than in LY-S cells; however, upon induction with selenium the activity increases 15.6-fold in LY-R cells and 50.3-fold in LY-S cells. Altogether, the sensitivity difference is related to the iron content, the amount of the initial DNA damage, as well as to the efficiency of the antioxidant defence system. Differential nuclear translocation of p65-NF-kappaB in LY sublines is due to the more efficient antioxidant defence in LY-S than in LY-R cells.

  18. FBI-1 enhances transcription of the nuclear factor-kappaB (NF-kappaB)-responsive E-selectin gene by nuclear localization of the p65 subunit of NF-kappaB.

    PubMed

    Lee, Dong-Kee; Kang, Jae-Eun; Park, Hye-Jin; Kim, Myung-Hwa; Yim, Tae-Hee; Kim, Jung-Min; Heo, Min-Kyu; Kim, Kyu-Yeun; Kwon, Ho Jeong; Hur, Man-Wook

    2005-07-29

    The POZ domain is a highly conserved protein-protein interaction motif found in many regulatory proteins. Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of a variety of genes in response to infection, inflammation, and stressful conditions. We found that the POZ domain of FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) interacted with the Rel homology domain of the p65 subunit of NF-kappaB in both in vivo and in vitro protein-protein interaction assays. FBI-1 enhanced NF-kappaB-mediated transcription of E-selectin genes in HeLa cells upon phorbol 12-myristate 13-acetate stimulation and overcame gene repression by IkappaB alpha or IkappaB beta. In contrast, the POZ domain of FBI-1, which is a dominant-negative form of FBI-1, repressed NF-kappaB-mediated transcription, and the repression was cooperative with IkappaB alpha or IkappaB beta. In contrast, the POZ domain tagged with a nuclear localization sequence polypeptide of FBI-1 enhanced NF-kappaB-responsive gene transcription, suggesting that the molecular interaction between the POZ domain and the Rel homology domain of p65 and the nuclear localization by the nuclear localization sequence are important in the transcription enhancement mediated by FBI-1. Confocal microscopy showed that FBI-1 increased NF-kappaB movement into the nucleus and increased the stability of NF-kappaB in the nucleus, which enhanced NF-kappaB-mediated transcription of the E-selectin gene. FBI-1 also interacted with IkappaB alpha and IkappaB beta.

  19. RelA/p65 Regulation of IκBβ

    PubMed Central

    Hertlein, Erin; Wang, Jingxin; Ladner, Katherine J.; Bakkar, Nadine; Guttridge, Denis C.

    2005-01-01

    IκB inhibitor proteins are the primary regulators of NF-κB. In contrast to the defined regulatory interplay between NF-κB and IκBα, much less is known regarding the regulation of IκBβ by NF-κB. Here, we describe in detail the regulation of IκBβ by RelA/p65. Using p65−/− fibroblasts, we show that IκBβ is profoundly reduced in these cells, but not in other NF-κB subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65−/− cells and tissues, IκBα is also reduced, but not nearly to the same extent as IκBβ, thus highlighting the degree to which IκBβ is dependent on p65. This dependence is based on the ability of p65 to stabilize IκBβ protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, IκBβ was found to exist in both a basally phosphorylated and a hyperphosphorylated form. While the hyperphosphorylated form is less abundant, it is also more stable and less dependent on p65 and its carboxyl domain. Finally, we show that in p65−/− fibroblasts, expression of a proteolysis-resistant form of IκBβ, but not IκBα, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of IκBβ protein by p65 is necessary for the maintenance of cellular homeostasis. PMID:15923614

  20. RelA/p65 regulation of IkappaBbeta.

    PubMed

    Hertlein, Erin; Wang, Jingxin; Ladner, Katherine J; Bakkar, Nadine; Guttridge, Denis C

    2005-06-01

    IkappaB inhibitor proteins are the primary regulators of NF-kappaB. In contrast to the defined regulatory interplay between NF-kappaB and IkappaBalpha, much less is known regarding the regulation of IkappaBbeta by NF-kappaB. Here, we describe in detail the regulation of IkappaBbeta by RelA/p65. Using p65(-/-) fibroblasts, we show that IkappaBbeta is profoundly reduced in these cells, but not in other NF-kappaB subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65(-/-) cells and tissues, IkappaBalpha is also reduced, but not nearly to the same extent as IkappaBbeta, thus highlighting the degree to which IkappaBbeta is dependent on p65. This dependence is based on the ability of p65 to stabilize IkappaBbeta protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, IkappaBbeta was found to exist in both a basally phosphorylated and a hyperphosphorylated form. While the hyperphosphorylated form is less abundant, it is also more stable and less dependent on p65 and its carboxyl domain. Finally, we show that in p65(-/-) fibroblasts, expression of a proteolysis-resistant form of IkappaBbeta, but not IkappaBalpha, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of IkappaBbeta protein by p65 is necessary for the maintenance of cellular homeostasis.

  1. Activation of the serum response factor by p65/NF-kappaB.

    PubMed Central

    Franzoso, G; Carlson, L; Brown, K; Daucher, M B; Bressler, P; Siebenlist, U

    1996-01-01

    This study demonstrates that the NF-kappaB subunit p65 can act like an accessory protein for the serum response factor (SRF) in transfection assays. p65 functionally synergizes with SRF to activate the transcription of a reporter construct dependent only on the serum response element (SRE). The synergy of the two factors requires neither a kappaB motif nor direct contact of p65 with DNA. Consistent with these results, a physical complex containing p65 and SRF is observed in vitro. Synergy of the factors is independent of the previously described activation domains present on p65, ruling out indirect effects of p65, but synergy is dependent on the activation domain of SRF. The complexing of p65 and SRF is mediated by a segment of the SRF DNA binding domain, a region of the protein which has also been reported to inhibit its own activation domain. We speculate that p65, upon direct or facilitated interaction with SRF, may relieve the inhibitory activity of this segment, thus enabling the activation domain of SRF to become fully functional. In contrast to p65, the p50 subunit of NF-kappaB does not interact significantly with SRF, either functionally or physically. The data suggest the intriguing possibility that NF-kappaB may participate in the regulation of SRE-dependent promoters, expanding the range of activities of this rapidly activatable transcription factor. Images PMID:8670842

  2. Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry.

    PubMed

    Maguire, Orla; O'Loughlin, Kieran; Minderman, Hans

    2015-08-01

    Aberrant activity of Nuclear Factor-kappaB (NF-κB) is associated with many diseases and is therapeutically targeted. Post-translational modifications, particularly phosphorylation of the RELA/p65 sub-unit, are essential for cytoplasmic to nuclear localization of NF-κB/p65 and initiation of transcription of downstream target genes. Immunoblot and phospho-flow cytometry have been used to study the relationship between phosphorylation motifs and NF-κB activation and microscopic analysis of nuclear localization of p65 is also used as a parameter for activation. The labor intensive nature of these approaches commonly limits the number of sampling points or replicates. Recent insights into the relationship between p65 phosphorylation motifs and their nuclear localization indicate that these parameters have different significances and should not be used interchangeably. In this study, we demonstrate feasibility and reproducibility of studying the relationship between p65 phosphorylation and nuclear translocation using imaging flow cytometry (IFC). TNFα- or PMA/Ionomycin-induced phosphorylation of p65 at serine 529 in cell line models and healthy donor lymphocytes served as the experimental model. IFC analysis demonstrated that expression of phosphorylated serine 529 (P-p65(s529)) increased rapidly following stimulation and that nuclear localization of P-p65(s529) followed the nuclear localization pattern of total p65. However, in the presence of tacrolimus, P-p65(s529) expression was inhibited without affecting nuclear localization of total p65. The data demonstrate the application of IFC to simultaneously assess phosphorylation of p65 and its cellular localization and the results obtained by this analysis corroborate current insights regarding the specific effect of tacrolimus on serine 529 phosphorylation.

  3. NF-κB p65 recruited SHP regulates PDCD5-mediated apoptosis in cancer cells.

    PubMed

    Murshed, Farhan; Farhana, Lulu; Dawson, Marcia I; Fontana, Joseph A

    2014-03-01

    Transcription factor NF-κB promotes cell proliferation in response to cell injury. Increasing evidence, however, suggests that NF-κB can also play an apoptotic role depending on the stimulus and cell type. We have previously demonstrated that novel retinoid 4-[3-Cl-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC)-mediated apoptosis in breast carcinoma cells requires activation of canonical and non-canonical NF-κB pathways. The mechanism NF-κB uses to induce apoptosis remains largely unknown. NF-κB subunit p65 (RelA) was identified as one potent transcriptional activator in 3-Cl-AHPC-mediated apoptosis in cells. Here we used ChIP-on-chip to identify NF-κB p65 genes activated in 3-Cl-AHPC mediated apoptosis. This paper focuses on one hit: pro-apoptotic protein programmed cell death 5 (PDCD5). 3-Cl-AHPC mediated apoptosis in MDA-MB-468 had three related effects on PDCD5: NF-κB p65 binding to the PDCD5 gene, enhanced PDCD5 promoter activity, and increased PDCD5 protein expression. Furthermore, 3-Cl-AHPC increased orphan nuclear receptor small heterodimer partner (SHP) mRNA expression, increased SHP protein bound to NF-κB p65, and found the SHP/NF-κB p65 complex attached to the PDCD5 gene. PDCD5 triggered apoptosis through increased Bax protein and release of cytochrome C from mitochondria to cytosol. Lastly, knockdown of PDCD5 protein expression blocked 3-Cl-AHPC mediated apoptosis, while over-expression of PDCD5 enhanced apoptosis, suggesting PDCD5 is necessary and sufficient for NF-κB p65 mediated apoptosis. Our results demonstrate a novel pathway for NF-κB p65 in regulating apoptosis through SHP and PDCD5.

  4. Negative regulation of NF-κB p65 activity by serine 536 phosphorylation

    PubMed Central

    Pradère, Jean-Philippe; Hernandez, Céline; Koppe, Christiane; Friedman, Richard A; Luedde, Tom; Schwabe, Robert F.

    2016-01-01

    Nuclear factor κB (NF-κB) is a master regulator of inflammation and cell death. Whereas most of the activity of NF-κB is regulated through the inhibitor of kB (IκB) kinase (IKK)–dependent degradation of IκB, IKK also phosphorylates subunits of NF-κB. Here, we investigated the contribution of the phosphorylation of the NF-κ B subunit p65 at the IKK phosphorylation site serine 536 (Ser536) in humans, which is thought to be required for the activation and nuclear translocation of NF-κB. Through experiments with knock-in mice (S534A mice) expressing a mutant p65 with an alanine-to-serine substitution at position 534 (the murine homolog of human Ser536), we observed increased expression of NF-κ B–dependent genes after injection of mice with the inflammatory stimulus lipopolysaccharide (LPS) or exposure to gamma irradiation, and the enhanced gene expression was most pronounced at late time points. Compared to wild-type mice, S534A mice displayed increased mortality after injection with LPS. Increased NF-κ B signaling in the S534A mice was at least in part explained by the increased stability of the S534A p65 protein compared to that of the Ser534-phosphorylated wild-type protein. Together, our results suggest that Ser534 phosphorylation of p65 in mice (and, by extension, Ser536 phosphorylation of human p65) is not required for its nuclear translocation, but instead inhibits NF-κ B signaling to prevent deleterious inflammation. PMID:27555662

  5. PHF20 regulates NF-κB signalling by disrupting recruitment of PP2A to p65

    PubMed Central

    Zhang, Tiejun; Park, Kyeong Ah; Li, Yuwen; Byun, Hee Sun; Jeon, Juhee; Lee, Yoonjung; Hong, Jang Hee; Kim, Jin Man; Huang, Song-Mei; Choi, Seung-Won; Kim, Sun-Hwan; Sohn, Kyung-Cheol; Ro, Hyunju; Lee, Ji Hoon; Lu, Tao; Stark, George R.; Shen, Han-Ming; Liu, Zheng-gang; Park, Jongsun; Hur, Gang Min

    2014-01-01

    Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that plant homeodomain finger protein 20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In plant homeodomain finger protein 20-overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between plant homeodomain finger protein 20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that plant homeodomain finger protein 20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies plant homeodomain finger protein 20 as a novel regulator of NF-κB activation and suggests that elevated expression of plant homeodomain finger protein 20 may drive constitutive NF-κB activation in some cancers. PMID:23797602

  6. Transcription factor NF-κB (p50/p50, p65/p65) controls porcine ovarian cells functions.

    PubMed

    Pavlová, Silvia; Klucska, Karolina; Vašíček, Dušan; Kotwica, Ján; Sirotkin, Alexander V

    2011-10-01

    The aim of these in vitro studies was to examine the involvement of transcription factor NF-κB (p50/p50, p65/p65) and FSH in control of porcine ovarian granulosa cells functions and the possible role of dimers p50/p50, p65/p65 in mediating FSH actions on these cells. Monolayer of primary granulosa cells was transfected with plasmids encoding human p50 cDNA and p65 cDNA, and cultured with or without addition of FSH (0, 1, 10 or 100 ng/ml). The accumulation of proteins p50 and p65, as well as of proliferation markers (PCNA and MAPK/ERK1,2) and marker of apoptosis (Bax) in cells was detected by using SDS-PAGE-Western immunoblotting and immunocytochemistry. DNA fragmentation was evaluated by TUNEL assay. Release of hormones insulin-like growth factor I (IGF-I), progesterone (P(4)), oxytocin (OT), prostaglandins E(2) (PGE(2)) and F(2α) (PGF(2α)) was measured by using RIA. We observed, that p50/p50 promoted the accumulation of PCNA, MAPK/ERK1,2, the release of OT, PGF(2α); inhibited the occurrence of TdT-positive cells, the release of IGF-I and P(4), and did not influence the accumulation of Bax and the release of PGE(2). p65/p65 enhanced the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, PGE(2) and PGF(2α); decreased the percentage of cell containing TdT and did not affect the release of P(4). FSH stimulated the accumulation of PCNA, MAPK/ERK1,2 and Bax, the release of IGF-I, OT, P(4), PGE(2); but reduced the proportion of TdT-positive cells and the release of PGF(2α). These observations suggest (1) the involvement of NF-κB (p50/p50) in stimulation of proliferation, inhibition of apoptosis and in either stimulation (OT, PGE(2)) or inhibition (IGF-I, P(4), but not PGF(2)) of hormones release by porcine ovarian granulosa cells; (2) the involvement of NF-κB (p65/p65) in stimulation of proliferation and mitochondrial/Bax-related apoptosis, inhibition of nuclear/TdT-related apoptosis and in stimulation of ovarian hormones (IGF-I, OT, PGE(2), PGF

  7. The glutaredoxin/glutathione system modulates NF-kappaB activity by glutathionylation of p65 in cinnamaldehyde-treated endothelial cells.

    PubMed

    Liao, Being-Chyuan; Hsieh, Chia-Wen; Lin, Yuan-Chun; Wung, Being-Sun

    2010-07-01

    Reversible protein glutathionylation is an important posttranslational modification that provides protection against oxidation. In endothelial cells (ECs), cinnamaldehyde is an electrophilic compound that can increase the intracellular glutathione (GSH) levels or reactive oxygen species (ROS) production depending on the treatment duration. ECs treated with GSH and H(2)O(2) show increased sulfhydryl modifications of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), which are responsible for NF-kappaB inactivation, and also a block in TNF-alpha-induced p65 nuclear translocation and inter-cellular adhesion molecule-1 (ICAM-1) expression. In our current study, we find that cinnamaldehyde induces p65 glutathionylation and inhibits TNF-alpha-induced p65 nuclear translocation and ICAM-1 expression within 12 h of treatment. Our analyses also reveal that p65 glutathionylation is suppressed by a GSH synthesis inhibitor, buthionine sulfoximine (BSO), and we further observed that the inhibitory effects of p65 nuclear translocation and ICAM-1 expression are also suppressed by BSO. NF-E2-related factor-2 small interfering RNA (siRNA) molecules not only inhibit glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) induction and increases in GSH but also abolish cinnamaldehyde-induced p65 glutathionylation and its inhibitory effects. The gene expression and activity of glutaredoxin-1 (Grx-1), which catalyzes the formation of protein-glutathione mixed disulfides (protein-SSG), were also found to be increased after cinnamaldehyde treatment. A knock down of endogenous Grx-1 by siRNA or pretreatment with an inhibitor of Grx-1 activity, CdCl(2), abolishes p65-SSG formation. In addition, Grx-1 siRNA blocks the inhibition of p65 nuclear translocation and ICAM-1 expression, suggesting that this enzyme is involved in the cinnamaldehyde-mediated NF-kappaB inhibition. Our current results thus

  8. S-Nitrosylation of NF-κB p65 Inhibits TSH-Induced Na(+)/I(-) Symporter Expression.

    PubMed

    Nicola, Juan Pablo; Peyret, Victoria; Nazar, Magalí; Romero, Jorge Miguel; Lucero, Ariel Maximiliano; Montesinos, María del Mar; Bocco, José Luis; Pellizas, Claudia Gabriela; Masini-Repiso, Ana María

    2015-12-01

    Nitric oxide (NO) is a ubiquitous signaling molecule involved in a wide variety of cellular physiological processes. In thyroid cells, NO-synthase III-endogenously produced NO reduces TSH-stimulated thyroid-specific gene expression, suggesting a potential autocrine role of NO in modulating thyroid function. Further studies indicate that NO induces thyroid dedifferentiation, because NO donors repress TSH-stimulated iodide (I(-)) uptake. Here, we investigated the molecular mechanism underlying the NO-inhibited Na(+)/I(-) symporter (NIS)-mediated I(-) uptake in thyroid cells. We showed that NO donors reduce I(-) uptake in a concentration-dependent manner, which correlates with decreased NIS protein expression. NO-reduced I(-) uptake results from transcriptional repression of NIS gene rather than posttranslational modifications reducing functional NIS expression at the plasma membrane. We observed that NO donors repress TSH-induced NIS gene expression by reducing the transcriptional activity of the nuclear factor-κB subunit p65. NO-promoted p65 S-nitrosylation reduces p65-mediated transactivation of the NIS promoter in response to TSH stimulation. Overall, our data are consistent with the notion that NO plays a role as an inhibitory signal to counterbalance TSH-stimulated nuclear factor-κB activation, thus modulating thyroid hormone biosynthesis.

  9. Efficient nuclear export of p65-IkappaBalpha complexes requires 14-3-3 proteins.

    PubMed

    Aguilera, Cristina; Fernández-Majada, Vanessa; Inglés-Esteve, Julia; Rodilla, Verónica; Bigas, Anna; Espinosa, Lluís

    2006-09-01

    IkappaB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFkappaB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFkappaB signaling pathway by physically interacting with p65 and IkappaBalpha proteins. We identify two functional 14-3-3 binding domains in the p65 protein involving residues 38-44 and 278-283, and map the interaction region of IkappaBalpha in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IkappaBalpha results in a predominantly nuclear distribution of both proteins. TNFalpha treatment promotes recruitment of 14-3-3 and IkappaBalpha to NFkappaB-dependent promoters and enhances the binding of 14-3-3 to p65. Disrupting 14-3-3 activity by transfection with a dominant-negative 14-3-3 leads to the accumulation of nuclear p65-IkappaBalpha complexes and the constitutive association of p65 with the chromatin. In this situation, NFkappaB-dependent genes become unresponsive to TNFalpha stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IkappaBalpha-p65 complexes and are required for the appropriate regulation of NFkappaB signaling.

  10. BZLF1, an Epstein-Barr virus immediate-early protein, induces p65 nuclear translocation while inhibiting p65 transcriptional function

    SciTech Connect

    Morrison, Thomas E.; Kenney, Shannon C. . E-mail: shann@med.unc.edu

    2004-10-25

    We have previously demonstrated that the Epstein-Barr virus immediate-early BZLF1 protein interacts with, and is inhibited by, the NF-{kappa}B family member p65. However, the effects of BZLF1 on NF-{kappa}B activity have not been intensively studied. Here we show that BZLF1 inhibits p65-dependent gene expression. BZLF1 inhibited the ability of IL-1, as well as transfected p65, to activate the expression of two different NF-{kappa}B-responsive genes, ICAM-1 and I{kappa}B-{alpha}. BZLF1 also reduced the constitutive level of I{kappa}B-{alpha} protein in HeLa and A549 cells, and increased the amount of nuclear NF-{kappa}B to a similar extent as tumor necrosis factor-alpha (TNF-{alpha}) treatment. In spite of this BZLF1-associated increase in the nuclear form of NF-{kappa}B, BZLF1 did not induce binding of NF-{kappa}B to NF-{kappa}B responsive promoters (as determined by chromatin immunoprecipitation assay) in vivo, although TNF-{alpha} treatment induced NF-{kappa}B binding as expected. Overexpression of p65 dramatically inhibited the lytic replication cycle of EBV in 293-EBV cells, confirming that NF-{kappa}B also inhibits BZLF1 transcriptional function. Our results are consistent with a model in which BZLF1 inhibits the transcriptional function of p65, resulting in decreased transcription of I{kappa}B-{alpha}, decreased expression of I{kappa}B-{alpha} protein, and subsequent translocation of NF-{kappa}B to the nucleus. This nuclear translocation of NF-{kappa}B may promote viral latency by negatively regulating BZLF1 transcriptional activity. In situations where p65 activity is limiting in comparison to BZLF1, the ability of BZLF1 to inhibit p65 transcriptional function may protect the virus from the host immune system during the lytic form of infection.

  11. Evidence for the Critical Roles of NF-κB p65 and Specificity Proteins in the Apoptosis-Inducing Activity of Proteasome Inhibitors in Leukemia Cells

    PubMed Central

    Reuter, Simone; Gupta, Subash C.; Kannappan, Ramaswamy; Aggarwal, Bharat B.

    2012-01-01

    Although proteasome inhibitors, such as Bortezomib, have been approved for the treatment of multiple myeloma and mantle cell lymphoma, the mechanism by which they induce apoptosis is still incompletely understood. In the present study, we demonstrate that genetic deletion of the NF-κB p65 subunit abolished the ability of Bortezomib to induce apoptosis, indicating that p65 is needed for apoptosis. Although Bortezomib inhibited TNF–induced NF-κB activation through suppression of IκBα degradation, it also induced proteolytic degradation of constitutive NF-κB proteins, including p65, IκBα and p105. These effects were also observed with two other proteasome inhibitors, N-acetyl-leucylleucyl-norleucinal (ALLN) and MG132. The p65 is known to be linked with Specific proteins (Sp), and we found that proteasome inhibition also induced degradation of Sp-1, Sp-3, and Sp-4 proteins. Bortezomib induced apoptosis in cells expressing caspase-3 but not in cells that lack caspase-3, indicating the critical role for this enzyme in the apoptotic action of Bortezomib. Furthermore, inhibition of pan-caspases abolished Bortezomib-induced degradation of p65, p105 and Sp proteins, but not that of IκBα. Overall, our results demonstrate for the first time a critical role for the degradation of NF-κB and Sp proteins by caspases in the apoptosis-inducing activity of proteasome inhibitors, such as Bortezomib.

  12. Hepatic MiR-291b-3p Mediated Glucose Metabolism by Directly Targeting p65 to Upregulate PTEN Expression

    PubMed Central

    Guo, Jun; Dou, Lin; Meng, Xiangyu; Chen, Zhenzhen; Yang, Weili; Fang, Weiwei; Yang, Chunxiao; Huang, Xiuqing; Tang, Weiqing; Yang, Jichun; Li, Jian

    2017-01-01

    Several studies have suggested an important role of miR-291b-3p in the development of embryonic stem cells. In previous study, we found that the expression of miR-291b-3p was significantly upregulated in the liver of db/db mice. However, the role of miR-291b-3p in glucose metabolism and its underlying mechanisms remain unknown. In the present study, we demonstrated that miR-291b-3p was abundantly expressed in the liver. Of note, hepatic miR-291b-3p expression was upregulated in HFD-fed mice and induced by fasting in C57BL/6 J normal mice. Importantly, hepatic inhibition miR-291b-3p expression ameliorated hyperglycemia and insulin resistance in HFD-fed mice, whereas hepatic overexpression of miR-291b-3p led to hyperglycemia and insulin resistance in C57BL/6 J normal mice. Further study revealed that miR-291b-3p suppressed insulin-stimulated AKT/GSK signaling and increased the expression of gluconeogenic genes in hepatocytes. Moreover, we identified that p65, a subunit of nuclear factor-κB (NF-κB), is a target of miR-291b-3p by bioinformatics analysis and luciferase reporter assay. Silencing of p65 significantly augmented the expression of PTEN and impaired AKT activation. In conclusion, we found novel evidence suggesting that hepatic miR-291b-3p mediated glycogen synthesis and gluconeogenesis through targeting p65 to regulate PTEN expression. Our findings indicate the therapeutic potential of miR-291b-3p inhibitor in hyperglycemia and insulin resistance. PMID:28054586

  13. Suppression of RelA/p65 transactivation activity by a lignoid manassantin isolated from Saururus chinensis.

    PubMed

    Lee, Jeong-Hyung; Hwang, Bang Yeon; Kim, Kyung-Sook; Nam, Jeong Beom; Hong, Young Soo; Lee, Jung Joon

    2003-11-15

    In our search for NF-kappaB inhibitors from natural resources, we have previously identified two structurally related dilignans, manassantin A and B as specific inhibitors of NF-kappaB activation from Saururus chinensis. However, their molecular mechanism of action remains unclear. We here demonstrate that manassantins A and B are potent inhibitors of NF-kappaB activation by the suppression of transciptional activity of RelA/p65 subunit of NF-kappaB. These compounds significantly inhibited the induced expression of NF-kappaB reporter gene by LPS or TNF-alpha in a dose-dependent manner. However, these compounds did not prevent the DNA-binding activity of NF-kappaB assessed by electrophoretic mobility shift assay as well as the induced-degradation of IkappaB-alpha protein by LPS or TNF-alpha. Further analysis revealed that manassantins A and B dose-dependently suppressed not only the induced NF-kappaB activation by overexpression of RelA/p65, but also transactivation activity of RelA/p65. Furthermore, treatment of cells with these compounds prevented the TNF-alpha-induced expression of anti-apoptotic NF-kappaB target genes Bfl-1/A1, a prosurvival Bcl-2 homologue, and resulted in sensitizing HT-1080 cells to TNF-alpha-induced cell death. Similarly, these compounds also suppressed the LPS-induced inducible nitric oxide synthase expression and nitric oxide production. Taken together, manassantins A and B could be valuable candidate for the intervention of NF-kappaB-dependent pathological condition such as inflammation and cancer.

  14. Recombinant adeno-associated virus serotype 9 with p65 ribozyme protects H9c2 cells from oxidative stress through inhibiting NF-κB signaling pathway

    PubMed Central

    SUN, Zhan; MA, Yi-Tong; CHEN, Bang-Dang; LIU, Fen

    2014-01-01

    Background Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. It can trigger inflammatory cascades which are primarily mediated via nuclear factor-κB (NF-κB). The NF-κB transcription factor family includes several subunits (p50, p52, p65, c-Rel, and Rel B) that respond to myocardial ischemia. It has been proved that persistent myocyte NF-κB p65 activation in heart failure exacerbates cardiac remodeling. Mechods A recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein and anti-NF-κB p65 ribozyme (AAV9-R65-CMV-eGFP) was constructed. The cells were assessed by MTT assay, Annexin V–propidium iodide dual staining to study apoptosis. The expression of P65 and P50 were assessed by Western blot to investigate the underlying molecular mechanisms. Results After stimulation with H2O2 for 6 h, H9c2 cells viability decreased significantly, a large fraction of cells underwent apoptosis. We observed a rescue of H9c2 cells from H2O2-induced apoptosis in pretreatment with AAV9-R65-CMV-eGFP. Moreover, AAV9-R65-CMV-eGFP decreased H2O2-induced P65 expression. Conclusions AAV9-R65-CMV-eGFP protects H9c2 cells from oxidative stress induced apoptosis through down-regulation of P65 expression. These observations indicate that AAV9-R65-CMV-eGFP has the potential to exert cardioprotective effects against oxidative stress, which might be of great importance to clinical efficacy for cardiovascular disease. PMID:25593580

  15. Epidermal p65/NF-κB signalling is essential for skin carcinogenesis.

    PubMed

    Kim, Chun; Pasparakis, Manolis

    2014-07-01

    The nuclear factor kappa B (NF-κB) signalling pathway exhibits both tumour-promoting and tumour-suppressing functions in different tissues and models of carcinogenesis. In particular in epidermal keratinocytes, NF-κB signalling was reported to exert primarily growth inhibitory and tumour-suppressing functions. Here, we show that mice with keratinocyte-restricted p65/RelA deficiency were resistant to 7, 12-dimethylbenz(a)anthracene (DMBA)-/12-O-tetra decanoylphorbol-13 acetate (TPA)-induced skin carcinogenesis. p65 deficiency sensitized epidermal keratinocytes to DNA damage-induced death in vivo and in vitro, suggesting that inhibition of p65-dependent prosurvival functions prevented tumour initiation by facilitating the elimination of cells carrying damaged DNA. In addition, lack of p65 strongly inhibited TPA-induced epidermal hyperplasia and skin inflammation by suppressing the expression of proinflammatory cytokines and chemokines by epidermal keratinocytes. Therefore, p65-dependent NF-κB signalling in keratinocytes promotes DMBA-/TPA-induced skin carcinogenesis by protecting keratinocytes from DNA damage-induced death and facilitating the establishment of a tumour-nurturing proinflammatory microenvironment.

  16. NF-κB (p65) negatively regulates myocardin-induced cardiomyocyte hypertrophy through multiple mechanisms.

    PubMed

    Liao, Xing-Hua; Wang, Nan; Zhao, Dong-Wei; Zheng, De-Liang; Zheng, Li; Xing, Wen-Jing; Zhou, Hao; Cao, Dong-Sun; Zhang, Tong-Cun

    2014-12-01

    Myocardin is well known to play a key role in the development of cardiomyocyte hypertrophy. But the exact molecular mechanism regulating myocardin stability and transactivity to affect cardiomyocyte hypertrophy has not been studied clearly. We now report that NF-κB (p65) can inhibit myocardin-induced cardiomyocyte hypertrophy. Then we explore the molecular mechanism of this response. First, we show that p65 can functionally repress myocardin transcriptional activity and also reduce the protein expression of myocardin. Second, the function of myocardin can be regulated by epigenetic modifications. Myocardin sumoylation is known to transactivate cardiac genes, but whether p65 can inhibit SUMO modification of myocardin is still not clear. Our data show that p65 weakens myocardin transcriptional activity through attenuating SUMO modification of myocardin by SUMO1/PIAS1, thereby impairing myocardin-mediated cardiomyocyte hypertrophy. Furthermore, the expression of myocardin can be regulated by several microRNAs, which play important roles in the development and function of the heart and muscle. We next investigated potential role of miR-1 in cardiac hypotrophy. Our results show that p65 can upregulate the level of miR-1 and miR-1 can decrease protein expression of myocardin in cardiac myocytes. Notably, miR-1 expression is also controlled by myocardin, leading to a feedback loop. These data thus provide important and novel insights into the function that p65 inhibits myocardin-mediated cardiomyocyte hypertrophy by downregulating the expression and SUMO modification of myocardin and enhancing the expression of miR-1.

  17. TDP-43 Inhibits NF-κB Activity by Blocking p65 Nuclear Translocation

    PubMed Central

    Zhu, Jingyan; Cynader, Max S.; Jia, William

    2015-01-01

    TDP-43 (TAR DNA binding protein 43) is a heterogeneous nuclear ribonucleoprotein (hnRNP) that has been found to play an important role in neurodegenerative diseases. TDP-43’s involvement in nuclear factor-kappaB pathways has been reported in both neurons and microglial cells. The NF-κB pathway targets hundreds of genes, many of which are involved in inflammation, immunity and cancer. p50/p65 (p50/RelA) heterodimers, as the major Rel complex in the NF-κB family, are induced by diverse external physiological stimuli and modulate transcriptional activity in almost all cell types. Both p65 and TDP-43 translocation occur through the classic nuclear transportation system. In this study, we report that TDP-43 overexpression prevents TNF-α induced p65 nuclear translocation in a dose dependent manner, and that this further inhibits p65 transactivation activity. The inhibition by TDP-43 does not occur through preventing IκB degradation but probably by competing for the nuclear transporter-importin α3 (KPNA4). This competition is dependent on the presence of the nuclear localization signal (NLS) in TDP-43. Silencing TDP-43 using a specific siRNA also increased p65 nuclear localization upon TNF-α stimulation, suggesting that endogenous TDP-43 may be a default suppressor of the NF-κB pathway. Our results indicate that TDP-43 may play an important role in regulating the levels of NF-κB activity by controlling the nuclear translocation of p65. PMID:26571498

  18. INHALED PARTICULATE MATTER CAUSES EXPRESSION OF NUCLEAR FACTOR (NF)-KAPPAB-RELATED GENES AND OXIDANT-DEPENDENT NF-KAPPAB ACTIVATION IN VITRO. (R826244)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  19. INHALED PARTICULATE MATTER CAUSES EXPRESSION OF NF-KAPPAB-RELATED GENES AND OXIDANT-DEPENDENT NF-KAPPAB ACTIVATION IN VITRO. (R827351C004)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  20. Effect of Huperzine A on Aβ-induced p65 of astrocyte in vitro.

    PubMed

    Xie, Lushuang; Jiang, Cen; Wang, Zhang; Yi, Xiaohong; Gong, Yuanyuan; Chen, Yunhui; Fu, Yan

    2016-12-01

    Alzheimer's disease (AD) is the most common cause of dementia. Its pathology often accompanies inflammatory action, and astrocytes play important roles in such procedure. Rela(p65) is one of significant message factors in NF-κB pathway which has been reported high expression in astrocyte treated by Aβ. HupA, an alkaloid isolated from Chinese herb Huperzia serrata, has been widely used to treat AD and observations reflected that it improves memory and cognitive capacity of AD patients. To reveal its molecular mechanisms on p65, we cultured astrocytes, built Aβ-induced AD model, treated astrocytes with HupA at different concentrations, assayed cell viability with MTT, and detected p65 expression by immunohistochemistry and PCR. Our results revealed that treatment with 10 μM Aβ1-42 for 24 h induced a significant increase of NF-κB in astrocytes; HupA significantly down-regulated p65 expression induced by Aβ in astrocytes. This study infers that HupA can regulate NF-κB pathway to treat AD.

  1. Proepithelin stimulates growth plate chondrogenesis via nuclear factor-kappaB-p65-dependent mechanisms.

    PubMed

    Wu, Shufang; Zang, Weijin; Li, Xu; Sun, Hongzhi

    2011-07-08

    Proepithelin, a previously unrecognized growth factor in cartilage, has recently emerged as an important regulator for cartilage formation and function. In the present study, we provide several lines of evidences in proepithelin-mediated induction of cell proliferation, differentiation, and apoptosis in the metatarsal growth plate. Proepithelin-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by pyrrolidine dithiocarbamate, a known NF-κB inhibitor. In rat growth plate chondrocytes, proepithelin induced NF-κB-p65 nuclear translocation, and nuclear NF-κB-p65 initiated its target gene cyclin D1 to regulate chondrocyte functions. The inhibition of NF-κB-p65 expression and activity (by p65 short interfering RNA (siRNA) and pyrrolidine dithiocarbamate, respectively) in chondrocytes reversed the proepithelin-mediated induction of cell proliferation and differentiation and the proepithelin-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of proepithelin on NF-κB activation. Finally, using siRNA and antisense strategies, we demonstrated that endogenously produced proepithelin by chondrocytes is important for chondrocyte growth in serum-deprived conditions. These results support the hypothesis that the induction of NF-κB activity of in growth plate chondrocytes is critical in proepithelin-mediated growth plate chondrogenesis and longitudinal bone growth.

  2. Inflammatory stimuli induce inhibitory S-nitrosylation of the deacetylase SIRT1 to increase acetylation and activation of p53 and p65

    PubMed Central

    Shinozaki, Shohei; Chang, Kyungho; Sakai, Michihiro; Shimizu, Nobuyuki; Yamada, Marina; Tanaka, Tomokazu; Nakazawa, Harumasa; Ichinose, Fumito; Yamada, Yoshitsugu; Ishigami, Akihito; Ito, Hideki; Ouchi, Yasuyoshi; Starr, Marlene E.; Saito, Hiroshi; Shimokado, Kentaro; Stamler, Jonathan S.; Kaneki, Masao

    2015-01-01

    Inflammation increases the abundance of inducible nitric oxide synthase (iNOS), leading to enhanced production of nitric oxide (NO), which can modify proteins by S-nitrosylation. Enhanced NO production increases the activities of the transcription factors p53 and nuclear factor κB (NF-κB) in several models of disease-associated inflammation. S-Nitrosylation inhibits the activity of the protein deacetylase SIRT1. SIRT1 limits apoptosis and inflammation by deacetylating p53 and p65 (also known as RelA), a subunit of NF-κB. We showed in multiple cultured mammalian cell lines that NO donors or inflammatory stimuli induced S-nitrosylation of SIRT1 within CXXC motifs, which inhibited SIRT1 by disrupting its ability to bind zinc. Inhibition of SIRT1 reduced deacetylation and promoted activation of p53 and p65, leading to apoptosis and increased expression of proinflammatory genes. In rodent models of systemic inflammation, Parkinson’s disease, or aging-related muscular atrophy, S-nitrosylation of SIRT1 correlated with increased acetylation of p53 and p65 and activation of p53 and NF-κB target genes, suggesting that S-nitrosylation of SIRT1 may represent a proinflammatory switch common to many diseases and aging. PMID:25389371

  3. Poxvirus Protein MC132 from Molluscum Contagiosum Virus Inhibits NF-κB Activation by Targeting p65 for Degradation

    PubMed Central

    Haas, Darya A.; Farrell, Paul J.; Pichlmair, Andreas

    2015-01-01

    ABSTRACT Molluscum contagiosum virus (MCV) is unique in being the only known extant, human-adapted poxvirus, yet to date, it is very poorly characterized in terms of host-pathogen interactions. MCV causes persistent skin lesions filled with live virus, but these are generally immunologically silent, suggesting the presence of potent inhibitors of human antiviral immunity and inflammation. Fewer than five MCV immunomodulatory genes have been characterized in detail, but it is likely that many more remain to be discovered given the density of such sequences in all well-characterized poxviruses. Following virus infection, NF-κB activation occurs in response to both pattern recognition receptor (PRR) signaling and cellular activation by virus-elicited proinflammatory cytokines, such as tumor necrosis factor (TNF). As such, NF-κB activation is required for virus detection, antiviral signaling, inflammation, and clearance of viral infection. Hence, we screened a library of MCV genes for effects on TNF-stimulated NF-κB activation. This revealed MC132, a unique protein with no orthologs in other poxviral genomes, as a novel inhibitor of NF-κB. Interestingly, MC132 also inhibited PRR- and virus-activated NF-κB, since MC132 interacted with the NF-κB subunit p65 and caused p65 degradation. Unbiased affinity purification to identify host targets of MC132 revealed that MC132 acted by targeting NF-κB p65 for ubiquitin-dependent proteasomal degradation by recruiting p65 to a host Cullin-5/Elongin B/Elongin C complex. These data reveal a novel mechanism for poxviral inhibition of human innate immunity and further clarify how the human-adapted poxvirus MCV can so effectively evade antiviral immunity to persist in skin lesions. IMPORTANCE How human cells detect and respond to viruses is incompletely understood, but great leaps in our understanding have been made by studying both the early innate immune response and the ways that viruses evade it. Poxviruses adapt to specific

  4. Small ubiquitin-related modifier 1 is involved in hepatocellular carcinoma progression via mediating p65 nuclear translocation

    PubMed Central

    Liu, Jun; Tao, Xiaofang; Zhang, Jin; Wang, Peng; Sha, Manqi; Ma, Yong; Geng, Xiaoping; Feng, Lijie; Shen, Yujun; Yu, Yifan; Wang, Siying; Fang, Shengyun; Shen, Yuxian

    2016-01-01

    Small ubiquitin-related modifier (SUMO) proteins participate in a post-translational modification called SUMOylation and regulate a variety of intracellular processes, such as targeting proteins for nuclear import. The nuclear transport of p65 results in the activation of NF-κB, and p65 contains several SUMO interacting motifs (SIMs). However, the relationship between p65 and SUMO1 in hepatocellular carcinoma (HCC) remains unclear. In this study, we demonstrated the potential roles of SUMO1 in HCC via the regulation of p65 subcellular localization. We found that either SUMO1- or p65-positive immunoreactivity was remarkably increased in the nuclei of tumor tissues in HCC patients compared with non-tumor tissues, and further analysis suggested a correlation between SUMO1- and nuclear p65-positive immunoreactivities (R = 0.851, P = 0.002). We also verified the interaction between p65 and SUMO1 in HCC by co-immunoprecipitation. TNF-α and hypoxia increased SUMO1 protein levels and enhanced SUMO1-modified p65 SUMOylation. Moreover, the knockdown of SUMO1 decreased p65 nuclear translocation and inhibited NF-κB transcriptional activity. Further the results of this study revealed that the knockdown of SUMO1 suppressed the proliferation and migration of hepatoma cells. These results suggest that SUMO1 contributes to HCC progression by promoting p65 nuclear translocation and regulating NF-κB activity. PMID:26993772

  5. Nuclear localisation of p65 in sputum macrophages but not in sputum neutrophils during COPD exacerbations

    PubMed Central

    Caramori, G; Romagnoli, M; Casolari, P; Bellettato, C; Casoni, G; Boschetto, P; Fan, C; Barnes, P; Adcock, I; Ciaccia, A; Fabbri, L; Papi, A

    2003-01-01

    Methods: Total and nuclear p65 immunoreactivity was measured by immunocytochemistry in the sputum cells of 11 smokers with moderate COPD during an exacerbation and after 6–8 weeks of clinical stability. Results: Total sputum cell count was significantly increased during exacerbations from a median (IQR) of 880 (510–1865) to 1914.5 (1065–3205) x 103/ml (p<0.05). The main inflammatory cells in the sputum were neutrophils (83.2 (75.4–92.3)%) and macrophages (14.7 (2.6–21.6)%) and their relative proportion did not change during exacerbations. Nuclear staining for p65 was absent in sputum neutrophils, both during exacerbations and in the stable phase. In contrast, the percentage of macrophages expressing nuclear p65 increased significantly during exacerbations from a median (IQR) of 16 (7–24)% to 41.4 (6–69)% (p<0.05). Conclusions: NF-κB appears to be activated in sputum macrophages but not in sputum neutrophils during exacerbations of COPD PMID:12668802

  6. HSF1 and NF-κB p65 participate in the process of exercise preconditioning attenuating pressure overload-induced pathological cardiac hypertrophy.

    PubMed

    Xu, Tongyi; Zhang, Ben; Yang, Fan; Cai, Chengliang; Wang, Guokun; Han, Qingqi; Zou, Liangjian

    2015-05-08

    Pathological cardiac hypertrophy, often accompanied by hypertension, aortic stenosis and valvular defects, is typically associated with myocyte remodeling and cardiac dysfunction. Exercise preconditioning (EP) has been proven to enhance the tolerance of the myocardium to cardiac ischemia-reperfusion injury. However, the effects of EP in pathological cardiac hypertrophy are rarely reported. 10-wk-old male Sprague-Dawley rats (n = 80) were randomly divided into four groups: sham, TAC, EP + sham and EP + TAC. Two EP groups were subjected to 4 weeks of treadmill training, and the EP + TAC and TAC groups were followed by TAC operations. The sham and EP + sham groups underwent the same operation without aortic constriction. Eight weeks after the surgery, we evaluated the effects of EP by echocardiography, morphology, and histology and observed the expressions of the associated proteins. Compared with the respective control groups, hypertrophy-related indicators were significantly increased in the TAC and EP + TAC groups (p < 0.05). However, between the TAC and EP + TAC groups, all of these changes were effectively inhibited by EP treatment (p < 0.05). Furthermore, EP treatment upregulated the expression of HSF1 and HSP70, increased the HSF1 levels in the nuclear fraction, inhibited the expression of the NF-κB p65 subunit, decreased the NF-κB p65 subunit levels in the nuclear fraction, and reduced the IL2 levels in the myocardia of rats. EP could effectively reduce the cardiac hypertrophic responses induced by TAC and may play a protective role by upregulating the expressions of HSF1 and HSP70, activating HSF1 and then inhibiting the expression of NF-κB p65 and nuclear translocation.

  7. 1,5-Anhydro-D-fructose attenuates lipopolysaccharide-induced cytokine release via suppression of NF-{kappa}B p65 phosphorylation

    SciTech Connect

    Meng Xiaojie; Kawahara, Ko-ichi; Nawa, Yuko; Miura, Naoki; Shrestha, Binita; Tancharoen, Salunya; Sameshima, Hisayo; Hashiguchi, Teruto; Maruyama, Ikuro

    2009-03-06

    Lipopolysaccharide (LPS) stimulates macrophages by activating NF-{kappa}B, which contributes to the release of tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-6. 1,5-anhydro-D-fructose (1,5-AF), a monosaccharide formed from starch and glycogen, exhibits anti-oxidant activity and enhances insulin secretion. This study examined the effects of 1,5-AF on LPS-induced inflammatory reactions and elucidated its molecular mechanisms. Before LPS challenge, mice were pretreated with 1,5-AF (38.5 mg/kg). We found that 1,5-AF pretreatment attenuated cytokine release into the serum, including TNF-{alpha}, IL-6 and macrophage chemoattractant protein (MCP)-1. Furthermore, pretreatment with 1,5-AF (500 {mu}g/ml) attenuated cytokine release, and 1,5-AF directly inhibited the nuclear translocalization of the NF-{kappa}B p65 subunit in LPS-stimulated murine macrophage-like RAW264.7 cells. This inhibition was responsible for decreased LPS-induced phosphorylation on Ser536 of the NF-{kappa}B p65 subunit, which is a posttranslational modification involved in the non-canonical pathway. Collectively, these findings indicate that the anti-inflammatory activity of 1,5-AF occurs via inactivation of NF-{kappa}B.

  8. Dynamics of NF kappa B and Ikappa Balpha studied with green fluorescent protein (GFP) fusion proteins. Investigation of GFP-p65 binding to DNa by fluorescence resonance energy transfer.

    PubMed

    Schmid, J A; Birbach, A; Hofer-Warbinek, R; Pengg, M; Burner, U; Furtmüller, P G; Binder, B R; de Martin, R

    2000-06-02

    We investigated the dynamics of nuclear transcription factor kappaB (NF-kappaB) by using fusion proteins of the p65 subunit with mutants of green fluorescent protein (GFP). GFP-NF-kappaB chimeras were functional both in vitro and in vivo, as demonstrated by electrophoretic mobility shift assays and reporter gene studies. GFP-p65 was regulated by IkappaBalpha similar to wild type p65 and associated with its inhibitor even if both proteins were linked to a GFP protein. This finding was also verified by fluorescence resonance energy transfer (FRET) microscopy and studies showing mutual regulation of the intracellular localization of both GFP chimerae. Incubation of GFP-p65 with fluorescently labeled NF-kappaB-binding oligonucleotides also resulted in FRET. This effect was DNA sequence-specific and exhibited saturation characteristics. Application of stopped-flow fluorometry to measure the kinetics of FRET between GFP-p65 and oligonucleotides revealed a fast increase of acceptor fluorescence with a plateau after about 10 ms. The observed initial binding rate showed a temperature-dependent linear correlation with the oligonucleotide concentration. The association constant calculated according to pre-steady state kinetics was 3 x 10(6) m(-1), although equilibrium binding studies implied significantly higher values. This observation suggests that the binding process involves a rapid association with a rather high off-rate followed by a conformational change resulting in an increase of the association constant.

  9. HSF1 and NF-κB p65 participate in the process of exercise preconditioning attenuating pressure overload-induced pathological cardiac hypertrophy

    SciTech Connect

    Xu, Tongyi; Zhang, Ben; Yang, Fan; Cai, Chengliang; Wang, Guokun; Han, Qingqi; Zou, Liangjian

    2015-05-08

    Pathological cardiac hypertrophy, often accompanied by hypertension, aortic stenosis and valvular defects, is typically associated with myocyte remodeling and cardiac dysfunction. Exercise preconditioning (EP) has been proven to enhance the tolerance of the myocardium to cardiac ischemia-reperfusion injury. However, the effects of EP in pathological cardiac hypertrophy are rarely reported. 10-wk-old male Sprague–Dawley rats (n = 80) were randomly divided into four groups: sham, TAC, EP + sham and EP + TAC. Two EP groups were subjected to 4 weeks of treadmill training, and the EP + TAC and TAC groups were followed by TAC operations. The sham and EP + sham groups underwent the same operation without aortic constriction. Eight weeks after the surgery, we evaluated the effects of EP by echocardiography, morphology, and histology and observed the expressions of the associated proteins. Compared with the respective control groups, hypertrophy-related indicators were significantly increased in the TAC and EP + TAC groups (p < 0.05). However, between the TAC and EP + TAC groups, all of these changes were effectively inhibited by EP treatment (p < 0.05). Furthermore, EP treatment upregulated the expression of HSF1 and HSP70, increased the HSF1 levels in the nuclear fraction, inhibited the expression of the NF-κB p65 subunit, decreased the NF-κB p65 subunit levels in the nuclear fraction, and reduced the IL2 levels in the myocardia of rats. EP could effectively reduce the cardiac hypertrophic responses induced by TAC and may play a protective role by upregulating the expressions of HSF1 and HSP70, activating HSF1 and then inhibiting the expression of NF-κB p65 and nuclear translocation. - Highlights: • EP could effectively reduce the cardiac hypertrophic responses induced by TAC. • EP may play a protective role by upregulating the expressions of HSF1 and HSP70 and then activating HSF1. • EP may play a protective role by inhibiting the expression

  10. Function of phosphorylation of NF-kB p65 ser536 in prostate cancer oncogenesis

    PubMed Central

    Creighton, Chad J.; Zhang, Yiqun; Xin, Li; Ittmann, Michael; Wang, Jianghua

    2015-01-01

    Majority of prostate cancer (PCa) patients carry TMPRSS2/ERG (T/E) fusion genes and there has been tremendous interest in understanding how the T/E fusion may promote progression of PCa. We showed that T/E fusion can activate NF-kB pathway by increasing phosphorylation of NF-kB p65 Ser536 (p536), but the function of p536 has never been studied in PCa. We report here that active p536 can significantly increase cell motility and transform PNT1a cells (an immortalized normal cell line), suggesting p536 plays a critical role in promoting PCa tumorigenesis. We have discovered a set of p536 regulated genes, among which we validated the regulation of CCL2 by p536. Based on all evidence, we favor that T/E fusion, NF-kB p536 and CCL2 form a signaling chain. Finally, PNT1a cells (not tumorigenic) can form tumors in SCID mice when overexpressing of either wild type or active p65 in the presence of activated AKT, demonstrating synergistic activities of NF-kB and AKT signals in promoting PCa tumorigenesis. These findings indicate that combination therapies targeting T/E fusion, NF-kB, CCL2 and/or AKT pathways may have efficacy in T/E fusion gene expressing PCa. If successful, such targeted therapy will benefit more than half of PCa patients who carry T/E fusions. PMID:25749044

  11. Nf-Kappab as a Critical Biological Link Between Psychological Stress and Breast Cancer

    DTIC Science & Technology

    2007-11-01

    reporter mice. Mice were restrained for 30 minutes, treated with 1mg/kg norepinephrine via IP injection or pretreated with 1mg/kg prazosin 45...can be inhibited by concomitant treatment with Prazosin , an adrenergic antagonist. Having confirmed that it was likely that NF-kB activity was

  12. C/EBPβ knockdown protects cardiomyocytes from hypertrophy via inhibition of p65-NFκB.

    PubMed

    Zou, Jian; Li, Hong; Chen, Xi; Zeng, Siyu; Ye, Jiantao; Zhou, Changhua; Liu, Min; Zhang, Luankun; Yu, Na; Gan, Xiaohong; Zhou, Houfeng; Xian, Zhiwei; Chen, Shaorui; Liu, Peiqing

    2014-06-05

    C/EBPβ, a member of the bHLH gene family of DNA-binding transcription factors, has been indicated as a central signal in physiologic hypertrophy. However, the role of C/EBPβ in pathological cardiac hypertrophy remains to be elucidated. In this study, we revealed that C/EBPβ is involved in cardiac hypertrophy, the expression of C/EBPβ were significantly increased in response to hypertrophic stimulation in vitro and in vivo. C/EBPβ knockdown inhibited PE-induced cardiac hypertrophy, and diminished the nuclear translocation and DNA binding activity of p65-NFκB. These results suggested that C/EBPβ knockdown protected cardiomyocytes from hypertrophy, which may be attributed to inhibition of NFκB-dependent transcriptional activity. These findings shed new light on the understanding of C/EBPβ-related cardiomyopathy, and suggest the potential application of C/EBPβ inhibitors in cardiac hypertrophy.

  13. Improvement of a p(65)+Be neutron beam for therapy at Cyclone, Louvain-la-Neuve.

    PubMed

    Vynckier, S; Pihet, P; Flémal, J M; Meulders, J P; Wambersie, A

    1983-06-01

    The variable energy cyclotron of the Catholic University of Louvain is used to produce intense neutron beams for neutron therapy purposes. As a first step, neutrons were produced by bombarding a Be target with 50 MeV deuterons; at present they are produced by 65 MeV protons. This paper describes the improvements to the target system. A new (17 mm) Be target together with the old (10 mm) Be target are inserted in a movable support which allows the production of neutrons either by 65 MeV protons or by 50 MeV deuterons. Both targets can be removed for proton beam therapy. The dosimetric characteristics of the p(65)+Be and d(50)+Be neutron beams are compared: dose rate, gamma-contribution, depth dose and room activation.

  14. PAFR activation of NF-κB p65 or p105 precursor dictates pro- and anti-inflammatory responses during TLR activation in murine macrophages

    PubMed Central

    Ishizuka, Edson K.; Filgueiras, Luciano Ribeiro; Rios, Francisco J.; Serezani, Carlos H.; Jancar, Sonia

    2016-01-01

    Platelet-activating factor receptor (PAFR) is a G protein-coupled receptor (GPCR) implicated in many diseases. Toll-like receptors (TLRs) play a critical role in shaping innate and adaptive immune responses. In this study, we investigated whether PAFR signaling changes the macrophages responsiveness to agonists of TLR2 (Pam3Cys), TLR4 (LPS), and TLR3 agonist Poly(I:C). Exogenous PAF inhibited the production of pro-inflammatory cytokines (IL-12p40, IL-6, and TNF-α) and increased anti-inflammatory IL-10 in macrophages challenged with Pam3Cys and LPS, but not with Poly (I:C). PAF did not affect mRNA expression of MyD88, suggesting that PAF acts downstream the adaptor. PAF inhibited LPS-induced phosphorylation of NF-κB p65 and increased NF-κB p105 phosphorylation, which is processed in the proteasome to generate p50 subunit. The PAF potentiation of IL-10 production was dependent on proteasome processing but independent of NF-κB transactivation domain. Inhibition of p50 abolished the PAF-induced IL-10 production. These findings indicate that the impaired transcriptional activity of the p65 subunit and the enhanced p105 phosphorylation induced by PAF are responsible for down regulation of pro-inflammatory cytokines and up regulation of IL-10, respectively, in LPS-challenged macrophages. Together, our data unveil a heretofore unrecognized role for PAFR in modulating activation of NF-κB in macrophages. PMID:27554194

  15. Interaction of Ash/Grb-2 via its SH3 domains with neuron-specific p150 and p65.

    PubMed Central

    Miura, K; Miki, H; Shimazaki, K; Kawai, N; Takenawa, T

    1996-01-01

    We found that 180 kDa, 150 kDa (p150), 110 kDa, 100 kDa and 65 kDa (p65) proteins comprise the major Ash/Grb-2-binding proteins in bovine brain. Among these proteins, 180 kDa and 100 kDa proteins have already been identified as Sos and dynamin respectively. Here, p150 and p65 were affinity-purified with glutathione S-transferase-Ash fusion protein and their partial amino acid sequences were determined. Analysis showed p150 and p65 to be new proteins. These two proteins bind to both the N-terminal SH3 domain and the C-terminal SH3 domain of Ash. It was found that p150 and p65 are expressed predominantly in brain, although Ash is widely distributed in all tissues examined by Western blots. Immunohistochemical staining of rat brain showed p150 and p65 to be localized in a variety of neurons in the cerebellum and hippocampus, with p65 being especially concentrated in the nerve terminal. When the Ash-binding-motif peptide of the epidermal growth factor receptor was used to detect complexes formed with Ash in vivo, 180 kDa, 150 kDa, 110 kDa, 100 kDa and 65 kDa proteins were also bound; this shows that these proteins form complexes with Ash in brain. In addition, p150 and p65 co-immunoprecipitated with Ash. All these results suggest that Ash may function as a regulator of synaptic vesicle transport through dynamin, p150 and p65. PMID:8687411

  16. In vitro proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

    PubMed

    Maxwell, S; Arlinghaus, R B

    1981-09-01

    Moloney murine leukemia virus, disrupted in concentrations of 0.1 to 0.5% Nonidet P-40, catalyzed the cleavage of p65, the gag gene polyprotein of the Gazdar strain of murine sarcoma virus, into polypeptides with sizes and antigenic determinants of murine leukemia virus-specified p30, p15, pp12, and p10. Cleavage performed in the presence of 0.15% Nonidet P-40 in water yielded polypeptides of approximately 40,000 (P40) and 25,000 (P25) Mr. In vitro cleavage performed in a buffered solution containing dithiothreitol in addition to 0.1% Nonidet P-40 allowed the efficient processing of P40 to p30 and a band migrating with p10. Immunoprecipitation with monospecific sera indicated that P40 contained p30 and p10, whereas P25 contained p15 and pp12 determinants. P40 and P25 are similar in size and antigenic properties to Pr40gag and Pr25gag observed in infected cells (Naso et al, J. Virol. 32:187-198, 1979).

  17. Baicalin attenuates focal cerebral ischemic reperfusion injury through inhibition of nuclear factor {kappa}B p65 activation

    SciTech Connect

    Xue, Xia; Qu, Xian-Jun; Yang, Ying; Sheng, Xie-Huang; Cheng, Fang; Jiang, E-Nang; Wang, Jian-hua; Bu, Wen; Liu, Zhao-Ping

    2010-12-17

    Research highlights: {yields} Permanent NF-{kappa}B p65 activation contributes to the infarction after ischemia-reperfusion injury in rats. {yields} Baicalin can markedly inhibit the nuclear NF-{kappa}B p65 expression and m RNA levels after ischemia-reperfusion injury in rats. {yields} Baicalin decreased the cerebral infarction area via inhibiting the activation of nuclear NF-{kappa}B p65. -- Abstract: Baicalin is a flavonoid compound purified from plant Scutellaria baicalensis Georgi. We aimed to evaluate the neuroprotective effects of baicalin against cerebral ischemic reperfusion injury. Male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 24 h. Baicalin at doses of 50, 100 and 200 mg/kg was intravenously injected after ischemia onset. Twenty-four hours after reperfusion, the neurological deficit was scored and infarct volume was measured. Hematoxylin and eosin (HE) staining was performed to analyze the histopathological changes of cortex and hippocampus neurons. We examined the levels of NF-{kappa}B p65 in ischemic cortexes by Western blot analysis and RT-PCR assay. The results showed that the neurological deficit scores were significantly decreased from 2.0 {+-} 0.7 to 1.2 {+-} 0.4 and the volume of infarction was reduced by 25% after baicalin injection. Histopathological examination showed that the increase of neurons with pycnotic shape and condensed nuclear in cortex and hippocampus were not observed in baicalin treated animals. Further examination showed that NF-{kappa}B p65 in cortex was increased after ischemia reperfusion injury, indicating the molecular mechanism of ischemia reperfusion injury. The level of NF-{kappa}B p65 was decreased by 73% after baicalin treatment. These results suggest that baicalin might be useful as a potential neuroprotective agent in stroke therapy. The neuroprotective effects of baicalin may relate to inhibition of NF-{kappa}B p65.

  18. Defining critical roles for NF-κB p65 and type I interferon in innate immunity to rhinovirus

    PubMed Central

    Bartlett, Nathan W; Slater, Louise; Glanville, Nicholas; Haas, Jennifer J; Caramori, Gaetano; Casolari, Paolo; Clarke, Deborah L; Message, Simon D; Aniscenko, Julia; Kebadze, Tatiana; Zhu, Jie; Mallia, Patrick; Mizgerd, Joseph P; Belvisi, Maria; Papi, Alberto; Kotenko, Sergei V; Johnston, Sebastian L; Edwards, Michael R

    2012-01-01

    The importance of NF-κB activation and deficient anti-viral interferon induction in the pathogenesis of rhinovirus-induced asthma exacerbations is poorly understood. We provide the first in vivo evidence in man and mouse that rhinovirus infection enhanced bronchial epithelial cell NF-κB p65 nuclear expression, NF-κB p65 DNA binding in lung tissue and NF-κB-regulated airway inflammation. In vitro inhibition of NF-κB reduced rhinovirus-induced pro-inflammatory cytokines but did not affect type I/III interferon induction. Rhinovirus-infected p65-deficient mice exhibited reduced neutrophilic inflammation, yet interferon induction, antiviral responses and virus loads were unaffected, indicating that NF-κB p65 is required for pro-inflammatory responses, but redundant in interferon induction by rhinoviruses in vivo. Conversely, IFNAR1−/− mice exhibited enhanced neutrophilic inflammation with impaired antiviral immunity and increased rhinovirus replication, demonstrating that interferon signalling was critical to antiviral immunity. We thus provide new mechanistic insights into rhinovirus infection and demonstrate the therapeutic potential of targeting NF-κB p65 (to suppress inflammation but preserve anti-viral immunity) and type I IFN signalling (to enhance deficient anti-viral immunity) to treat rhinovirus-induced exacerbations of airway diseases. PMID:23165884

  19. Defining critical roles for NF-κB p65 and type I interferon in innate immunity to rhinovirus.

    PubMed

    Bartlett, Nathan W; Slater, Louise; Glanville, Nicholas; Haas, Jennifer J; Caramori, Gaetano; Casolari, Paolo; Clarke, Deborah L; Message, Simon D; Aniscenko, Julia; Kebadze, Tatiana; Zhu, Jie; Mallia, Patrick; Mizgerd, Joseph P; Belvisi, Maria; Papi, Alberto; Kotenko, Sergei V; Johnston, Sebastian L; Edwards, Michael R

    2012-12-01

    The importance of NF-κB activation and deficient anti-viral interferon induction in the pathogenesis of rhinovirus-induced asthma exacerbations is poorly understood. We provide the first in vivo evidence in man and mouse that rhinovirus infection enhanced bronchial epithelial cell NF-κB p65 nuclear expression, NF-κB p65 DNA binding in lung tissue and NF-κB-regulated airway inflammation. In vitro inhibition of NF-κB reduced rhinovirus-induced pro-inflammatory cytokines but did not affect type I/III interferon induction. Rhinovirus-infected p65-deficient mice exhibited reduced neutrophilic inflammation, yet interferon induction, antiviral responses and virus loads were unaffected, indicating that NF-κB p65 is required for pro-inflammatory responses, but redundant in interferon induction by rhinoviruses in vivo. Conversely, IFNAR1(-/-) mice exhibited enhanced neutrophilic inflammation with impaired antiviral immunity and increased rhinovirus replication, demonstrating that interferon signalling was critical to antiviral immunity. We thus provide new mechanistic insights into rhinovirus infection and demonstrate the therapeutic potential of targeting NF-κB p65 (to suppress inflammation but preserve anti-viral immunity) and type I IFN signalling (to enhance deficient anti-viral immunity) to treat rhinovirus-induced exacerbations of airway diseases.

  20. Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation.

    PubMed

    Ferreira, R M; Franco, E; Teixeira, A R

    1996-08-15

    The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (< 1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose bisphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues.

  1. Development of a blocking ELISA for detection of Mycoplasma hyopneumoniae infection based on a monoclonal antibody against protein P65

    PubMed Central

    LIU, Maojun; DU, Gaimei; ZHANG, Yue; WU, Yuzi; WANG, Haiyan; LI, Bin; BAI, Yun; FENG, Zhixin; XIONG, Qiyan; BAI, Fangfang; BROWNING, Glenn F; SHAO, Guoqing

    2016-01-01

    Mycoplasma hyopneumoniae causes porcine enzootic pneumonia, an economically important disease of swine. A more sensitive and reliable method for detection of serum antibodies is needed for epidemiological investigations and to evaluate the effect of immunization. We expressed the M. hyopneumoniae protein P65 in Escherichia coli and produced a monoclonal antibody (mAb) that bound specifically to recombinant P65. Using this mAb, a blocking enzyme linked immunosorbent assay (ELISA) was developed. The blocking ELISA had similar specificity to and sensitivity with the commercial ELISA produced by IDEXX. Thus, this blocking ELISA is a useful test for serological confirmation of M. hyopneumoniae infection. PMID:27075114

  2. Increases in nuclear p65 activation in dystrophic skeletal muscle are secondary to increases in the cellular expression of p65 and are not solely produced by increases in IkappaB-alpha kinase activity.

    PubMed

    Singh, Rajvir; Millman, Gregory; Turin, Eric; Polisiakeiwicz, Lucasz; Lee, Brian; Gatti, Francesca; Berge, Jonas; Smith, Emily; Rutter, John; Sumski, Chris; Winders, W Tyler; Samadi, Abbas; Carlson, C George

    2009-10-15

    Dystrophin-deficient muscle exhibits substantial increases in nuclear NF-kappaB activation. To examine potential mechanisms for this enhanced activation, the present study employs conventional Western blot techniques to provide the first determination of the relative expression of NF-kappaB signaling molecules in adult nondystrophic and dystrophic (mdx) skeletal muscle. The results indicate that dystrophic muscle is characterized by increases in the whole cell expression of IkappaB-alpha, p65, p50, RelB, p100, p52, IKK, and TRAF-3. The proportion of phosphorylated IkappaB-alpha, p65, NIK, and IKKbeta, and the level of cytosolic IkappaB-alpha, were also increased in the mdx diaphragm. Proteasomal inhibition using MG-132 increased the proportion of phosphorylated IkappaB-alpha in nondystrophic diaphragm, but did not significantly increase this proportion in the mdx diaphragm. This result suggests that phosphorylated IkappaB-alpha accumulates in dystrophic cytosol because the rate of IkappaB-alpha degradation is lower than the effective rate of IkappaB-alpha synthesis and phosphorylation. Dystrophic increases in SUMO-1 (small ubiquitin modifier-1) protein and in Akt activation were also observed. The results indicate that increases in nuclear p65 activation in dystrophic muscle are not produced solely by increases in the activity of IkappaB-alpha kinase (IKK), but are due primarily to increases in the expression of p65 and other NF-kappaB signaling components.

  3. Simulated ischemia/reperfusion-induced p65-Beclin 1-dependent autophagic cell death in human umbilical vein endothelial cells

    PubMed Central

    Zeng, Min; Wei, Xin; Wu, Zhiyong; Li, Wei; Zheng, Yin; Li, Bing; Meng, Xuqing; Fu, Xiuhong; Fei, Yi

    2016-01-01

    Myocardial ischemia/reperfusion (I/R) injury detrimentally alters the prognosis of patients undergoing revascularization after acute myocardial infarction. Our previous study demonstrated that NF-κB-induced autophagy plays a detrimental role in cardiac I/R injury using a rabbit myocardial I/R model. In this study, we sought to explore the specific mechanism of this autophagy-mediated cell damage in an in vitro simulated ischemia/reperfusion (sI/R) model using human umbilical vein endothelial cells. Our current study demonstrates that simulated I/R induces autophagy in a p65-Beclin 1-dependent manner, which can be suppressed with the inhibition of NF-κB. Furthermore, rapamycin which promotes autophagy, exacerbates sI/R-induced cell death. While 3-methyladenine rescues cell damage. Our data thus suggest that I/R promotes NF-κB p65 activity mediated Beclin 1-mediated autophagic flux, thereby exacerbating myocardial injury. PMID:27857190

  4. Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation

    PubMed Central

    Li, Jasmine; Hardy, Kristine; Phetsouphanh, Chan; Tu, Wen Juan; Sutcliffe, Elissa L.; McCuaig, Robert; Sutton, Christopher R.; Zafar, Anjum; Munier, C. Mee Ling; Zaunders, John J.; Xu, Yin; Theodoratos, Angelo; Tan, Abel; Lim, Pek Siew; Knaute, Tobias; Masch, Antonia; Zerweck, Johannes; Brezar, Vedran; Milburn, Peter J.; Dunn, Jenny; Casarotto, Marco G.; Turner, Stephen J.; Seddiki, Nabila; Kelleher, Anthony D.

    2016-01-01

    ABSTRACT Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4+ T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4+ T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells. PMID:27149922

  5. A RelA(p65) Thr505 phospho-site mutation reveals an important mechanism regulating NF-κB-dependent liver regeneration and cancer

    PubMed Central

    Moles, A; Butterworth, J A; Sanchez, A; Hunter, J E; Leslie, J; Sellier, H; Tiniakos, D; Cockell, S J; Mann, D A; Oakley, F; Perkins, N D

    2016-01-01

    Post-translational modifications of nuclear factor (NF)-κB subunits provide a mechanism to differentially regulate their activity in response to the many stimuli that induce this pathway. However, the physiological significance of these modifications is largely unknown, and it remains unclear if these have a critical role in the normal and pathological functions of NF-κB in vivo. Among these, phosphorylation of the RelA(p65) Thr505 residue has been described as an important regulator of NF-κB activity in cell lines, but its physiological significance was not known. Therefore, to learn more about the role of this pathway in vivo, we generated a knockin mouse with a RelA T505A mutation. Unlike RelA knockout mice, the RelA T505A mice develop normally but exhibit aberrant hepatocyte proliferation following liver partial hepatectomy or damage resulting from carbon tetrachloride (CCl4) treatment. Consistent with these effects, RelA T505A mice exhibit earlier onset of cancer in the N-nitrosodiethylamine model of hepatocellular carcinoma. These data reveal a critical pathway controlling NF-κB function in the liver that acts to suppress the tumour-promoting activities of RelA. PMID:26853469

  6. Stepwise phosphorylation of p65 promotes NF-κB activation and NK cell responses during target cell recognition

    PubMed Central

    Kwon, Hyung-Joon; Choi, Go-Eun; Ryu, Sangryeol; Kwon, Soon Jae; Kim, Sun Chang; Booth, Claire; Nichols, Kim E.; Kim, Hun Sik

    2016-01-01

    NF-κB is a key transcription factor that dictates the outcome of diverse immune responses. How NF-κB is regulated by multiple activating receptors that are engaged during natural killer (NK)-target cell contact remains undefined. Here we show that sole engagement of NKG2D, 2B4 or DNAM-1 is insufficient for NF-κB activation. Rather, cooperation between these receptors is required at the level of Vav1 for synergistic NF-κB activation. Vav1-dependent synergistic signalling requires a separate PI3K-Akt signal, primarily mediated by NKG2D or DNAM-1, for optimal p65 phosphorylation and NF-κB activation. Vav1 controls downstream p65 phosphorylation and NF-κB activation. Synergistic signalling is defective in X-linked lymphoproliferative disease (XLP1) NK cells entailing 2B4 dysfunction and required for p65 phosphorylation by PI3K-Akt signal, suggesting stepwise signalling checkpoint for NF-κB activation. Thus, our study provides a framework explaining how signals from different activating receptors are coordinated to determine specificity and magnitude of NF-κB activation and NK cell responses. PMID:27221592

  7. Examination of CK2α and NF-κB p65 expression in human benign prostatic hyperplasia and prostate cancer tissues.

    PubMed

    Qaiser, Fatima; Trembley, Janeen H; Sadiq, Sarah; Muhammad, Iqbal; Younis, Rubina; Hashmi, Shoaib Naiyar; Murtaza, Badar; Rector, Thomas S; Naveed, Abdul Khaliq; Ahmed, Khalil

    2016-09-01

    Protein kinase CK2 plays a critical role in cell growth, proliferation, and suppression of cell death. CK2 is overexpressed, especially in the nuclear compartment, in the majority of cancers, including prostate cancer (PCa). CK2-mediated activation of transcription factor nuclear factor kappa B (NF-κB) p65 is a key step in cellular proliferation, resulting in translocation of NF-κB p65 from the cytoplasm to the nucleus. As CK2 expression and activity are also elevated in benign prostatic hyperplasia (BPH), we sought to increase the knowledge of CK2 function in benign and malignant prostate by examination of the relationships between nuclear CK2 and nuclear NF-κB p65 protein expression. The expression level and localization of CK2α and NF-κB p65 proteins in PCa and BPH tissue specimens was determined. Nuclear CK2α and NF-κB p65 protein levels are significantly higher in PCa compared with BPH, and these proteins are positively correlated with each other in both diseases. Nuclear NF-κB p65 levels correlated with Ki-67 or with cytoplasmic NF-κB p65 expression in BPH, but not in PCa. The findings provide information that combined analysis of CK2α and NF-κB p65 expression in prostate specimens relates to the disease status. Increased nuclear NF-κB p65 expression levels in PCa specifically related to nuclear CK2α levels, indicating a possible CK2-dependent relationship in malignancy. In contrast, nuclear NF-κB p65 protein levels related to both Ki-67 and cytoplasmic NF-κB p65 levels exclusively in BPH, suggesting a potential separate impact for NF-κB p65 function in proliferation for benign disease as opposed to malignant disease.

  8. MiR-138 indirectly regulates the MDR1 promoter by NF-κB/p65 silencing.

    PubMed

    Requenez-Contreras, J L; López-Castillejos, E S; Hernández-Flores, R; Moreno-Eutimio, M A; Granados-Riveron, J T; Martinez-Ruiz, G U; Aquino-Jarquin, G

    2017-03-11

    MicroRNAs (miRNAs) are known to mediate post-transcriptional gene silencing in the cytoplasm and recent evidence indicates that may also possess nuclear roles in regulating gene expression. A previous study showed that miR-138 is involved in the multidrug resistance of leukemia cells through down-regulation of the drug efflux pump P-glycoprotein (P-gp), the protein encoded by the human multidrug-resistant ABCB1/MDR1 gene. However, the transcriptional regulatory mechanisms responsible remain to be elucidated. To deepen the description of the mechanism of transcriptional gene silencing on the MDR1 promoter, we initially performed a bioinformatics search for potential miR-138 binding sites in the MDR1 gene promoter sequence. Interestingly, we did not find miR-138 binding sites in this region, suggesting an indirect regulation. From six representative transcriptional factors involved in MDR1 gene regulation, an in silico analysis revealed that NF-κB/p65 has a specific binding site for miR-138. The results of luciferase reporter assay, western blot and flow cytometry shown here suggest that miR-138 might modulate the human MDR1 expression by inhibiting NF-κB/p65 as an indirect mechanism of MDR1 regulation. Furthermore, employing the human macrophage-like cell line U937 we observed comparable results with NF-κB/p65 down-regulation and we also observed a significant reduction in the IL-6 and TNF-α mRNA, as well as in their secreted pro-inflammatory cytokines following miR-138 expression, suggesting that canonical NF-κB target genes might also be potential targets for miR-138 in leukemia cells.

  9. RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A.

    PubMed Central

    Neumann, M; Grieshammer, T; Chuvpilo, S; Kneitz, B; Lohoff, M; Schimpl, A; Franza, B R; Serfling, E

    1995-01-01

    Stimulation of the protein kinase A (PKA) signalling pathway exerts an inhibitory effect on the proliferation of numerous cells, including T lymphocytes. In CD4+ T helper cells, stimulation of PKA leads to suppression of interleukin 2 (IL-2) induction, while induction of the genes coding for the lymphokines IL-4 and IL-5 is enhanced. We show that the differential effect of PKA activity on induction of the IL-2 and IL-4 genes is mediated through their promoters. One major target of the suppressive effect of PKA is the kappa B site in the IL-2 promoter. A kappa B site is missing in the IL-4 promoter. Mutations preventing factor binding to the IL-2 kappa B site result in a loss of PKA-mediated suppression of IL-2 promoter activity. Furthermore, activation of the PKA signalling pathway impairs the inducible activity of multiple kappa B sites of the IL-2 promoter, but not of other factor binding sites. The reduction in activity of kappa B sites in activated and PKA-stimulated T cells is accompanied by changes in the concentration and DNA binding of Rel/NF-kappa B factors. Stimulation of the PKA pathway in Jurkat T cells with the PKA activator forskolin leads to an increase in synthesis of c-Rel and p105/p50, while synthesis of p65/RelA remains unchanged. However, nuclear translocation and DNA binding of p65 is distinctly impaired, probably due to a retarded degradation of I kappa B-alpha. In a similar way, stimulation of the PKA signalling pathway inhibits nuclear translocation of p65 and generation of nuclear kappa B complexes in peripheral T lymphocytes from murine lymph nodes. These results indicate that PKA-mediated suppression of NF-kappa B activity plays an important role in the control of activation of peripheral T lymphocytes. Images PMID:7744006

  10. Glycyrrhizic acid prevents enteritis through reduction of NF‑κB p65 and p38MAPK expression in rat.

    PubMed

    Wang, Yi-Ming; Du, Guo-Qiang

    2016-04-01

    Glycyrrhizic acid has a variety of biological properties, including a protective function in the liver, and anti‑inflammatory, anti‑ulcer, anti‑anaphylaxis, anti‑oxidant, immunoregulatory, antiviral and anticancer activities. The efficacy of glycyrrhizic acid can be increased when combined with other medicines. In the present study, the potential protective effects of glycyrrhizic acid against enteritis in rats, and its role in regulating anti‑inflammation, anti‑oxidation, angiogenic and apoptotic mechanisms were investigated using enzyme‑linked immunosorbent and bicinchoninic acid assays, and reverse transcription‑quantitative polymerase chain reaction and western blotting analyses. Adult male Sprague‑Dawley rats were injected with 20 mg/kg methotrexate (MTX) to establish enteritis. Additionally, rats with MTX‑induced enteritis were peritoneally injected with 200 mg glycyrrhizic acid for 9 weeks. The current study demonstrated that glycyrrhizic acid could alleviate MTX‑induced increases of tumor necrosis factor‑α, interleukin (IL)‑1β and IL‑6 levels, and raise IL‑10 levels, in rats with enteritis. Treatment with glycyrrhizic acid significantly reduced D‑lactate and intercellular adhesion molecule‑1 gene expression (P<0.01), but did not inhibit diamine oxidase activity in MTX‑induced enteritis. Pretreatment with glycyrrhizic acid significantly suppressed the promotion of p38 mitogen‑activated protein kinase (p38MAPK), nuclear factor‑κB p65 (NF‑κB p65) protein expression, interferon‑γ protein concentration, and caspase‑3 and cycloxygenase‑2 activity in MTX‑induced enteritis (P<0.01). The findings of the current study suggest that glycyrrhizic acid may prevent enteritis by reducing NF‑κB p65 and p38MAPK expression levels, which may inform future therapeutic strategies for the treatment of enteritis.

  11. Coal dust contiguity-induced changes in the concentration of TNF- and NF- B p65 on the ocular surface

    SciTech Connect

    Sun, Z.Y.; Hong, J.; Liu, Z.Y.; Jin, X.D.; Gu, C.H.

    2009-07-01

    To observe the influence of coal dust on ocular surface of coal miners and rabbits with coal dust contiguity on expression TNF- and NF- Bp65 and dry eye occurrence. Expression TNF- and NF- Bp65 in ocular surface were determined. Results showed tear production, BUT and lysozyme decreased for coal miners and rabbits with coal dust contiguity. Coal dust exposure was linked to development of xerophthalmia, and induced a higher expression of NF- B p65 and TNF- perhaps as a mechanism to resist coal dust ocular surface injury.

  12. Allo-antigen stimulated CD8+ T-cells suppress NF-κB and Ets-1 DNA binding activity, and inhibit phosphorylated NF-κB p65 nuclear localization in CD4+ T-cells.

    PubMed

    Nagashima, Ryuichi; Kawakami, Fumitaka; Takahashi, Shinichiro; Obata, Fumiya; Kubo, Makoto

    2014-08-01

    CD8+ T-cells of asymptomatic HIV-1 carriers (AC) suppress human immunodeficiency virus type 1 (HIV-1) replication in a class I major histocompatibility complex (MHC-I)-restricted and -unrestricted manner. In order to investigate the mechanism of MHC-I-unrestricted CD8+ T-cell-mediated HIV-1 suppression, we previously established allo-antigen stimulated CD8+T-cells from HIV-1-uninfected donors. These allo-antigen stimulated CD8+ T-cells suppressed HIV-1 replication in acutely infected autologous CD4+ T-cells when directly co-cultured. To elucidate the mechanism of HIV-1 replication suppression, we analyzed DNA-binding activity and phosphorylation of transcriptional factors associated with HIV-1 replication by electrophoresis mobility shift assay and Western blotting. When CD4+ T-cells were cultured with allo-antigen stimulated CD8+ T-cells, the reduction of NF-κB and Ets-1 DNA-binding activity was observed. Nuclear localization of NF-κB p65 and Ets-1 was suppressed in CD4+ T-cells. Although NF-κB p65 and Ets-1 are known to be regulated by protein kinase A (PKA), no difference was observed in the expression and phosphorylation of the PKA catalytic subunit in CD4+ T-cells cultured with PHA-treated CD8+ T-cells or allo-antigen stimulated CD8+ T-cells. Cyclic AMP is also known to enter through gap junctions, but the suppression of HIV-1 replication mediated by allo-antigen stimulated CD8+ T-cells was not affected by the gap junction inhibitor. The nuclear transport of phosphorylated NF-κB p65 (Ser276) was inhibited only in CD4+ T-cells cultured with allo-antigen stimulated CD8+ T-cells. Our results indicate that allo-antigen stimulated CD8+ T-cells suppress the transcriptional activity of NF-κB p65 or Ets-1 in an antigen-nonspecific manner, and inhibit the nuclear transport of phosphorylated NF-κB p65 (Ser276).

  13. Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

    SciTech Connect

    Tiwari, Manisha; Mikuni, Shintaro; Muto, Hideki; Kinjo, Masataka

    2013-07-05

    Highlights: •We used two-laser-beam FCCS to determine the dissociation constant (K{sub d}) of IPT domain of p50/p65 heterodimer in living cell. •Interaction of p50 and p65 was analyzed in the cytoplasm and nucleus of single living cell. •Binding affinity of p50/p65 heterodimer is higher in cytoplasm than that of nucleus. -- Abstract: Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K{sub d}, for the heterodimer in living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K{sub d} values of mCherry{sub 2}- and EGFP-fused p50 and p65 were determined to be 0.46 μM in the cytoplasm and 1.06 μM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.

  14. The NF-κB p65/miR-23a-27a-24 cluster is a target for leukemia treatment.

    PubMed

    Zhang, Yong-Chang; Ye, Hui; Zeng, Zhi; Chin, Y Eugene; Huang, Yu-Ning; Fu, Guo-Hui

    2015-10-20

    p65 is a transcription factor that is involved in many physiological and pathologic processes. Here we report that p65 strongly binds to the miR-23a-27a-24 cluster promoter to up-regulate its expression. As bone marrow-derived cells differentiate into red blood cells in vitro, p65/miR-23a-27a-24 cluster expression increases sharply and then declines before the appearance of red blood cells, suggesting that this cluster is negatively related to erythroid terminal differentiation. Bioinformatic and molecular biology experiments confirmed that the miR-23a-27a-24 cluster inhibited the expression of the erythroid proteome and contributed to erythroleukemia progression. In addition, high level of the p65/miR-23a-27a-24 cluster was found in APL and AML cell lines and in nucleated peripheral blood cells from leukemia patients. Furthermore, anti-leukemia drugs significantly inhibited the expression of the p65/miR-23a-27a-24 cluster in leukemia cells. Administration of the p65 inhibitor parthenolide significantly improved hematology and myelogram indices while prolonging the life span of erythroleukemia mice. Meanwhile, stable overexpression of these three miRNAs in mouse erythroleukemia cells enhanced cell malignancy. Our findings thus connect a novel regulation pathway of the p65/miR-23a-27a-24 cluster with the erythroid proteome and provide an applicable approach for treating leukemia.

  15. Identification of an NF-κB p50/p65-responsive site in the human MIR155HG promoter

    PubMed Central

    2013-01-01

    Background MicroRNA-155 (miR-155) is the diced product of the MIR155HG gene. miR-155 regulates the expression of many immune-specific transcripts, is overexpressed in many human lymphomas, and has oncogenic activity in mouse transgenic models. MIR155HG has been proposed to be a target gene for transcription factor NF-κB largely due to the positive correlation between high nuclear NF-κB activity and increased miR-155 expression following treatment with NF-κB inducers or in subsets of hematopoietic cancers. Nevertheless, direct regulation of the human MIR155HG promoter by NF-κB has not been convincingly demonstrated previously. Results This report shows that induction of NF-κB activity rapidly leads to increased levels of both primary MIR155HG mRNA and mature miR-155 transcripts. We have mapped an NF-κB-responsive element to a position approximately 178 nt upstream of the MIR155HG transcription start site. The -178 site is specifically bound by the NF-κB p50/p65 heterodimer and is required for p65-induced reporter gene activation. Moreover, the levels of miR-155 in nine human B-lymphoma cell lines generally correlate with increased nuclear NF-κB proteins. Conclusion Overall, the identification of an NF-κB-responsive site in the MIR155HG proximal promoter suggests that MIR155HG is a direct NF-κB target gene in vivo. Understanding NF-κB-mediated regulation of miR-155 could lead to improved immune cell-related diagnostic tools and targeted therapies. PMID:24059932

  16. P65 inactivation in adipocytes and macrophages attenuates adipose inflammatory response in lean but not in obese mice.

    PubMed

    Gao, Zhanguo; Zhang, Jin; Henagan, Tara M; Lee, Jong Han; Ye, Xin; Wang, Hui; Ye, Jianping

    2015-03-15

    NF-κB induces transcriptional expression of proinflammatory genes and antiapoptotic genes. The two activities of NF-κB remain to be characterized in the mechanism of chronic inflammation in obesity. To address this issue, we inactivated NF-κB in adipose tissue by knocking out p65 (RelA) in mice (F-p65-KO) and examined the inflammation in lean and obese conditions. In the lean condition, KO mice exhibited a reduced inflammation in adipose tissue with a decrease in macrophage infiltration, M1 polarization, and proinflammatory cytokine expression. In the obese condition, KO mice had elevated inflammation with more macrophage infiltration, M1 polarization, and cytokine expression. In the mechanism of enhanced inflammation, adipocytes and macrophages exhibited an increase in cellular apoptosis, which was observed with more formation of crown-like structures (CLS) in fat tissue of KO mice. Body weight, glucose metabolism, and insulin sensitivity were not significantly altered in KO mice under the lean and obese conditions. A modest but significant reduction in body fat mass was observed in KO mice on HFD with an elevation in energy expenditure. The data suggest that in the control of adipose inflammation, NF-κB exhibits different activities in the lean vs. obese condition. NF-κB is required for expression of proinflammatory genes in the lean but not in the obese condition. NF-κB is required for inhibition of apoptosis in the obese condition, in which proinflammation is enhanced by NF-κB inactivation.

  17. Pentoxifylline and the proteasome inhibitor MG132 induce apoptosis in human leukemia U937 cells through a decrease in the expression of Bcl-2 and Bcl-XL and phosphorylation of p65

    PubMed Central

    2013-01-01

    Background In Oncology, the resistance of the cancerous cells to chemotherapy continues to be the principal limitation. The nuclear factor-kappa B (NF-κB) transcription factor plays an important role in tumor escape and resistance to chemotherapy and this factor regulates several pathways that promote tumor survival including some antiapoptotic proteins such as Bcl-2 and Bcl-XL. In this study, we investigated, in U937 human leukemia cells, the effects of PTX and the MG132 proteasome inhibitor, drugs that can disrupt the NF-κB pathway. For this, we evaluated viability, apoptosis, cell cycle, caspases-3, -8, -9, cytochrome c release, mitochondrial membrane potential loss, p65 phosphorylation, and the modification in the expression of pro- and antiapoptotic genes, and the Bcl-2 and Bcl-XL antiapoptotic proteins. Results The two drugs affect the viability of the leukemia cells in a time-dependent manner. The greatest percentage of apoptosis was obtained with a combination of the drugs; likewise, PTX and MG132 induce G1 phase cell cycle arrest and cleavage of caspases -3,-8, -9 and cytochrome c release and mitochondrial membrane potential loss in U937 human leukemia cells. In these cells, PTX and the MG132 proteasome inhibitor decrease p65 (NF-κB subunit) phosphorylation and the antiapoptotic proteins Bcl-2 and Bcl-XL. We also observed, with a combination of these drugs overexpression of a group of the proapoptotic genes BAX, DIABLO, and FAS while the genes BCL-XL, MCL-1, survivin, IκB, and P65 were downregulated. Conclusions The two drugs used induce apoptosis per se, this cytotoxicity was greater with combination of both drugs. These observations are related with the caspases -9, -3 cleavage and G1 phase cell cycle arrest, and a decrease in p65 phosphorylation and Bcl-2 and Bcl-XL proteins. As well as this combination of drugs promotes the upregulation of the proapoptotic genes and downregulation of antiapoptotic genes. These observations strongly confirm

  18. The Apoptogenic Toxin AIP56 Is a Metalloprotease A-B Toxin that Cleaves NF-κb P65

    PubMed Central

    Silva, Daniela S.; Pereira, Liliana M. G.; Moreira, Ana R.; Ferreira-da-Silva, Frederico; Brito, Rui M.; Faria, Tiago Q.; Zornetta, Irene; Montecucco, Cesare; Oliveira, Pedro; Azevedo, Jorge E.; Pereira, Pedro J. B.; Macedo-Ribeiro, Sandra; do Vale, Ana; dos Santos, Nuno M. S.

    2013-01-01

    AIP56 (apoptosis-inducing protein of 56 kDa) is a major virulence factor of Photobacterium damselae piscicida (Phdp), a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys39-Glu40 peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol. PMID:23468618

  19. Expression of RKIP, E-cadherin and NF-kB p65 in esophageal squamous cell carcinoma and their correlations.

    PubMed

    Ping, Fu-Min; Liu, Gui-Jing; Liu, Zhi-Jun; Li, Hai-Bin; Zhai, Jian-Wen; Li, Shu-Xia; Liu, Yue-Mei; Li, Bao-Wei; Wei, Hong

    2015-01-01

    To detect the expression of RKIP, E-cadherin and NF-kB p65 in esophageal squamous cell carcinoma (ESCC) and study their correlations. Steptavidin-peroxidase (S-P) method was employed to detect the expressions of RKIP, E-cadherin and NF-kB p65 in ESCC tissues from 77 cases and paracancerous tissues from 77 cases. The correlations between their expressions and clinicopathological indices and between the expressions of these proteins themselves were analyzed. The expressions of RKIP and E-cadherin in ESCC tissues were obviously lower than those in the paracancerous tissues (P<0.01); the expressions in ESCC tissues from cases with lymph node metastasis were lower than those from cases without lymph node metastasis (P<0.01); the expression of RKIP was positively correlated with the expression of E-cadherin in ESCC tissues (P<0.01). The expression of NF-kB p65 in ESCC tissues was correlated with clinical staging, lymph node metastasis and tumor differentiation (P<0.01); the expression of RKIP was negatively correlated with the expression of NF-kB p65 in ESCC tissues (P<0.05). Downregulation or depletion of RKIP was related to the onset and progression of ESCC, and facilitated the invasion and metastasis of ESCC by downregulating E-cadherin and upregulating NF-kB p65.

  20. Stimulatory effects of insulin-like growth factor-I on growth plate chondrogenesis are mediated by nuclear factor-kappaB p65.

    PubMed

    Wu, Shufang; Fadoju, Doris; Rezvani, Geoffrey; De Luca, Francesco

    2008-12-05

    Insulin-like growth factor-I (IGF-I) is an important regulator of endochondral ossification. However, little is known about the signaling pathways activated by IGF-I in growth plate chondrocytes. We have previously shown that NF-kappaB-p65 facilitates growth plate chondrogenesis. In this study, we first cultured rat metatarsal bones with IGF-I and/or pyrrolidine dithiocarbamate (PDTC), a known NF-kappaB inhibitor. The IGF-I-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by PDTC. In rat growth plate chondrocytes, IGF-I induced NF-kappaB-p65 nuclear translocation. The inhibition of NF-kappaB-p65 expression and activity (by p65 short interfering RNA and PDTC, respectively) in chondrocytes reversed the IGF-I-mediated induction of cell proliferation and differentiation and the IGF-I-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of IGF-I on NF-kappaB activation. In conclusion, our findings indicate that IGF-I stimulates growth plate chondrogenesis by activating NF-kappaB-p65 in chondrocytes.

  1. Reduced nuclear translocation of nuclear factor (NF)-kappaB p65 in the footpad epidermis of dogs infected with distemper virus.

    PubMed

    Friess, M; Engelhardt, P; Dobbelaere, D; Zurbriggen, A; Gröne, A

    2005-01-01

    Infection of canine footpads with the canine distemper virus (CDV) can cause massive epidermal thickening (hard pad disease), as a consequence of increased proliferation of keratinocytes and hyperkeratosis. Keratinocytes of canine footpad epidermis containing detectable CDV nucleoprotein antigen and CDV mRNA were shown previously to have increased proliferation indices. Because various proteins that play a role in the proliferation of epidermal cells are viral targets, the potential participation of such proteins in CDV-associated keratinocyte proliferation was investigated. Transforming growth factor-alpha (TGF-alpha), cell cycle regulatory proteins p21, p27 and p53, and nuclear factor (NF)-kappaB transcription factor components p50 and p65 were studied in the footpad epidermis from the following groups of dogs inoculated with CDV: group 1, consisting of seven dogs with clinical distemper and CDV in the footpad epidermis; group 2, consisting of four dogs with clinical distemper but no CDV in the footpad epidermis; group 3, consisting of eight dogs with neither clinical distemper nor CDV in the footpad epithelium. Group 4 consisted of two uninoculated control dogs. The expression of TGF-alpha, p21, p27 and p53, and p50 in the basal layer, lower and upper spinous layers, and in the granular layer did not differ statistically between CDV-positive (group 1) and CDV-negative (groups 2-4) footpad epidermis. However, there were differences in the levels of nuclear and cytoplasmic p65 expression between group 1 dogs and the other three groups. Thus, footpads from group 1 dogs had more keratinocytes containing p65 in the cytoplasm and, conversely, fewer nuclei that were positive for p65. These findings indicate that p65 translocation into the nucleus is reduced in CDV-infected footpad epidermis. Such decreased translocation of p65 may help to explain increased keratinocyte proliferation in hard pad disease and suggests interference of CDV with the NF-kappaB pathway.

  2. Annexin A1 nuclear translocation induces retinal ganglion cell apoptosis after ischemia-reperfusion injury through the p65/IL-1β pathway.

    PubMed

    Zhao, Yin; Li, Xing; Gong, Jieling; Li, Lu; Chen, Liwen; Zheng, Lu; Chen, Zhiqi; Shi, Jing; Zhang, Hong

    2017-04-04

    The degeneration of retinal ganglion cells (RGCs) has been identified as a major problem in glaucoma. Previous studies have indicated an association between annexin A1 (ANXA1) and neuronal cell apoptosis, and RGCs apoptosis in acute ischemia-reperfusion was attributed to an increased production of IL-1β. We found that the expression and nuclear translocation of ANXA1 were upregulated in models of acute ischemia-reperfusion in RGCs in vivo. ANXA1 was found to have a promoting effect on the expression of IL-1β in primary cultured RGCs, which could be inhibited by treatment with ANXA1 shRNA or the p65 inhibitor BAY 11-7082. ANXA1 interacted with p65, and recruited it into the nucleus. Chromatin immunoprecipitation assay revealed that ANXA1 accumulated at the IL-1β gene promoter. The reduction of p65 nuclear translocation using a membrane-permeable ANXA1 peptide containing a Ser5Ala mutation led to a decrease in the expression of IL-1β, and acute ischemia-reperfusion induced RGCs apoptosis in vivo. These results indicate that in RGCs, ANXA1 increases IL-1β expression by recruiting p65 to the nucleus, which induces cell apoptosis. The obtained results may help the development of a novel treatment strategy against RGCs apoptosis in acute ischemia-reperfusion injury.

  3. Suppressor of cytokine signaling 1 (SOCS1) limits NFkappaB signaling by decreasing p65 stability within the cell nucleus.

    PubMed

    Strebovsky, Julia; Walker, Patrick; Lang, Roland; Dalpke, Alexander H

    2011-03-01

    Suppressor of cytokine signaling (SOCS) proteins are inhibitors of cytoplasmic Janus kinases (Jak) and signal transducer and activator of transcription (STAT) signaling pathways. Previously the authors surprisingly observed that SOCS1 translocated into the nucleus, which was because of the presence of a nuclear localization sequence. This report now hypothesizes that SOCS1 mediates specific functions within the nuclear compartment because it is instantly transported into the nucleus, as shown by photoactivation and live cell imaging in human HEK293 cells. The NFκB component p65 is identified as an interaction partner for SOCS1 but not for other members of the SOCS family. SOCS1 bound to p65 only within the nucleus. By means of its SOCS box domain, SOCS1 operated as a ubiquitin ligase, leading to polyubiquitination and proteasomal degradation of nuclear p65. Thus, SOCS1 limited prolonged p65 signaling and terminated expression of NFκB inducible genes. Using mutants that lack either nuclear translocation or a functional SOCS box, this report identifies genes that are regulated in a manner dependent on the nuclear availability of SOCS1. Data show that beyond its receptor-proximal function in Jak/STAT signaling, SOCS1 also regulates the duration of NFκB signaling within the cell nucleus, thus exerting a heretofore unrecognized function.

  4. Radiobiological intercomparison of p(45)+Be and p(65)+Be neutron beams for lung tolerance in mice after single and fractionated irradiation.

    PubMed

    Grégoire, V; Beauduin, M; Gueulette, J; De Coster, B M; Octave-Prignot, M; Vynckier, S; Wambersie, A

    1993-01-01

    The lung tolerance in mice after single and fractionated irradiations with p(45)+Be and p(65)+Be neutrons produced at the isochronous cyclotron "CYCLONE" of Louvain-la-Neuve (Belgium) was studied. Cobalt-60 gamma rays were used for control irradiations. The end point was the dose which was lethal to 50% of the mice by 180 days (LD50/180). On a log-log plot, the slope (+/- SE) of the relationship between total isoeffect dose and fraction number decreases from 0.34 +/- 0.01 for gamma rays to 0.19 +/- 0.01 for p(65)+Be and 0.12 +/- 0.01 for p(45)+Be neutrons. The data have been analyzed using the linear-quadratic (LQ) model. The alpha/beta ratio (+95% confidence interval) increases from 5.3 (4.3-6.4) for gamma rays to 20.7 (16.7-24.9) for p(65)+Be and 37.9 (25.8-65.8) for p(45)+Be. The RBEs of neutrons relative to gamma rays were estimated from the LQ parameters, to 1.15 and 1.19 for a dose of 14 Gy gamma rays and 2.02 and 2.47 for a dose of 2 Gy gamma rays for p(65)+Be and p(45)+Be neutrons, respectively. The neutron RBE of the p(45)+Be relative to the p(65)+Be calculated from the ratio of their respective RBEs relative to gamma rays reaches 1.03 and 1.23 for doses of 14 and 2 Gy gamma-ray equivalent, respectively. These data are compared with other published data on lung tolerance after irradiation with lower-energy neutrons and with data obtained previously in our laboratory on mouse jejunum and Vicia faba.

  5. RelA NF-κB subunit activation as a therapeutic target in diffuse large B-cell lymphoma

    PubMed Central

    Manyam, Ganiraju C.; Visco, Carlo; Tzankov, Alexandar; Wang, Jing; Montes-Moreno, Santiago; Dybkaer, Karen; Chiu, April; Orazi, Attilio; Zu, Youli; Bhagat, Govind; Richards, Kristy L.; Hsi, Eric D.; Choi, William W.L.; Han van Krieken, J.; Huh, Jooryung; Ponzoni, Maurilio; Ferreri, Andrés J.M.; Møller, Michael B.; Parsons, Ben M.; Winter, Jane N.; Piris, Miguel A.; Jeffrey Medeiros, L.; Pham, Lan V.; Young, Ken H.

    2016-01-01

    It has been well established that nuclear factor kappa-B (NF-κB) activation is important for tumor cell growth and survival. RelA/p65 and p50 are the most common NF-κB subunits and involved in the classical NF-κB pathway. However, the prognostic and biological significance of RelA/p65 is equivocal in the field. In this study, we assessed RelA/p65 nuclear expression by immunohistochemistry in 487 patients with de novo diffuse large B-cell lymphoma (DLBCL), and studied the effects of molecular and pharmacological inhibition of NF-κB on cell viability. We found RelA/p65 nuclear expression, without associations with other apparent genetic or phenotypic abnormalities, had unfavorable prognostic impact in patients with stage I/II DLBCL. Gene expressionprofiling analysis suggested immune dysregulation and antiapoptosis may be relevant for the poorer prognosis associated with p65 hyperactivation in germinal center B-cell–like (GCB) DLBCL and in activated B-cell–like (ABC) DLBCL, respectively. We knocked down individual NF-κB subunits in representative DLBCL cells in vitro, and found targeting p65 was more effective than targeting other NF-κB subunits in inhibiting cell growth and survival. In summary, RelA/p65 nuclear overexpression correlates with significant poor survival in early-stage DLBCL patients, and therapeutic targeting RelA/p65 is effective in inhibiting proliferation and survival of DLBCL with NF-κB hyperactivation. PMID:27941215

  6. Chemopreventive activity of GEN-27, a genistein derivative, in colitis-associated cancer is mediated by p65-CDX2-β-catenin axis.

    PubMed

    Du, Qianming; Wang, Yajing; Liu, Chao; Wang, Hong; Fan, Huimin; Li, Yan; Wang, Jianing; Zhang, Xu; Lu, Jinrong; Ji, Hui; Hu, Rong

    2016-04-05

    Nonresolving inflammation in the intestine predisposes individuals to colitis-associated colorectal cancer (CAC), which leads to high morbidity and mortality. Here we show that genistein-27 (GEN-27), a derivative of genistein, inhibited proliferation of human colorectal cancer cells through inhibiting β-catenin activity. Our results showed that GEN-27 increased expressions of adenomatous polyposis coli (APC) and axis inhibition protein 2 (AXIN2), and reduced β-catenin nuclear localization, which resulted from the inhibition of NF-κB/p65 nuclear localization and up-regulation of caudal-related homeobox transcription factor 2 (CDX2). Furthermore, GEN-27 decreased binding of p65 to the silencer region of CDX2 and increased binding of CDX2 to the promoter regions of APC and AXIN2, thus inhibiting the activation of β-catenin induced by TNF-α. Importantly, GEN-27 protected mice from azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon carcinogenesis, with reduced mortality, tumor number and tumor volume. Histopathology, immunohistochemistry and flow cytometry revealed that dietary GEN-27 significantly decreased secretion of proinflammatory cytokines and macrophage infiltration. Moreover, GEN-27 inhibited AOM/DSS-induced p65 and β-catenin nuclear translocation, while promoted the expression of CDX2, APC, and AXIN2. Taken together, our findings demonstrate that the anti-proliferation effect of GEN-27 in vitro and the prevention of CAC in vivo is mediated by p65-CDX2-β-catenin axis via inhibiting β-catenin target genes. Our results imply that GEN-27 could be a promising candidate for the chemoprevention of CAC.

  7. Prolyl-4-hydroxylase Domain Protein 2 Controls NF-κB/p65 Transactivation and Enhances the Catabolic Effects of Inflammatory Cytokines on Cells of the Nucleus Pulposus*

    PubMed Central

    Li, Jun; Yuan, Wen; Jiang, Shuai; Ye, Wei; Yang, Hao; Shapiro, Irving M.; Risbud, Makarand V.

    2015-01-01

    Prolyl-4-hydroxylase (PHD) proteins are key in sensing tissue hypoxia. In nucleus pulposus (NP) cells, our previous work demonstrated that PHD isoforms have a differential contribution in controlling hypoxia-inducible factor (HIF)-α degradation and activity. Recently we have shown that a regulatory relationship exists between PHD3 and inflammatory cytokines in NP cells. With respect to PHD2, the most abundant PHD isoform in NP cells, very little is known concerning its function and regulation under inflammatory conditions that characterize intervertebral disc degeneration. Here, we show that PHD2 is a potent regulator of the catabolic activities of TNF-α; silencing of PHD2 significantly decreased TNF-α-induced expression of catabolic markers including SDC4, MMP-3, MMP-13, and ADAMTS5, as well as several inflammatory cytokines and chemokines, while partially restoring aggrecan and collagen II expression. Use of NF-κB reporters with ShPHD2, SiHIF-1α, as well as p65−/−, PHD2−/−, and PHD3−/− cells, shows that PHD2 serves as a co-activator of NF-κB/p65 signaling in HIF-1-independent fashion. Immunoprecipitation of endogenous and exogenously expressed tagged proteins, as well as fluorescence microscopy, indicates that following TNF-α treatment, PHD2 interacts and co-localizes with p65. Conversely, loss of function experiments using lentivirally delivered Sh-p65, Sh-IKKβ, and NF-κB inhibitor confirmed that cytokine-dependent PHD2 expression in NP cells requires NF-κB signaling. These findings clearly demonstrate that PHD2 forms a regulatory circuit with TNF-α via NF-κB and thereby plays an important role in enhancing activity of this cytokine. We propose that during disc degeneration PHD2 may offer a therapeutic target to mitigate the deleterious actions of TNF-α, a key proinflammatory cytokine. PMID:25635047

  8. Human Bocavirus NS1 and NS1-70 Proteins Inhibit TNF-α-Mediated Activation of NF-κB by targeting p65

    PubMed Central

    Liu, Qingshi; Zhang, Zhenfeng; Zheng, Zhenhua; Zheng, Caishang; Liu, Yan; Hu, Qinxue; Ke, Xianliang; Wang, Hanzhong

    2016-01-01

    Human bocavirus (HBoV), a parvovirus, is a single-stranded DNA etiologic agent causing lower respiratory tract infections in young children worldwide. Nuclear factor kappa B (NF-κB) transcription factors play crucial roles in clearance of invading viruses through activation of many physiological processes. Previous investigation showed that HBoV infection could significantly upregulate the level of TNF-α which is a strong NF-κB stimulator. Here we investigated whether HBoV proteins modulate TNF-α–mediated activation of the NF-κB signaling pathway. We showed that HBoV NS1 and NS1-70 proteins blocked NF-κB activation in response to TNF-α. Overexpression of TNF receptor-associated factor 2 (TRAF2)-, IκB kinase alpha (IKKα)-, IκB kinase beta (IKKβ)-, constitutively active mutant of IKKβ (IKKβ SS/EE)-, or p65-induced NF-κB activation was inhibited by NS1 and NS1-70. Furthermore, NS1 and NS1-70 didn’t interfere with TNF-α-mediated IκBα phosphorylation and degradation, nor p65 nuclear translocation. Coimmunoprecipitation assays confirmed the interaction of both NS1 and NS1-70 with p65. Of note, NS1 but not NS1-70 inhibited TNF-α-mediated p65 phosphorylation at ser536. Our findings together indicate that HBoV NS1 and NS1-70 inhibit NF-κB activation. This is the first time that HBoV has been shown to inhibit NF-κB activation, revealing a potential immune-evasion mechanism that is likely important for HBoV pathogenesis. PMID:27329558

  9. Anti-inflammatory activity of chitosan nanoparticles carrying NF-κB/p65 antisense oligonucleotide in RAW264.7 macropghage stimulated by lipopolysaccharide.

    PubMed

    Ma, Li; Shen, Chuan-an; Gao, Lei; Li, Da-wei; Shang, Yu-ru; Yin, Kai; Zhao, Dong-xu; Cheng, Wen-feng; Quan, Dong-qin

    2016-06-01

    The purpose of this present study is to prepare NF-κB/p65 antisense oligonucleotide loaded chitosan nanoparticles (NPs) and evaluate their physicochemical characterization and antisense effects in RAW264.7 macrophages. Condensed nanoparticles with mean particle size of 128±16nm, average Zeta potential of 19.6±6.3mV and high entrapment efficiency (EE) of 98.6±0.11% were formed between NF-κB/p65 antisense gene (NAG) and chitosan by complex coacervation method. Trypan blue staining and MTT tests showed that NAG chitosan NPs had no toxic effect on RAW264.7 macrophages when the dose was no more than 20μg/mL. Confocal microscopy images showed that NAG chitosan NPs were capable to deliver NAG into cytoplasm of RAW264.7 macrophages and finally into nucleus. Real-time PCR tests verified that NAG chitosan NPs could significantly decrease the mRNA expression level of NF-κB/p65 and inflammatory cytokines including TNF-ɑ, IL-1 and IL-6. Accordingly, western blot study showed that NAG NPs uptaken in the cells could efficiently reversed the expression of NF-κB/p65 protein induced by LPS. At last, downstream release level of inflammatory factors including TNF-ɑ, IL-1 and IL-6 in LPS stimulated RAW264.7 macrophages was significantly decreased after treated by NAG chitosan NPs. It could be concluded that chitosan NPs were excellent delivery vectors to ferry the NAG into the cytoplasm and nucleus of macrophages. The NAG chitosan NPs might be a novel therapeutic apparatus for the treatment of LPS induced sepsis by inhibiting NF-κB-related pro-inflammatory cytokines secretion.

  10. p65-Dependent production of interleukin-1β by osteolytic prostate cancer cells causes an induction of chemokine expression in osteoblasts.

    PubMed

    Schulze, Jochen; Weber, Kristoffer; Baranowsky, Anke; Streichert, Thomas; Lange, Tobias; Spiro, Alexander Simon; Albers, Joachim; Seitz, Sebastian; Zustin, Josef; Amling, Michael; Fehse, Boris; Schinke, Thorsten

    2012-04-01

    Skeletal metastases are a frequent complication of prostate, breast and lung cancer, and the interactions of tumor cells with bone-forming osteoblasts and bone-resorbing osteoclasts have been suggested to play critical roles in disease progression. We have previously shown that treatment of primary murine osteoblasts with conditioned medium of the human osteolytic prostate cancer cell line PC-3 results in a rapid induction of chemokine expression, thereby providing further evidence for a molecular crosstalk between bone and tumor cells. The aim of our current study was to identify PC-3-derived molecules mediating this effect. Using Affymetrix Gene Chip hybridization followed by qRT-PCR we were able to confirm that the expression of chemokine-encoding genes is markedly induced in human primary osteoblasts following incubation with PC-3-conditioned medium. Since this induction was significantly affected upon alteration of p65-levels in PC-3 cells, we performed a second genome-wide expression analysis to identify p65-regulated cytokines, which were then tested for their ability to induce chemokine expression. Here we observed that interleukin-1β (IL-1B) did not only increase the expression of chemokines in osteoblasts, but also the phosphorylation of p65 and thereby its own expression. Since immunohistochemistry on bone biopsy sections from prostate cancer metastases demonstrated IL-1B expression in both, tumor cells and osteoblasts, our data suggest that IL-1B is one of the relevant cytokines involved in the skeletal complications of cancer metastases.

  11. B7-H3 Augments Inflammatory Responses and Exacerbates Brain Damage via Amplifying NF-κB p65 and MAPK p38 Activation during Experimental Pneumococcal Meningitis

    PubMed Central

    Chen, Xuqin; Li, Yan; Blankson, Siobhan; Liu, Min; Huang, Danping; Redmond, H. Paul; Huang, Jing; Wang, Jiang Huai; Wang, Jian

    2017-01-01

    The costimulatory protein B7-H3 has been shown to play a contributory role in the development and progression of experimental pneumococcal meningitis by augmentation of the innate immunity-associated inflammatory response via a TLR2-dependent manner. This study aimed to clarify the component(s) of TLR2-mediated signal transduction pathways responsible for B7-H3-augmented inflammatory response and subsequent brain damage during experimental pneumococcal meningitis. Administration of B7-H3 did not augment expression of TLR2 and other TLR2 upstream components, but led to an enhanced formation of MyD88-IRAK immunocomplex in the brain of S. pneumoniae-infected mice. Furthermore, B7-H3 substantially augmented S. pneumoniae-induced activation of TLR2 downstream NF-κB p65 and MAPK p38 pathways in the brain of S. pneumoniae-infected mice. Notably, blockage of NF-κB p65 and/or MAPK p38 with their specific inhibitors strongly attenuated B7-H3-amplified inflammatory response with significantly reduced proinflammatory cytokine and chemokine production, and markedly ameliorated B7-H3-exacerbated disruption of blood-brain barrier and severity of disease status in S. pneumoniae-infected mice. These results indicate that targeting NF-κB p65 and/or MAPK p38 may represent a promising therapeutic option for amelioration of overwhelming inflammatory response-associated brain injury frequently observed during pneumococcal meningitis. PMID:28141831

  12. Inactivation of NF-κB p65 (RelA) in Liver Improves Insulin Sensitivity and Inhibits cAMP/PKA Pathway.

    PubMed

    Ke, Bilun; Zhao, Zhiyun; Ye, Xin; Gao, Zhanguo; Manganiello, Vincent; Wu, Bin; Ye, Jianping

    2015-10-01

    The transcription factor nuclear factor-κB (NF-κB) mediates inflammation and stress signals in cells. To test NF-κB in the control of hepatic insulin sensitivity, we inactivated NF-κB in the livers of C57BL/6 mice through deletion of the p65 gene, which was achieved by crossing floxed-p65 and Alb-cre mice to generate L-p65-knockout (KO) mice. KO mice did not exhibit any alterations in growth, reproduction, and body weight while on a chow diet. However, the mice on a high-fat diet (HFD) exhibited an improvement in systemic insulin sensitivity. Hepatic insulin sensitivity was enhanced as indicated by increased pyruvate tolerance, Akt phosphorylation, and decreased gene expression in hepatic gluconeogenesis. In the liver, a decrease in intracellular cAMP was observed with decreased CREB phosphorylation. Cyclic nucleotide phosphodiesterase-3B (PDE3B), a cAMP-degrading enzyme, was increased in mRNA and protein as a result of the absence of NF-κB activity. NF-κB was found to inhibit PDE3B transcription through three DNA-binding sites in the gene promoter in response to tumor necrosis factor-α. Body composition, food intake, energy expenditure, and systemic and hepatic inflammation were not significantly altered in KO mice on HFD. These data suggest that NF-κB inhibits hepatic insulin sensitivity by upregulating cAMP through suppression of PDE3B gene transcription.

  13. Andrographolide Inhibits Nuclear Factor-κB Activation through JNK-Akt-p65 Signaling Cascade in Tumor Necrosis Factor-α-Stimulated Vascular Smooth Muscle Cells

    PubMed Central

    Chen, Yu-Ying; Hsieh, Cheng-Ying; Lee, Lin-Wen; Sheu, Joen-Rong

    2014-01-01

    Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Andrographolide is the most active and critical constituent isolated from the leaves of Andrographis paniculata, a herbal medicine widely used for treating anti-inflammation in Asia. In this study, we investigated the mechanisms of the inhibitory effects of andrographolide in vascular smooth muscle cells (VSMCs) exposed to a proinflammatory stimulus, tumor necrosis factor-α (TNF-α). Treating TNF-α-stimulated VSMCs with andrographolide suppressed the expression of inducible nitric oxide synthase in a concentration-dependent manner. A reduction in TNF-α-induced c-Jun N-terminal kinase (JNK), Akt, and p65 phosphorylation was observed in andrographolide-treated VSMCs. However, andrographolide affected neither IκBα degradation nor p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 phosphorylation under these conditions. Both treatment with LY294002, a phosphatidylinositol 3-kinase/Akt inhibitor, and treatment with SP600125, a JNK inhibitor, markedly reversed the andrographolide-mediated inhibition of p65 phosphorylation. In addition, LY294002 and SP600125 both diminished Akt phosphorylation, whereas LY294002 had no effects on JNK phosphorylation. These results collectively suggest that therapeutic interventions using andrographolide can benefit the treatment of vascular inflammatory diseases, and andrographolide-mediated inhibition of NF-κB activity in TNF-α-stimulated VSMCs occurs through the JNK-Akt-p65 signaling cascade, an IκBα-independent mechanism. PMID:25114952

  14. Naringenin ameliorates daunorubicin induced nephrotoxicity by mitigating AT1R, ERK1/2-NFκB p65 mediated inflammation.

    PubMed

    Karuppagounder, Vengadeshprabhu; Arumugam, Somasundaram; Thandavarayan, Rajarajan Amirthalingam; Pitchaimani, Vigneshwaran; Sreedhar, Remya; Afrin, Rejina; Harima, Meilei; Suzuki, Hiroshi; Suzuki, Kenji; Nakamura, Masahiko; Ueno, Kazuyuki; Watanabe, Kenichi

    2015-09-01

    Inflammation and oxidative stress play important roles in the progression of renal damage. The natural polyphenol naringenin is known to exert potent antioxidant and anti-inflammatory effects. In this study, we have investigated the effect of naringenin on kidney dysfunction, fibrosis, endoplasmic reticulum (ER) stress, angiotensin II type I receptor (AT1R) expression and inflammation in daunorubicin (DNR) induced nephrotoxicity model. Nephrotoxicity was induced in rats by intravenous injection of DNR at a cumulative dose of 9 mg/kg. After 1 week, naringenin (20mg/kg/day. p.o) was administered daily for 6 weeks. Biochemical studies were performed to evaluate renal function. Western blotting was performed to measure the protein levels of AT1R, endothelin (ET)1, ET receptor type A (ETAR), extracellular signal-regulated kinase (ERK)1/2, nuclear factor (NF)κB p65, peroxisome proliferator activated receptor (PPAR)γ, oxidative/ER stress, apoptosis, and inflammatory markers in the kidney of DNR treated rats. Histopathological analysis was done using hemotoxylin eosin and Masson trichrome stained renal sections to investigate the structural abnormalities and fibrosis. DNR treated rats suffered from nephrotoxicity as evidenced by worsened renal function, increased blood urea nitrogen, serum creatinine levels in renal tissues and histopathogical abnormalities. Treatment with naringenin mitigated these changes. Furthermore, naringenin up regulated PPARγ and down regulated AT1R, ET1, ETAR, p-ERK1/2, p-NFκB p65, ER stress, apoptosis, and inflammatory markers. Our results suggest that naringenin has an ability to improve renal function and attenuates AT1R, ERK1/2-NFκB p65 signaling pathway in DNR induced nephrotoxicity in rats.

  15. Hyperosmotic stress activates p65/RelB NFkappaB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death.

    PubMed

    Eisner, Verónica; Quiroga, Clara; Criollo, Alfredo; Eltit, José Miguel; Chiong, Mario; Parra, Valentina; Hidalgo, Karla; Toro, Barbra; Díaz-Araya, Guillermo; Lavandero, Sergio

    2006-06-12

    NFkappaB is a participant in the process whereby cells adapt to stress. We have evaluated the activation of NFkappaB pathway by hyperosmotic stress in cultured cardiomyocytes and its role in the activation of caspase and cell death. Exposure of cultured rat cardiomyocytes to hyperosmotic conditions induced phosphorylation of IKKalpha/beta as well as degradation of IkappaBalpha. All five members of the NFkappaB family were identified in cardiomyocytes. Analysis of the subcellular distribution of NFkappaB isoforms in response to hyperosmotic stress showed parallel migration of p65 and RelB from the cytosol to the nucleus. Measurement of the binding of NFkappaB to the consensus DNA kappaB-site binding by EMSA revealed an oscillatory profile with maximum binding 1, 2 and 6h after initiation of the hyperosmotic stress. Supershift analysis revealed that p65 and RelB (but not p50, p52 or cRel) were involved in the binding of NFkappaB to DNA. Hyperosmotic stress also resulted in activation of the NFkappaB-lux reporter gene, transient activation of caspases 9 and 3 and phosphatidylserine externalization. The effect on cell viability was not prevented by ZVAD (a general caspase inhibitor). Blockade of NFkappaB with AdIkappaBalpha, an IkappaBalpha dominant negative overexpressing adenovirus, prevented activation of caspase 9 (more than that caspase 3) but did not affect cell death in hyperosmotically stressed cardiomyocytes. We conclude that hyperosmotic stress activates p65 and RelB NFkappaB isoforms and NFkappaB mediates caspase 9 activation in cardiomyocytes. However cell death triggered by hyperosmotic stress was caspase- and NFkappaB-independent.

  16. Silencing of the DEK gene induces apoptosis and senescence in CaSki cervical carcinoma cells via the up-regulation of NF-κB p65.

    PubMed

    Liu, Kuiran; Feng, Tianda; Liu, Jie; Zhong, Ming; Zhang, Shulan

    2012-06-01

    The human DEK proto-oncogene has been found to play an important role in autoimmune disease, viral infection and human carcinogenesis. Although it is transcriptionally up-regulated in cervical cancer, its intracellular function and regulation is still unexplored. In the present study, DEK and IκBα [inhibitor of NF-κB (nuclear factor κB) α] shRNAs (short hairpin RNAs) were constructed and transfected into CaSki cells using Lipofectamine™. The stable cell line CaSki-DEK was obtained after G418 selection. CaSki-IκB cells were observed at 48 h after psiRNA-IκB transfection. The inhibitory efficiency of shRNAs were detected by RT (reverse transcription)-PCR and Western blot analysis. The proliferation activity of cells were measured using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, cell apoptosis was measured using an Annexin V/PI (propidium iodide) kit, the cell cycle was analysed by flow cytometry and cell senescence was detected using senescence β-galactosidase staining. The intracellular expression of NF-κB p65 protein was studied by cytochemistry. The expression levels of NF-κB p65, p50, c-Rel, IκBα and phospho-IκBα protein were analysed by immunoblotting in whole-cell lysates, cytosolic fractions and nuclear extracts. The protein expression and activity of p38 and JNK (c-Jun N-terminal kinase) were also assayed. In addition, the NF-κB p65 DNA-binding activity was measured by ELISA. Following the silencing of DEK and IκBα, cell proliferation was inhibited, apoptosis was increased, the cell cycle was blocked in the G0/G1-phase with a corresponding decrease in the G2/M-phase, and cell senescence was induced. All of these effects may be related to the up-regulation of NF-κB p65 expression and its nuclear translocation.

  17. Zinc finger protein 91 (ZFP91) activates HIF-1α via NF-κB/p65 to promote proliferation and tumorigenesis of colon cancer

    PubMed Central

    Ma, Juan; Mi, Chunliu; Wang, Ke Si; Lee, Jung Joon; Jin, Xuejun

    2016-01-01

    Zinc finger protein 91 (ZFP91) has been reported to be involved in various biological processes. However, the clinical significance and biological role of ZFP91 in colon cancer remains unknown. Here, we show that ZFP91 expression is upregulated in patients with colon cancer. We found that ZFP91 upregulated HIF-1α at the levels of promoter and protein in colon cancer cells. Using chromatin immunoprecipitation, electrophoretic mobility shift assay and luciferase reporter gene assay, we found that NF-κB/p65 is required for the binding of ZFP91 to the HIF-1α promoter at −197/−188 base pairs and for the transcriptional activation of HIF-1α gene mediated by ZFP91. Flow cytometry, 5-ethynyl-2′-deoxyuridine (EdU) incorporation and tumor xenograft assay demonstrated that ZFP91 enhanced cell proliferation of colon cancer through upregulating HIF-1α in vitro and in vivo. Furthermore, ZFP91 is positively associated with HIF-1α in human colon cancer. Thus, we concluded that ZFP91 activates transcriptional coregulatory protein HIF-1α through transcription factor NF-κB/p65 in the promotion of proliferation and tumorigenesis in colon cancer cell. ZFP91 may serve as a driver gene to activate HIF-1α transcription in the development of cancer. PMID:27144516

  18. Inhibition of Nuclear Transport of NF-ĸB p65 by the Salmonella Type III Secretion System Effector SpvD

    PubMed Central

    Rolhion, Nathalie; Furniss, R. Christopher D.; Grabe, Grzegorz; Ryan, Aindrias; Liu, Mei; Matthews, Sophie A.; Holden, David W.

    2016-01-01

    Salmonella enterica replicates in macrophages through the action of effector proteins translocated across the vacuolar membrane by a type III secretion system (T3SS). Here we show that the SPI-2 T3SS effector SpvD suppresses proinflammatory immune responses. SpvD prevented activation of an NF-ĸB-dependent promoter and caused nuclear accumulation of importin-α, which is required for nuclear import of p65. SpvD interacted specifically with the exportin Xpo2, which mediates nuclear-cytoplasmic recycling of importins. We propose that interaction between SpvD and Xpo2 disrupts the normal recycling of importin-α from the nucleus, leading to a defect in nuclear translocation of p65 and inhibition of activation of NF-ĸB regulated promoters. SpvD down-regulated pro-inflammatory responses and contributed to systemic growth of bacteria in mice. This work shows that a bacterial pathogen can manipulate host cell immune responses by interfering with the nuclear transport machinery. PMID:27232334

  19. Protective effect of tea polyphenols on renal ischemia/reperfusion injury via suppressing the activation of TLR4/NF-κB p65 signal pathway.

    PubMed

    Li, Yan-Wei; Zhang, Yan; Zhang, Ling; Li, Xu; Yu, Jian-Bo; Zhang, Hong-Tao; Tan, Bin-Bin; Jiang, Lian-Hao; Wang, Ya-Xin; Liang, Yu; Zhang, Xiu-Shan; Wang, Wen-Sheng; Liu, Hai-Gen

    2014-05-25

    Tea polyphenols (TP) was investigated in rats for its protective effect on renal ischemia/reperfusion injury (RIRI). Rats were randomized into groups as follows: (I) sham group (n=10); (II) RIRI group (n=10); (III) RIRI+TP (100mg/kg) group (n=5); (IV) RIRI+TP (200mg/kg) group (n=5); (V) RIRI+TP+ Astragalus mongholicus aqueous extract (AMAE) (300 mg/kg+100mg/kg) group (n=5). For the IRI+TP groups, rats were orally given with tea polyphenols (100, 200 and 300 mg/kg body weight) once daily 10 days before induction of ischemia, followed by renal IRI. For the sham group and RIRI group, rats were orally given with equal volume of saline once daily 10 days before induction of ischemia, followed by renal IRI. Results showed that tea polyphenol pretreatment significantly suppressed ROS level and MDA release. On the other hand, in rats subjected to ischemia-reperfusion, the activities of endogenous antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) showed recovery, whereas the levels of urea nitrogen and serum creatinine were reduced by administration of tea polyphenols orally for 10 days prior to ischemia-reperfusion. Moreover, tea polyphenol pretreatment significantly decreased TLR4 and NF-κB p65 protein expression levels in RIRI rats. At the same time, tea polyphenol pretreatment attenuated the increased level of serum IL-1β, IL-6, ICAM-1 and TNF-α, and enhanced IL-10 production in RIRI rats. Furthermore, tea polyphenol pretreatment significantly decreased renal epithelial tubular cell apoptosis induced by renal ischemia/reperfusion, alleviating renal ischemia/reperfusion injury. These results cumulatively indicate that tea polyphenol pretreatment could suppress the TLR4/NF-κB p65 signaling pathway, protecting renal tubular epithelial cells against ischemia/reperfusion-induced apoptosis, which implies that antioxidants may be a potential and effective agent for prevention of the

  20. Hyperforin attenuates microglia activation and inhibits p65-Ser276 NFκB phosphorylation in the rat piriform cortex following status epilepticus.

    PubMed

    Lee, Sang-Kyu; Kim, Ji-Eun; Kim, Yeon-Joo; Kim, Min-Ju; Kang, Tae-Cheon

    2014-08-01

    Hyperforin, a lipophilic constituent of medicinal herb St. John's Wort, has neurobiological effects including antidepressant activity, antibiotic potency, anti-inflammatory activity and anti-tumoral properties. Furthermore, hyperforin activates transient receptor potential conical channel-6 (TRPC6), a nonselective cation channel. To elucidate the roles of hyperforin and TRPC6 in neuroinflammation in vivo, we investigated the effect of hyperforin on neuroinflammatory responses and its related events in the rat piriform cortex (PC) following status epilepticus (SE). Hyperforin attenuated microglial activation, p65-serine 276 NFκB phosphorylation, and suppressed TNF-α expression in the PC following SE. Hyperforin also effectively alleviated SE-induced vasogenic edema formation, neuronal damage, microglial TRPC6 induction and blood-derived monocyte infiltration. Our findings suggest that hyperforin may effectively attenuate microglia-mediated neuroinflammation in the TRPC6-independent manner.

  1. [Construction and influence of human nuclear factor-κB p65 shRNA lentiviral 
vector on malignant biological behavior of lung cancer cells].

    PubMed

    Guo, Hongjuan; Zhu, Guangfa; Wu, Chunting

    2014-05-01

    背景与目的 核因子-κB作为重要转录因子,与多种恶性肿瘤的发生发展有着密切联系,直接或间接抑制NF-κBp65的表达可逆转肿瘤细胞生物学行为。构建人核因子-κBp65基因shRNA重组质粒,感染A549细胞并筛选出稳定细胞株,对其迁移、粘附能力进行鉴定。方法 设计并合成人核因子-κBp65的乱序对照序列(scramble)和干扰序列(shRNA)构建重组质粒,在5’端引入一个BamHI位点,3’端引入一个XhoI位点和EcoRI位点;感染A549细胞并由嘌呤霉素做稳转株的筛选;应用Western blot、qRT-PCR技术检测NF-κBp65的干扰效果及IκBα蛋白表达水平的变化;Transwell、MTT法分析其对A549细胞迁移、粘附能力的影响。结果 成功构建重组质粒并筛选出A549/NF-κB p65 scramble稳转株和A549/NF-κB p65 shRNA稳转株;A549/NF-κB p65 shRNA稳转细胞株与A549/NF-κB p65 scramble稳转细胞株及A549细胞相比NF-κBp65的mRNA、蛋白表达水平均下调;IκBα蛋白水平明显下调;细胞迁移能力及粘附能力均降低。结论 本实验通过RNA干扰技术构建的重组慢病毒可有效抑制NF-κBp65 mRNA和蛋白的表达水平;抑制NF-κBp65可明显降低A549细胞的迁移和粘附能力。

  2. Coal dust contiguity-induced changes in the concentration of TNF-alpha and NF-kappa B p65 on the ocular surface.

    PubMed

    Sun, Zhaoyi; Hong, Jing; Liu, Zaiying; Jin, Xiudong; Gu, Changhong

    2009-01-01

    To observe the influence of coal dust on ocular surface of coal miners and rabbits with coal dust contiguity on expression TNF-alpha and NF-kappa Bp65 and dry eye occurrence. Tear production, BUT and lysozyme decreased for coal miners and rabbits with coal dust contiguity. Expression TNF-alpha and NF-kappa Bp65 in ocular surface were determined. Results showed tear production, BUT and lysozyme decreased for coal miners and rabbits with coal dust contiguity. Coal dust exposure was linked to development of xerophthalmia, and induced a higher expression of NF-kappa B p65 and TNF-alpha perhaps as a mechanism to resist coal dust ocular surface injury.

  3. Immunohistochemical analysis of the expression of cellular transcription NFκB (p65), AP-1 (c-Fos and c-Jun), and JAK/STAT in leprosy.

    PubMed

    Silva, Luciana Mota; Hirai, Kelly Emi; de Sousa, Jorge Rodrigues; de Souza, Juarez; Fuzii, Hellen Thais; Dias, Leonidas Braga; Carneiro, Francisca Regina Oliveira; de Souza Aarão, Tinara Leila; Quaresma, Juarez Antonio Simões

    2015-05-01

    Leprosy is a disease whose clinical spectrum depends on the cytokine patterns produced during the early stages of the immune response. The main objective of this study was to describe the activation pattern of cellular transcription factors and to correlate these factors with the clinical forms of leprosy. Skin samples were obtained from 16 patients with the tuberculoid (TT) form and 14 with the lepromatous (LL) form. The histologic sections were immunostained with anti-c-Fos and anti-c-Jun monoclonal antibodies for investigation of AP-1, anti-NFκB p65 for the study of NFκB, and anti-JAK2, STAT1, STAT3, and STAT4 for investigation of the JAK/STAT pathway. Cells expressing STAT1 were more frequent in the TT form than in LL lesions (P = .0096), in agreement with the protective immunity provided by IFN-γ. STAT4 was also more highly expressed in the TT form than in the LL form (P = .0098). This transcription factor is essential for the development of a Th1 response because it is associated with interleukin-12. NFκB (p65) and STAT4 expression in the TT form showed a strong and significant correlation (r = 0.7556 and P = .0007). A moderate and significant correlation was observed between JAK2 and STAT4 in the TT form (r = 0.6637 and P = .0051), with these factors responding to interleukin-12 in Th1 profiles. The results suggest that STAT1, JAK2, and NFκB, together with STAT4, contribute to the development of cell-mediated immunity, which is able to contain the proliferation of Mycobacterium leprae.

  4. Upregulation of mGlu2 receptors via NF-κB p65 acetylation is involved in the Proneurogenic and antidepressant effects of acetyl-L-carnitine.

    PubMed

    Cuccurazzu, Bruna; Bortolotto, Valeria; Valente, Maria Maddalena; Ubezio, Federica; Koverech, Aleardo; Canonico, Pier Luigi; Grilli, Mariagrazia

    2013-10-01

    Acetyl-L-carnitine (ALC) is a naturally occurring molecule with an important role in cellular bioenergetics and as donor of acetyl groups to proteins, including NF-κB p65. In humans, exogenously administered ALC has been shown to be effective in mood disturbances, with a good tolerability profile. No current information is available on the antidepressant effect of ALC in animal models of depression and on the putative mechanism involved in such effect. Here we report that ALC is a proneurogenic molecule, whose effect on neuronal differentiation of adult hippocampal neural progenitors is independent of its neuroprotective activity. The in vitro proneurogenic effects of ALC appear to be mediated by activation of the NF-κB pathway, and in particular by p65 acetylation, and subsequent NF-κB-mediated upregulation of metabotropic glutamate receptor 2 (mGlu2) expression. When tested in vivo, chronic ALC treatment could revert depressive-like behavior caused by unpredictable chronic mild stress, a rodent model of depression with high face validity and predictivity, and its behavioral effect correlated with upregulated expression of mGlu2 receptor in hippocampi of stressed mice. Moreover, chronic, but not acute or subchronic, drug treatment significantly increased adult born neurons in hippocampi of stressed and unstressed mice. We now propose that this mechanism could be potentially involved in the antidepressant effect of ALC in humans. These results are potentially relevant from a clinical perspective, as for its high tolerability profile ALC may be ideally employed in patient subpopulations who are sensitive to the side effects associated with classical antidepressants.

  5. Endothelial lipase is upregulated by interleukin-6 partly via the p38 MAPK and p65 NF-κB signaling pathways

    PubMed Central

    Yue, Xin; Wu, Minghui; Jiang, Hong; Hao, Jing; Zhao, Qinghao; Zhu, Qing; Saren, Gaowa; Zhang, Yun; Zhang, Xiaoli

    2016-01-01

    To investigate the effects of inflammatory factor interleukin (IL)-6 on the expression of endothelial lipase (EL) and its potential signaling pathways in atherosclerosis, a primary culture of human umbilical vein endothelial cells (HUVECs) was established and treated as follows: i) Control group without any treatment; ii) recombinant human (rh)IL-6 treatment (10 ng/ml) for 0, 4, 8, 12 and 24 h; iii) p38 mitogen-activated protein kinases (MAPKs) inhibitor (SB203580, 10 µmol/l) pretreatment for 1 h prior to rhIL-6 (10 ng/ml) treatment; iv) nuclear factor (NF)-κB activation inhibitor (pyrrolidine dithiocarbamate, 10 mmol/l) pretreatment for 1 h prior to rhIL-6 (10 ng/ml) treatment. EL levels were detected by immunocytochemical staining and western blot analysis. Proliferation of HUVECs was detected by immunostaining of proliferating cell nuclear antigen (PCNA) and an MTT assay. p38 MAPK and NF-κB p65 levels were detected by western blotting. The results showed that rhIL-6 treatment increased EL expression and proliferation of HUVECs. NF-κB p65 and MAPK p38 protein levels also increased in a time-dependent manner in HUVECs after rhIL-6 treatment. NF-κB inhibitor and MAPK p38 inhibitor prevented the effects of rhIL-6 on EL expression. In conclusion, inflammatory factor IL-6 may participate in the pathogenesis of atherosclerosis by increasing EL expression and the proliferation of endothelial cells via the p38 MAPK and NF-κB signaling pathways. PMID:27430252

  6. Morris Water Maze Training in Mice Elevates Hippocampal Levels of Transcription Factors Nuclear Factor (Erythroid-derived 2)-like 2 and Nuclear Factor Kappa B p65

    PubMed Central

    Snow, Wanda M.; Pahlavan, Payam S.; Djordjevic, Jelena; McAllister, Danielle; Platt, Eric E.; Alashmali, Shoug; Bernstein, Michael J.; Suh, Miyoung; Albensi, Benedict C.

    2015-01-01

    Research has identified several transcription factors that regulate activity-dependent plasticity and memory, with cAMP-response element binding protein (CREB) being the most well-studied. In neurons, CREB activation is influenced by the transcription factor nuclear factor kappa B (NF-κB), considered central to immunity but more recently implicated in memory. The transcription factor early growth response-2 (Egr-2), an NF-κB gene target, is also associated with learning and memory. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an antioxidant transcription factor linked to NF-κB in pathological conditions, has not been studied in normal memory. Given that numerous transcription factors implicated in activity-dependent plasticity demonstrate connections to NF-κB, this study simultaneously evaluated protein levels of NF-κB, CREB, Egr-2, Nrf2, and actin in hippocampi from young (1 month-old) weanling CD1 mice after training in the Morris water maze, a hippocampal-dependent spatial memory task. After a 6-day acquisition period, time to locate the hidden platform decreased in the Morris water maze. Mice spent more time in the target vs. non-target quadrants of the maze, suggestive of recall of the platform location. Western blot data revealed a decrease in NF-κB p50 protein after training relative to controls, whereas NF-κB p65, Nrf2 and actin increased. Nrf2 levels were correlated with platform crosses in nearly all tested animals. These data demonstrate that training in a spatial memory task results in alterations in and associations with particular transcription factors in the hippocampus, including upregulation of NF-κB p65 and Nrf2. Training-induced increases in actin protein levels caution against its use as a loading control in immunoblot studies examining activity-dependent plasticity, learning, and memory. PMID:26635523

  7. L-arginine and aminoguanidine reduce colonic damage of acetic acid-induced colitis in rats: potential modulation of nuclear factor-κB/p65.

    PubMed

    Farghaly, Hanan S M; Thabit, Romany H

    2014-10-01

    The transcription factor, nuclear factor-κB (NF-κB) is a key inducer of inducible nitric oxide synthase (iNOS) gene expression. The aim of the present study was to investigate the potential protective effect of l-arginine (Arg; nitric oxide precursor) and aminoguanidine (inducible nitric oxide synthase inhibitor) against acetic acid (AA)-induced colitis in rats, and the potential role of NF-κB. Colitis was induced by intrarectal inoculation of rats with 4% acetic acid for three consecutive days. The effect of Arg and aminoguanidine on nitric oxide levels was assessed by Greiss assay and protein expression of NF-κB/p65, and inducible nitric oxide synthase was also investigated by immunohistochemistry. Slides were examined using ImageJ, and results reported as the percent area positive for each marker. Intrarectal AA caused a significant increase in bodyweight loss and colon weights. Arg at 100 mg/day for 7 days before induction of colitis diminished the changes in both bodyweight loss and colon weights. Furthermore, Arg attenuated the colonic tissues macroscopic and microscopic damage induced by acetic acid. In addition, i.p. AG 100 mg/kg given during and after induction of colitis recovered the colonic ulcerative lesion induced by AA. Arg can protect against colonic inflammation; an effect that probably be attributed to its nitric oxide-donating property, resulting in modulatory effects on the expression of NF-κB/p65 in the colon tissues. The results suggested that Arg might reduce the inflammation associated with colitis as confirmed by histopathological investigations. Arg might inhibit AA-induced colitis through the NF-κB/nitric oxide pathway.

  8. Sodium channel auxiliary subunits.

    PubMed

    Tseng, Tsai-Tien; McMahon, Allison M; Johnson, Victoria T; Mangubat, Erwin Z; Zahm, Robert J; Pacold, Mary E; Jakobsson, Eric

    2007-01-01

    Voltage-gated ion channels are well known for their functional roles in excitable tissues. Excitable tissues rely on voltage-gated ion channels and their auxiliary subunits to achieve concerted electrical activity in living cells. Auxiliary subunits are also known to provide functional diversity towards the transport and biogenesis properties of the principal subunits. Recent interests in pharmacological properties of these auxiliary subunits have prompted significant amounts of efforts in understanding their physiological roles. Some auxiliary subunits can potentially serve as drug targets for novel analgesics. Three families of sodium channel auxiliary subunits are described here: beta1 and beta3, beta2 and beta4, and temperature-induced paralytic E (TipE). While sodium channel beta-subunits are encoded in many animal genomes, TipE has only been found exclusively in insects. In this review, we present phylogenetic analyses, discuss potential evolutionary origins and functional data available for each of these subunits. For each family, we also correlate the functional specificity with the history of evolution for the individual auxiliary subunits.

  9. IDENTIFICATION OF NF-KAPPAB IN THE MARINE FISH STENOTOMUS CHRYSOPS AND EXAMINATION OF ITS ACTIVATION BY ARYL HYDROCARBON RECEPTOR AGONISTS. (R827102)

    EPA Science Inventory

    Abstract

    Members of the Rel family of proteins have been identified in Drosophila, an echinoderm, Xenopus, birds and mammals. Dimers of Rel proteins form the transcription factor nuclear factor Microglial CX3CR1 promotes adult neurogenesis by inhibiting Sirt 1/p65 signaling independent of CX3CL1.

    PubMed

    Sellner, Sabine; Paricio-Montesinos, Ricardo; Spieß, Alena; Masuch, Annette; Erny, Daniel; Harsan, Laura A; Elverfeldt, Dominik V; Schwabenland, Marius; Biber, Knut; Staszewski, Ori; Lira, Sergio; Jung, Steffen; Prinz, Marco; Blank, Thomas

    2016-09-17

    Homo and heterozygote cx3cr1 mutant mice, which harbor a green fluorescent protein (EGFP) in their cx3cr1 loci, represent a widely used animal model to study microglia and peripheral myeloid cells. Here we report that microglia in the dentate gyrus (DG) of cx3cr1 (-/-) mice displayed elevated microglial sirtuin 1 (SIRT1) expression levels and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 activation, despite unaltered morphology when compared to cx3cr1 (+/-) or cx3cr1 (+/+) controls. This phenotype was restricted to the DG and accompanied by reduced adult neurogenesis in cx3cr1 (-/-) mice. Remarkably, adult neurogenesis was not affected by the lack of the CX3CR1-ligand, fractalkine (CX3CL1). Mechanistically, pharmacological activation of SIRT1 improved adult neurogenesis in the DG together with an enhanced performance of cx3cr1 (-/-) mice in a hippocampus-dependent learning and memory task. The reverse condition was induced when SIRT1 was inhibited in cx3cr1 (-/-) mice, causing reduced adult neurogenesis and lowered hippocampal cognitive abilities. In conclusion, our data indicate that deletion of CX3CR1 from microglia under resting conditions modifies brain areas with elevated cellular turnover independent of CX3CL1.

  10. Green tea catechins alone or in combination alter functional parameters of human neutrophils via suppressing the activation of TLR-4/NFκB p65 signal pathway.

    PubMed

    Marinovic, M P; Morandi, A C; Otton, R

    2015-10-01

    The purpose of this study was to evaluate the potential of a mixture containing the four main catechins found in green tea, as well it separately, as modulators of the functional parameters of human neutrophils. The cells were obtained from peripheral blood of healthy individuals isolated and cultured with a mix: 30 μM of EGCG, 3 μM of EGC, 2 μM of ECG and 1.4 μM of EC, as well as each one alone. We evaluated the cytotoxicity of catechins, production of several reactive oxygen species (ROS), antioxidant enzymes (SOD, CAT, GPx and GR), Nrf2, TLR4/IKK/NFκB, CD11b mRNA levels, intracellular calcium release, chemotactic and phagocytic capacity, myeloperoxidase (MPO), and G6PDH activities, hypochlorous acid (HOCl) and pro-inflammatory cytokines release, protein levels of TLR4, p38 MAPK, iNOS and p-65 NFκB. The actions of the catechins were evidenced by the reduction in inflammatory parameters, including the suppression of TLR4, NFκB and iNOS protein expression, decreased release of TNF-α, IL-1β and IL-6, migration capacity, MPO activity and HOCl production and the suppression of ROS, nitric oxide and peroxynitrite production, while inducing antioxidant enzyme activities and Nrf2 mRNA levels, phagocytic capacity and calcium release. Our results demonstrate that catechins present marked immunomodulatory actions, either alone or in combination.

  11. Astaxanthin Inhibits Proliferation and Induces Apoptosis of Human Hepatocellular Carcinoma Cells via Inhibition of Nf-Κb P65 and Wnt/Β-Catenin in Vitro

    PubMed Central

    Li, Jingjing; Dai, Weiqi; Xia, Yujing; Chen, Kan; Li, Sainan; Liu, Tong; Zhang, Rong; Wang, Jianrong; Lu, Wenxia; Zhou, Yuqing; Yin, Qin; Abudumijiti, Huerxidan; Chen, Rongxia; Zheng, Yuanyuan; Wang, Fan; Lu, Jie; Zhou, Yingqun; Guo, Chuanyong

    2015-01-01

    Hepatocellular carcinoma (HCC) is a malignant tumor that can cause systemic invasion; however, the exact etiology and molecular mechanism are unknown. Astaxanthin (ASX), a powerful antioxidant, has efficient anti-oxidant, anti-inflammatory, and other activities, and has great research prospects in cancer therapy. We selected the human hepatoma cell lines, LM3 and SMMC-7721, to study the anti-tumor effect and related mechanisms of ASX. The cell lines were treated with different concentrations of ASX, and its solvent DMSO as a control, for different time periods and the results were determined using CCK8, qRT-PCR, WB, apoptotic staining, and flow cytometry. ASX induced significant apoptosis of HCC cells, and its effect may have been caused by NF-κB p65 and Wnt/β-catenin down-regulation via negative activation of PI3K/Akt and ERK. Antitumor research on ASX has provided us with a potential therapy for patients with hepatomas. PMID:26404320

  12. Hyperinvasive Meningococci Induce Intra-nuclear Cleavage of the NF-κB Protein p65/RelA by Meningococcal IgA Protease

    PubMed Central

    Besbes, Anissa; Le Goff, Salomé; Antunes, Ana; Terrade, Aude; Hong, Eva; Giorgini, Dario; Taha, Muhamed-Kheir; Deghmane, Ala-Eddine

    2015-01-01

    Differential modulation of NF-κB during meningococcal infection is critical in innate immune response to meningococcal disease. Non-invasive isolates of Neisseria meningitidis provoke a sustained NF-κB activation in epithelial cells. However, the hyperinvasive isolates of the ST-11 clonal complex (ST-11) only induce an early NF-κB activation followed by a sustained activation of JNK and apoptosis. We show that this temporal activation of NF-κB was caused by specific cleavage at the C-terminal region of NF-κB p65/RelA component within the nucleus of infected cells. This cleavage was mediated by the secreted 150 kDa meningococcal ST-11 IgA protease carrying nuclear localisation signals (NLS) in its α-peptide moiety that allowed efficient intra-nuclear transport. In a collection of non-ST-11 healthy carriage isolates lacking NLS in the α-peptide, secreted IgA protease was devoid of intra-nuclear transport. This part of iga polymorphism allows non-invasive isolates lacking NLS, unlike hyperinvasive ST-11 isolates of N. meningitides habouring NLS in their α-peptide, to be carried asymptomatically in the human nasopharynx through selective eradication of their ability to induce apoptosis in infected epithelial cells. PMID:26241037

  13. Thiamet G mediates neuroprotection in experimental stroke by modulating microglia/macrophage polarization and inhibiting NF-κB p65 signaling.

    PubMed

    He, Yating; Ma, Xiaofeng; Li, Daojing; Hao, Junwei

    2016-01-01

    Inflammatory responses are accountable for secondary injury induced by acute ischemic stroke (AIS). Previous studies indicated that O-GlcNAc modification (O-GlcNAcylation) is involved in the pathology of AIS, and increase of O-GlcNAcylation by glucosamine attenuated the brain damage after ischemia/reperfusion. Inhibition of β-N-acetylglucosaminidase (OGA) with thiamet G (TMG) is an alternative option for accumulating O-GlcNAcylated proteins. In this study, we investigate the neuroprotective effect of TMG in a mouse model of experimental stroke. Our results indicate that TMG administration either before or after middle cerebral artery occlusion (MCAO) surgery dramatically reduced infarct volume compared with that in untreated controls. TMG treatment ameliorated the neurological deficits and improved clinical outcomes in neurobehavioral tests by modulating the expression of pro-inflammatory and anti-inflammatory cytokines. Additionally, TMG administration reduced the number of Iba1(+) cells in MCAO mice, decreased expression of the M1 markers, and increased expression of the M2 markers in vivo. In vitro, M1 polarization of BV2 cells was inhibited by TMG treatment. Moreover, TMG decreased the expression of iNOS and COX2 mainly by suppressing NF-κB p65 signaling. These results suggest that TMG exerts a neuroprotective effect and could be useful as an anti-inflammatory agent for ischemic stroke therapy.

  14. The proximal promoter of a novel interleukin-8-encoding gene in rainbow trout (Oncorhynchus mykiss) is strongly induced by CEBPA, but not NF-κB p65.

    PubMed

    Rebl, Alexander; Rebl, Henrike; Korytář, Tomáš; Goldammer, Tom; Seyfert, Hans-Martin

    2014-10-01

    Interleukin-8 (IL8) is an immediate-early chemokine that has been well characterized in several fish species. Ten IL8 gene variants have already been described in rainbow trout, but none of their promoters has structurally been defined or functionally characterized in teleost fish. To uncover key factors regulating IL8 expression, we intended to functionally characterize an IL8 promoter from rainbow trout. Incidentally, we isolated a novel IL8 gene variant (IL8-G). It is structurally highly similar to the other trout IL8 gene variants and its mRNA concentration increased significantly in secondary lymphoid tissues after infecting healthy fish with Aeromonas salmonicida. The proximal promoter sequence of the IL8-G-encoding gene features in close proximity two consensus elements for CEBP attachment. The proximal site overlaps with a NF-κB-binding site. Cotransfection of an IL8-G promoter-driven reporter gene together with vectors expressing various mammalian CEBP or NF-κB factors revealed in human HEK-293 cells that CEBPA and NF-κB p50, but not NF-κB p65 activate this promoter. The stimulatory effect of NF-κB p50 is likely conveyed by synergizing with CEBPA. Deletion or mutation of either the distal or the proximal CEBP-binding site, respectively, caused a significant decrease in IL8-G promoter activation. We confirmed the significance of the CEBPA factor for IL8-G expression by comparing the stimulatory capacity of the trout CEBPA and -B factors, thereby reducing the evolutionary distance in the inter-species expression assays. Similar promoter induction potential and intracellular localization of the mammalian and teleostean CEBPA and -B factors suggests their functional conservation throughout evolution.

  15. TRAF7 protein promotes Lys-29-linked polyubiquitination of IkappaB kinase (IKKgamma)/NF-kappaB essential modulator (NEMO) and p65/RelA protein and represses NF-kappaB activation.

    PubMed

    Zotti, Tiziana; Uva, Antonio; Ferravante, Angela; Vessichelli, Mariangela; Scudiero, Ivan; Ceccarelli, Michele; Vito, Pasquale; Stilo, Romania

    2011-07-01

    Tumor necrosis factor receptor-associated factor (TRAF) proteins are cytoplasmic regulatory molecules that function as signal transducers for receptors involved in both innate and adaptive humoral immune responses. In this study, we show that TRAF7, the unique noncanonical member of the TRAF family, physically associates with IκB kinase/NF-κB essential modulator (NEMO) and with the RelA/p65 (p65) member of the NF-κB transcription factor family. TRAF7 promotes Lys-29-linked polyubiquitination of NEMO and p65 that results in lysosomal degradation of both proteins and altered activation. TRAF7 also influences p65 nuclear distribution. Microarray expression data are consistent with an inhibitory role for TRAF7 on NF-κB and a positive control of AP-1 transcription factor. Finally, functional data indicate that TRAF7 promotes cell death. Thus, this study identifies TRAF7 as a NEMO- and p65-interacting molecule and brings important information on the ubiquitination events that control NF-κB transcriptional activity.

  16. TRAF7 Protein Promotes Lys-29-linked Polyubiquitination of IκB Kinase (IKKγ)/NF-κB Essential Modulator (NEMO) and p65/RelA Protein and Represses NF-κB Activation

    PubMed Central

    Zotti, Tiziana; Uva, Antonio; Ferravante, Angela; Vessichelli, Mariangela; Scudiero, Ivan; Ceccarelli, Michele; Vito, Pasquale; Stilo, Romania

    2011-01-01

    Tumor necrosis factor receptor-associated factor (TRAF) proteins are cytoplasmic regulatory molecules that function as signal transducers for receptors involved in both innate and adaptive humoral immune responses. In this study, we show that TRAF7, the unique noncanonical member of the TRAF family, physically associates with IκB kinase/NF-κB essential modulator (NEMO) and with the RelA/p65 (p65) member of the NF-κB transcription factor family. TRAF7 promotes Lys-29-linked polyubiquitination of NEMO and p65 that results in lysosomal degradation of both proteins and altered activation. TRAF7 also influences p65 nuclear distribution. Microarray expression data are consistent with an inhibitory role for TRAF7 on NF-κB and a positive control of AP-1 transcription factor. Finally, functional data indicate that TRAF7 promotes cell death. Thus, this study identifies TRAF7 as a NEMO- and p65-interacting molecule and brings important information on the ubiquitination events that control NF-κB transcriptional activity. PMID:21518757

  17. Experimental autoimmune encephalomyelitis: Association with mutual regulation of RelA (p65)/NF-{kappa}B and phospho-I{kappa}B in the CNS

    SciTech Connect

    Hwang, Insun; Ha, Danbee; Ahn, Ginnae; Park, Eunjin; Joo, Haejin; Jee, Youngheun

    2011-07-29

    Highlights: {yields} The phosphorylation of RelA's inhibitory factor I{kappa}B and subsequent RelA activation are important to the disease process of EAE. {yields} The expression of RelA and phospho-I{kappa}B was markedly increased in the initiation and during the progression of EAE. {yields} TPCK-treated EAE mice showed lower incidence of EAE with less severe symptoms and quicker recovery than vehicle-treated EAE mice. {yields} TPCK significantly suppressed the MOG{sub 35-55}-specific T cell proliferation by reducing the production of IFN-{gamma} and IL-17 cytokines in EAE. {yields} The NF-{kappa}B cascade's activity increased gradually with the development of symptoms and brain pathology of EAE. -- Abstract: Recently emerging evidence that the NF-{kappa}B family plays an important role in autoimmune disease has produced very broad and sometimes paradoxical conclusions. In the present study, we elucidated that the activation of RelA (p65) of NF-{kappa}B and I{kappa}B dissociation assumes a distinct role in experimental autoimmune encephalomyelitis (EAE) progression by altering I{kappa}B phosphorylation and/or degradation. In the present study of factors that govern EAE, the presence and immunoreactivity of nuclear RelA and phospho-I{kappa}B were recorded at the initiation and peak stage, and degradation of I{kappa}B{alpha} progressed rapidly at an early stage then stabilized during recovery. The immunoreactivity to RelA and phospho-I{kappa}B occurred mainly in inflammatory cells and microglial cells but only slightly in astrocytes. Subsequently, the blockade of I{kappa}B dissociation from NF-{kappa}B reduced the severity of disease by decreasing antigen-specific T cell response and production of IL-17 in EAE. Thus, blocking the dissociation of I{kappa}B from NF-{kappa}B can be utilized as a strategy to inhibit the NF-{kappa}B signal pathway thereby to reduce the initiation, progression, and severity of EAE.

  18. HAT-P-65b and HAT-P-66b: Two Transiting Inflated Hot Jupiters and Observational Evidence for the Reinflation of Close-in Giant Planets

    NASA Astrophysics Data System (ADS)

    Hartman, J. D.; Bakos, G. Á.; Bhatti, W.; Penev, K.; Bieryla, A.; Latham, D. W.; Kovács, G.; Torres, G.; Csubry, Z.; de Val-Borro, M.; Buchhave, L.; Kovács, T.; Quinn, S.; Howard, A. W.; Isaacson, H.; Fulton, B. J.; Everett, M. E.; Esquerdo, G.; Béky, B.; Szklenar, T.; Falco, E.; Santerne, A.; Boisse, I.; Hébrard, G.; Burrows, A.; Lázár, J.; Papp, I.; Sári, P.

    2016-12-01

    We present the discovery of the transiting exoplanets HAT-P-65b and HAT-P-66b, with orbital periods of 2.6055 and 2.9721 days, masses of 0.527+/- 0.083 {M}{{J}} and 0.783+/- 0.057 {M}{{J}}, and inflated radii of 1.89+/- 0.13 {R}{{J}} and {1.59}-0.10+0.16 {R}{{J}}, respectively. They orbit moderately bright (V=13.145+/- 0.029 and V=12.993+/- 0.052) stars of mass 1.212+/- 0.050 {M}⊙ and {1.255}-0.054+0.107 {M}⊙ . The stars are at the main-sequence turnoff. While it is well known that the radii of close-in giant planets are correlated with their equilibrium temperatures, whether or not the radii of planets increase in time as their hosts evolve and become more luminous is an open question. Looking at the broader sample of well-characterized close-in transiting giant planets, we find that there is a statistically significant correlation between planetary radii and the fractional ages of their host stars, with a false-alarm probability of only 0.0041%. We find that the correlation between the radii of planets and the fractional ages of their hosts is fully explained by the known correlation between planetary radii and their present-day equilibrium temperatures; however, if the zero-age main-sequence equilibrium temperature is used in place of the present-day equilibrium temperature, then a correlation with age must also be included to explain the planetary radii. This suggests that, after contracting during the pre-main-sequence, close-in giant planets are reinflated over time due to the increasing level of irradiation received from their host stars. Prior theoretical work indicates that such a dynamic response to irradiation requires a significant fraction of the incident energy to be deposited deep within the planetary interiors. Based on observations obtained with the Hungarian-made Automated Telescope Network. Based on observations obtained at the W. M. Keck Observatory, which is operated by the University of California and the California Institute of Technology

  19. Acupuncture Decreases NF-κB p65, miR-155, and miR-21 and Increases miR-146a Expression in Chronic Atrophic Gastritis Rats

    PubMed Central

    Zhang, Jialing; Huang, Kangbai; Zhong, Guoxin; Huang, Yong; Li, Suhe; Qu, Shanshan; Zhang, Jiping

    2016-01-01

    Acupuncture has been used to treat chronic atrophic gastritis (CAG) in traditional Chinese medicine (TCM) for centuries. In this study, we evaluated the effect of acupuncture at Zusanli (ST36), Zhongwan (CV12), and Pishu (BL20) acupoints on weight changes of rats, histological changes of gastric glands, and expressions changes of nuclear factor-kappa B (NF-κB) p65, microRNA- (miR-) 155, miR-21, and miR-146a in CAG rats induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) combined with irregular diet. Consequently, we found that acupuncture treatment elevated body weight of rats significantly when compared to the model group. By observing histological changes, we found that the acupuncture group showed better improvement of gastric mucosa injury than the model group. Our results also demonstrated upregulation of NF-κB p65, miR-155, and miR-21 in gastric tissue of CAG rats and a positive correlation between miR-155 and miR-21. Relatively, expression of miR-146a was downregulated and negative correlation relationships between miR-146a and miR-155/miR-21 in CAG rats were observed. Additionally, expressions of NF-κB p65, miR-155, and miR-21 were downregulated and miR-146a was upregulated after acupuncture treatment. Taken together, our data imply that acupuncture can downregulate NF-κB p65, miR-155, and miR-21 and upregulate miR-146a expression in CAG rats. NF-κB p65, miR-155, miR-21, and miR-146a may play important roles in therapeutic effect of acupuncture in treating CAG. PMID:27293468

  1. Salt-induced stability and serum-resistance of polyglutamate polyelectrolyte brushes/nuclear factor-κB p65 siRNA Polyplex enhance the apoptosis and efficacy of doxorubicin.

    PubMed

    Zhao, Yuefang; Qin, Yuting; Liang, Yang; Zou, Haijuan; Peng, Xiao; Huang, Huan; Lu, Ming; Feng, Min

    2013-06-10

    Short interfering RNAs (siRNAs) as chemotherapeutic RNAi agents hold great promise for a significant improvement in cancer therapy. Despite the promise, effective transport of siRNA with minimal side effects remains a challenge. The common problem associated with the low delivery efficiencies of current polycation-based gene delivery systems is their low stability in the presence of salt and serum. In the present study we developed the polyglutamate derivatives (PGS) polyelectrolyte brushes for NF-κB p65 siRNA delivery. The PGS polyelectrolyte brushes/siRNA polyplex was colloidally stable (150 nm diameter) in physiological saline (150 mM NaCl), likely due to the osmotic brushes of PGS. The size-controlled siRNA/PGS polyplex also showed the serum resistance resulting in their efficient cellular uptake was not negatively influenced by the presence of serum. The endothermic profile of ITC, their low values of Gibbs free energy and binding constants Kb under salt conditions provided the direct evidence that PGS polyelectrolyte brushes had a much lower binding affinity for serum proteins, compared with PEI 25KDa. PGS polyelectrolyte brushes delivering NF-κB p65 siRNA achieved efficient down-regulation of NF-κB p65 protein in HeLa cells. The NF-κB p65 down-regulation mediated by PGS polyelectrolyte brushes was more significant than PEI 25KDa and comparable to Lipofectamine 2000. Furthermore, the combination treatment with PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex and doxorubicin demonstrated synergistic apoptotic and cytotoxic effects on HeLa cancer cells. The high stability in physiological saline and salt-induced serum resistance of PGS polyelectrolyte brushes/siRNA polyplex has potential applications together with standard chemotherapies such as doxorubicin to be a viable method to improve the clinical outcomes in cancer therapies.

  2. Retinoic acid induction of major histocompatibility complex class I genes in NTera-2 embryonal carcinoma cells involves induction of NF-kappa B (p50-p65) and retinoic acid receptor beta-retinoid X receptor beta heterodimers.

    PubMed Central

    Segars, J H; Nagata, T; Bours, V; Medin, J A; Franzoso, G; Blanco, J C; Drew, P D; Becker, K G; An, J; Tang, T

    1993-01-01

    Retinoic acid (RA) treatment of human embryonal carcinoma (EC) NTera-2 (NT2) cells induces expression of major histocompatibility complex (MHC) class I and beta-2 microglobulin surface molecules. We found that this induction was accompanied by increased levels of MHC class I mRNA, which was attributable to the activation of the two conserved upstream enhancers, region I (NF-kappa B like) and region II. This activation coincided with the induction of nuclear factor binding activities specific for the two enhancers. Region I binding activity was not present in undifferentiated NT2 cells, but binding of an NF-kappa B heterodimer, p50-p65, was induced following RA treatment. The p50-p65 heterodimer was produced as a result of de novo induction of p50 and p65 mRNAs. Region II binding activity was present in undifferentiated cells at low levels but was greatly augmented by RA treatment because of activation of a nuclear hormone receptor heterodimer composed of the retinoid X receptor (RXR beta) and the RA receptor (RAR beta). The RXR beta-RAR beta heterodimer also bound RA responsive elements present in other genes which are likely to be involved in RA triggering of EC cell differentiation. Furthermore, transfection of p50 and p65 into undifferentiated NT2 cells synergistically activated region I-dependent MHC class I reporter activity. A similar increase in MHC class I reporter activity was demonstrated by cotransfection of RXR beta and RAR beta. These data show that following RA treatment, heterodimers of two transcription factor families are induced to bind to the MHC enhancers, which at least partly accounts for RA induction of MHC class I expression in NT2 EC cells. Images PMID:8413217

  3. iNOS as a Driver of Inflammation and Apoptosis in Mouse Skeletal Muscle after Burn Injury: Possible Involvement of Sirt1 S-Nitrosylation-Mediated Acetylation of p65 NF-κB and p53

    PubMed Central

    Yasukawa, Takashi; Ishimaru, Kazuhiro; Yasuhara, Shingo; Yu, Yong-Ming; Martyn, J. A. Jeevendra; Tompkins, Ronald. G.; Shimokado, Kentaro; Kaneki, Masao

    2017-01-01

    Inflammation and apoptosis develop in skeletal muscle after major trauma, including burn injury, and play a pivotal role in insulin resistance and muscle wasting. We and others have shown that inducible nitric oxide synthase (iNOS), a major mediator of inflammation, plays an important role in stress (e.g., burn)-induced insulin resistance. However, it remains to be determined how iNOS induces insulin resistance. Moreover, the interrelation between inflammatory response and apoptosis is poorly understood, although they often develop simultaneously. Nuclear factor (NF)-κB and p53 are key regulators of inflammation and apoptosis, respectively. Sirt1 inhibits p65 NF-κB and p53 by deacetylating these transcription factors. Recently, we have shown that iNOS induces S-nitrosylation of Sirt1, which inactivates Sirt1 and thereby increases acetylation and activity of p65 NF-κB and p53 in various cell types, including skeletal muscle cells. Here, we show that iNOS enhances burn-induced inflammatory response and apoptotic change in mouse skeletal muscle along with S-nitrosylation of Sirt1. Burn injury induced robust expression of iNOS in skeletal muscle and gene disruption of iNOS significantly inhibited burn-induced increases in inflammatory gene expression and apoptotic change. In parallel, burn increased Sirt1 S-nitrosylation and acetylation and DNA-binding capacity of p65 NF-κB and p53, all of which were reversed or ameliorated by iNOS deficiency. These results indicate that iNOS functions not only as a downstream effector but also as an upstream enhancer of burn-induced inflammatory response, at least in part, by Sirt1 S-nitrosylation-dependent activation (acetylation) of p65 NF-κB. Our data suggest that Sirt1 S-nitrosylation may play a role in iNOS-mediated enhanced inflammatory response and apoptotic change, which, in turn, contribute to muscle wasting and supposedly to insulin resistance after burn injury. PMID:28099528

  4. Highly conserved small subunit residues influence rubisco large subunit catalysis.

    PubMed

    Genkov, Todor; Spreitzer, Robert J

    2009-10-30

    The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO(2) fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O(2), it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO(2)/O(2) specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO(2)/O(2) specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit alpha-helix 8, which is a structural element of the alpha/beta-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site alpha-helix 8 and the highly variable small subunit loop between beta-strands A and B, which can also influence Rubisco CO(2)/O(2) specificity.

  5. Fumaric Acid Esters Do Not Reduce Inflammatory NF-κB/p65 Nuclear Translocation, ICAM-1 Expression and T-Cell Adhesiveness of Human Brain Microvascular Endothelial Cells.

    PubMed

    Haarmann, Axel; Nehen, Mathias; Deiß, Annika; Buttmann, Mathias

    2015-08-13

    Dimethyl fumarate (DMF) is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS) by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF) modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 µM blocked the IL-1β-induced nuclear translocation of NF-κB/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1β-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1β-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells.

  6. 1,25-Dihydroxyvitamin D3 preserves intestinal epithelial barrier function from TNF-α induced injury via suppression of NF-kB p65 mediated MLCK-P-MLC signaling pathway.

    PubMed

    Chen, Shanwen; Zhu, Jing; Chen, Guowei; Zuo, Shuai; Zhang, Junling; Chen, Ziyi; Wang, Xin; Li, Junxia; Liu, Yucun; Wang, Pengyuan

    2015-05-08

    Substantial studies have demonstrated the protective effect of 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) on intestinal barrier function, but the mechanisms are not fully illustrated. In this study, the effect of 1,25(OH)2D3 on TNF-α induced barrier dysfunction was further investigated in Caco-2 cell monolayers. The barrier function of Caco-2 monolayers was evaluated by measuring trans-epithelial electrical resistance (TEER) and FITC-Dextran 40,000 Da (FD-40) trans-membrane flux. ZO-1 and Occludin were chosen as markers of the localization of tight junction (TJ) proteins for immunofluorescence. The expression of MLCK and phosphorylation level of myosin light chain (MLC) were measured by immunoblotting. The activation of NF-kB p65 was analyzed by EMSA and immunofluorescence. The results suggest that 1,25(OH)2D3 preserves intestinal epithelial barrier function from TNF-α induced injury via suppression of NF-kB p65 mediated activation of MLCK-P-MLC signaling pathway.

  7. Surgical removal of endometrioma decreases the NF-kB1 (p50/105) and NF-kB p65 (Rel A) expression in the eutopic endometrium during the implantation window.

    PubMed

    Celik, Onder; Celik, Ebru; Turkcuoglu, Ilgin; Yilmaz, Ercan; Ulas, Mustafa; Simsek, Yavuz; Karaer, Abdullah; Celik, Nilufer; Aydin, Nasuhi Engin; Ozerol, Ibrahim; Unlu, Cihat

    2013-07-01

    We aimed to investigate whether the surgical removal of endometrioma alters the nuclear factor-kappa B1 (NF-kB1; p50/105) and NF-kB p65 (Rel A) expression in the eutopic endometrium of infertile women with endometrioma before and after laparoscopic removal of the ovarian endometrioma during the mid-secretory phase. Infertile women with endometrioma (n = 15) were enrolled. Infertile patients with nonendometriotic ovarian cyst (n = 10) and healthy fertile women (n = 10) were recruited as controls. Endometrial samples were obtained before and 3 months after the laparoscopic cystectomy. The NF-kB1 (p50/105) levels were analyzed by enzyme-linked immunosorbent assay (ELISA) in the endometrium of all groups before and after laparoscopic ovarian cystectomy during implantation window. Expression of NF-kB1 (p50/105) in eutopic endometrium was significantly higher in infertile women with endometrioma compared to nonendometriotic cyst and fertile controls (P < .05). Laparoscopic cystectomy resulted in a significant decrease in NF-kB1 expression in women with endometrioma. The NF-kB p65 (Rel A) immunoreactivity of eutopic endometrium decreased significantly subsequent to the surgical removal of the endometrioma. In conclusion, increased endometrial NF-kB expression may contribute to endometriosis-associated infertility.

  8. Lipoxin A4 attenuates radicular pain possibly by inhibiting spinal ERK, JNK and NF-κB/p65 and cytokine signals, but not p38, in a rat model of non-compressive lumbar disc herniation.

    PubMed

    Miao, G-S; Liu, Z-H; Wei, S-X; Luo, J-G; Fu, Z-J; Sun, T

    2015-08-06

    Inflammatory response induced by protrused nucleus pulposus (NP) has been shown to play a crucial role in the process of radicular pain. Lipoxins represent a unique class of lipid mediators that have anti-inflammatory and pro-resolving action. The present study was undertaken to investigate if intrathecal lipoxin A4 (LXA4) could alleviate mechanical allodynia in the rat models of application of NP to the L5 dorsal root ganglion (DRG). Non-compressive models of application of NP to L5 DRG were established and intrathecal catheterization for drug administration was performed in rats. Daily intrathecal injection of vehicle or LXA4 (10ng or 100ng) was performed for three successive days post-operation. Mechanical thresholds were tested and the ipsilateral lumbar (L4-L6) segment of spinal dorsal horns were removed for the determination of tumor necrosis factor-α (TNF-α), IL-1β, transforming growth factor-β1 (TGF-β1) and IL-10 expression and NF-κB/p65, extracellular signal-regulated kinase (ERK), C-Jun N-terminal kinase (JNK) and P38 expression. Application of NP to DRG in rats induced mechanical allodynia, increased the expression of pro-inflammatory factors (TNF-α and IL-1β), NF-κB/p65, the phosphorylated-ERK (p-ERK), -JNK (p-JNK) and -P38 (p-p38) and decreased the expression of anti-inflammatory cytokines (TGF-β1 and IL-10) in the ipsilateral lumbar (L4-L6) segment of spinal dorsal horns. Intrathecal injection of LXA4 alleviated the development of neuropathic pain, inhibited the upregulation of pro-inflammatory cytokines (TNF-α and IL-1β), upregulated the expression of anti-inflammatory cytokines (TGF-β1 and IL-10) and attenuated the activation of NF-κB/p65, p-ERK, p-JNK, but not p-p38, in a dose-dependent manner. In this study, we have demonstrated that LXA4 potently alleviate radicular pain in a rat model of non-compressive lumbar disc herniation. The anti-inflammatory and pro-resolution properties of LXA4 have shown a great promise for the management

  9. Pretreatment of Mouse Neural Stem Cells with Carbon Monoxide-Releasing Molecule-2 Interferes with NF-κB p65 Signaling and Suppresses Iron Overload-Induced Apoptosis.

    PubMed

    Xie, Zhengxing; Han, Ping; Cui, Zhenwen; Wang, Baofeng; Zhong, Zhihong; Sun, Yuhao; Yang, Guoyuan; Sun, Qingfang; Bian, Liuguan

    2016-11-01

    Neural stem cell (NSC) transplantation is a promising approach to repair the damaged brain after hemorrhagic stroke; however, it is largely limited by the poor survival of donor cells. Breakdown products of the hematoma and subsequent iron overload contribute to the impairment of survival of neural cells. There is little information regarding the mechanism involved in the death of grafted cells. Furthermore, therapeutic research targeted to improving the survival of grafted neural stem cells (NSCs) is strikingly lacking. Here, we showed that iron overload induced apoptosis of C17.2 cells, a cell line originally cloned from mouse NSCs and immortalized by v-myc. Pretreatment with carbon monoxide-releasing molecule-2 (CORM-2) markedly protected C17.2 cells against iron overload in a dose-dependent manner. Moreover, CORM-2 interfered with NF-κB signaling, including inhibition of nuclear translocation and down-regulation of NF-κB p65. TUNEL staining showed that preconditioning C17.2 cells with CORM-2 enhanced their resistance to apoptosis induced by iron overload, which was concomitant with down-regulation of the pro-apoptotic proteins (Bax and cleaved caspase-3) and up-regulation of the anti-apoptotic protein Bcl2. The protective effect of CORM-2 could be simulated by BAY11-7082, a special inhibitor of NF-κB p65. These results provide a novel and effective strategy to enhance the survival of NSCs after transplantation and, therefore, their efficacy in repairing brain injury due to hemorrhagic stroke.

  10. U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III

    PubMed Central

    Singh, Jagjit; Sikand, Kavleen; Conrad, Heike; Will, Cindy L.; Komar, Anton A.; Shukla, Girish C.

    2016-01-01

    Formation of catalytic core of the U12-dependent spliceosome involves U6atac and U12 interaction with the 5′ splice site and branch site regions of a U12-dependent intron, respectively. Beyond the formation of intermolecular helix I region between U6atac and U12 snRNAs, several other regions within these RNA molecules are predicted to form stem-loop structures. Our previous work demonstrated that the 3′ stem-loop region of U6atac snRNA contains a U12-dependent spliceosome-specific targeting activity. Here, we show a detailed structure-function analysis and requirement of a substructure of U6atac 3′ stem-loop in U12-dependent in vivo splicing. We show that the C-terminal RNA recognition motif of p65, a U12 snRNA binding protein, also binds to the distal 3′ stem-loop of U6atac. By using a binary splice site mutation suppressor assay we demonstrate that p65 protein-binding apical stem-loop of U12 snRNA can be replaced by this U6atac distal 3′ stem-loop. Furthermore, we tested the compatibility of the U6atac 3′ end from phylogenetically distant species in a human U6atac background, to establish the evolutionary relatedness of these structures and in vivo function. In summary, we demonstrate that RNA-RNA and RNA-protein interactions in the minor spliceosome are highly plastic as compared to the major spliceosome. PMID:27510544

  11. Secretome of tumor-associated leukocytes augment epithelial-mesenchymal transition in positive lymph node breast cancer patients via activation of EGFR/Tyr845 and NF-κB/p65 signaling pathway.

    PubMed

    Elghonaimy, Eslam A; Ibrahim, Sherif A; Youns, Amal; Hussein, Zeinab; Nouh, Mohamed Akram; El-Mamlouk, Tahani; El-Shinawi, Mohamed; Mostafa Mohamed, Mona

    2016-09-01

    Epithelial-mesenchymal transition (EMT) is an essential process in breast cancer metastasis. The aim of the present study was to determine the role of secretions of tumor-associated leukocytes (TALs) isolated from negative and positive lymph nodes (nLNs and pLNs, respectively) breast cancer patients in regulating EMT mechanism and the associated signaling pathways. We found an increased infiltration of TALs, which was associated with downregulation of E-cadherin and over-expression of vimentin in the breast carcinoma tissues of pLNs as compared to nLNs patients and normal breast tissues obtained from healthy volunteers during mammoplasty. Furthermore, TALs isolated from pLNs breast cancer patients secreted an elevated panel of cytokines by up to 2-5-fold when compared with those isolated from nLNs patients. Secretome of TALs of pLNs possessed higher TARC, IGF-1, IL-3, TNF-β, IL-5, G-CSF, IL-4, and IL-1α with more than a fivefold compared to those of nLNs. Using the human breast cancer cell lines MCF-7 and MDA-MB-231, we found that cytokines secreted by TALs isolated from nLNs and pLNs breast cancer patients promoted EMT via upregulation of TGF-β and vimentin and downregulation of E-cadherin at messenger RNA (mRNA) levels in both cell lines and at protein level in MCF-7. While TGF-β is over-expressed by MDA-MB-231 seeded in media conditioned by secretome of TALs isolated from nLNs and pLNs breast cancer patients. The downstream TGF-β signaling transcription factors, Snail, Slug, and Twist, known to be associated with EMT mechanism were over-expressed by MCF-7 and MDA-MB-231 seeded in media conditioned by secretome of TALs isolated from nLNs and pLNs breast cancer patients. Acquisition of EMT in MCF-7 cells is mechanistically attributed to the activation of EGFR((Tyr845)) and NF-κB/p65((Ser276)) signaling which are significantly highly expressed by MCF-7 cells seeded in media conditioned by secretome of TALs isolated from pLNs compared to nLNs patients. Overall

  12. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  13. Tumor Necrosis Factor Receptor Associated Factors (TRAFs) 2 and 3 Form a Transcriptional Complex with Phosho-RNA Polymerase II and p65 in CD40 Ligand Activated Neuro2a Cells.

    PubMed

    El Hokayem, Jimmy; Brittain, George C; Nawaz, Zafar; Bethea, John R

    2017-03-01

    The tumor necrosis factor receptor-associated factors (TRAFs) have been classically described as adaptor proteins that function as solely cytosolic signaling intermediates for the TNF receptor superfamily, Toll-like receptors (TLRs), NOD, like receptors (NLRs), cytokine receptors, and others. In this study, we show for the first time that TRAFs are present within the cytoplasm and nucleus of Neuro2a cells and primary cortical neurons, and that TRAF2 and TRAF3 translocate into the nucleus within minutes of CD40L stimulation. Analysis of the transcriptional regulatory potential of TRAFs by luciferase assay revealed that each of the TRAFs differentially functions as a transcriptional activator or repressor in a cell-specific manner. Interestingly, ChIP-qPCR data demonstrate that TRAFs 2/3, p65, and pRNAPol II form part of a transcriptional complex on the Icam-1 gene promoter upon CD40L stimulation. We further determined that TRAF2 recruitment to the nucleus is critical for the ubiquitination of H2b, a transcription permissive epigenetic modification. Our findings demonstrate for the first time that TRAFs 2/3 participate in the formation of a CD40L-induced transcriptional complex in neuronal cells.

  14. Rotating proton pumping ATPases: subunit/subunit interactions and thermodynamics.

    PubMed

    Nakanishi-Matsui, Mayumi; Sekiya, Mizuki; Futai, Masamitsu

    2013-03-01

    In this article, we discuss single molecule observation of rotational catalysis by E. coli ATP synthase (F-ATPase) using small gold beads. Studies involving a low viscous drag probe showed the stochastic properties of the enzyme in alternating catalytically active and inhibited states. The importance of subunit interaction between the rotor and the stator, and thermodynamics of the catalysis are also discussed. "Single Molecule Enzymology" is a new trend for understanding enzyme mechanisms in biochemistry and physiology.

  15. The Subunit Structure of Benzylsuccinate Synthase†

    PubMed Central

    Li, Lei; Patterson, Dustin P.; Fox, Christel C.; Lin, Brian; Coschigano, Peter W.; Marsh, E. Neil G.

    2010-01-01

    Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits α, β and γ that in Thauera Aromatica T1 strain are encoded by the tutD, tutG and tutF genes respectively. The large α-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small β- and γ-subunits has remained unclear. We have over-expressed all three subunits of benzylsuccinate synthase in E. coli, both individually and in combination. Co-expression of the γ-subunit (but not the β-subunit) is essential for efficient expression of the α-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an α2β2γ2 hexamer by nickel-affinity chromatography through a ‘His6’ affinity tag engineered onto the C-terminus of the α-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the β-subunit and the other with the γ-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly a mutant γ-subunit lacking the [4Fe-4S] cluster is unable to stabilize the α-subunit when the proteins are co-expressed. PMID:19159265

  16. Role of the Rubisco Small Subunit

    SciTech Connect

    Spreitzer, Robert Joseph

    2016-11-05

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. However, it is a slow enzyme, and O2 competes with CO2 at the active site. Oxygenation initiates the photorespiratory pathway, which also results in the loss of CO2. If carboxylation could be increased or oxygenation decreased, an increase in net CO2 fixation would be realized. Because Rubisco provides the primary means by which carbon enters all life on earth, there is much interest in engineering Rubisco to increase the production of food and renewable energy. Rubisco is located in the chloroplasts of plants, and it is comprised of two subunits. Much is known about the chloroplast-gene-encoded large subunit (rbcL gene), which contains the active site, but much less is known about the role of the nuclear-gene-encoded small subunit in Rubisco function (rbcS gene). Both subunits are coded by multiple genes in plants, which makes genetic engineering difficult. In the eukaryotic, green alga Chlamydomonas reinhardtii, it has been possible to eliminate all the Rubisco genes. These Rubisco-less mutants can be maintained by providing acetate as an alternative carbon source. In this project, focus has been placed on determining whether the small subunit might be a better genetic-engineering target for improving Rubisco. Analysis of a variable-loop structure (βA-βB loop) of the small subunit by genetic selection, directed mutagenesis, and construction of chimeras has shown that the small subunit can influence CO2/O2 specificity. X-ray crystal structures of engineered chimeric-loop enzymes have indicated that additional residues and regions of the small subunit may also contribute to Rubisco function. Structural dynamics of the small-subunit carboxyl terminus was also investigated. Alanine-scanning mutagenesis of the most-conserved small-subunit residues has identified a

  17. 28 CFR 51.6 - Political subunits.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Political subunits. 51.6 Section 51.6 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) PROCEDURES FOR THE ADMINISTRATION OF SECTION 5 OF THE VOTING RIGHTS ACT OF 1965, AS AMENDED General Provisions § 51.6 Political subunits. All...

  18. Subunit mass analysis for monitoring antibody oxidation

    PubMed Central

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J.; Hu, Ping

    2017-01-01

    ABSTRACT Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd’ and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation. PMID:28106519

  19. Subunit mass analysis for monitoring antibody oxidation.

    PubMed

    Sokolowska, Izabela; Mo, Jingjie; Dong, Jia; Lewis, Michael J; Hu, Ping

    2017-04-01

    Methionine oxidation is a common posttranslational modification (PTM) of monoclonal antibodies (mAbs). Oxidation can reduce the in-vivo half-life, efficacy and stability of the product. Peptide mapping is commonly used to monitor the levels of oxidation, but this is a relatively time-consuming method. A high-throughput, automated subunit mass analysis method was developed to monitor antibody methionine oxidation. In this method, samples were treated with IdeS, EndoS and dithiothreitol to generate three individual IgG subunits (light chain, Fd' and single chain Fc). These subunits were analyzed by reversed phase-ultra performance liquid chromatography coupled with an online quadrupole time-of-flight mass spectrometer and the levels of oxidation on each subunit were quantitated based on the deconvoluted mass spectra using the UNIFI software. The oxidation results obtained by subunit mass analysis correlated well with the results obtained by peptide mapping. Method qualification demonstrated that this subunit method had excellent repeatability and intermediate precision. In addition, UNIFI software used in this application allows automated data acquisition and processing, which makes this method suitable for high-throughput process monitoring and product characterization. Finally, subunit mass analysis revealed the different patterns of Fc methionine oxidation induced by chemical and photo stress, which makes it attractive for investigating the root cause of oxidation.

  20. Gene targeting of CK2 catalytic subunits

    PubMed Central

    Lou, David Y.; Toselli, Paul; Landesman-Bollag, Esther; Dominguez, Isabel

    2013-01-01

    Protein kinase CK2 is a highly conserved and ubiquitous serine–threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2β subunits and two catalytic subunits, either CK2α/CK2α, CK2α/ CK2α′, or CK2α′/CK2α′. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2α′is essential for male germ cell development, and we now demonstrate that CK2α has an essential role in embryogenesis, as mice lacking CK2α die in mid-embryogenesis, with cardiac and neural tube defects. PMID:18594950

  1. Anthranilate synthase subunit organization in Chromobacterium violaceum.

    PubMed

    Carminatti, C A; Oliveira, I L; Recouvreux, D O S; Antônio, R V; Porto, L M

    2008-09-16

    Tryptophan is an aromatic amino acid used for protein synthesis and cellular growth. Chromobacterium violaceum ATCC 12472 uses two tryptophan molecules to synthesize violacein, a secondary metabolite of pharmacological interest. The genome analysis of this bacterium revealed that the genes trpA-F and pabA-B encode the enzymes of the tryptophan pathway in which the first reaction is the conversion of chorismate to anthranilate by anthranilate synthase (AS), an enzyme complex. In the present study, the organization and structure of AS protein subunits from C. violaceum were analyzed using bioinformatics tools available on the Web. We showed by calculating molecular masses that AS in C. violaceum is composed of alpha (TrpE) and beta (PabA) subunits. This is in agreement with values determined experimentally. Catalytic and regulatory sites of the AS subunits were identified. The TrpE and PabA subunits contribute to the catalytic site while the TrpE subunit is involved in the allosteric site. Protein models for the TrpE and PabA subunits were built by restraint-based homology modeling using AS enzyme, chains A and B, from Salmonella typhimurium (PDB ID 1I1Q).

  2. Anti-inflammatory effects of 1,25-dihydroxyvitamin D3 in monocytes cultured in serum from patients with type 2 diabetes mellitus and diabetic nephropathy with uremia via Toll-like receptor 4 and nuclear factor-κB p65.

    PubMed

    Yang, Mengxue; Xu, Jie; Yu, Jie; Yang, Bo; Gan, Hua; Li, Sicheng; Li, Xianwen

    2015-12-01

    Type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) seriously affect the quality of human life. 1,25-dihydroxyvitamin D3 (VD3) is known to exert anti‑inflammatory and immunomodulatory effects. The present study investigated the effects of VD3 on Toll‑like receptor 4 (TLR4) and nuclear factor (NF)‑κB p65 expression in monocytes from patients with T2DM and investigated the underlying mechanisms. Serum from subjects of the control, T2DM and DN with uremia groups was isolated, and THP‑1 monocytes were cultured in these sera with or without VD3. After treatment with lipopolysaccharide (LPS) and interleukin (IL)‑15, monocytes and culture supernatants were collected. The expression of TLR4, TLR9 and IL‑15 mRNA was detected using reverse‑transcription polymerase chain reaction analysis. Furthermore, the protein expression of TLR4, NF‑κB p65 and inhibitor of NF‑κB (IκB) was determined using western blot analysis. The levels of IL‑6 and monocyte chemotactic protein in culture supernatants were detected using ELISAs. The impact of LPS, IL‑15 and VD3 on the TLR/NF‑κB signaling pathway in the T2DM and DN uremia groups was also investigated. In the T2DM and DN uremia groups, LPS and IL‑15 downregulated the expression of IκB and upregulated levels of proteins, including TLR4, NF‑κB p65, IL‑6 and monocyte chemoattractant protein 1, as well as TLR4 and IL‑15 mRNA. There was no significant difference in TLR9 mRNA protein expression among the three groups. VD3 partially blocked the above effects; However, pre‑treatment with VD3 had no significant effect on TLR4 mRNA expression, protein expression of NF-κB p65 and IκB, or the levels of associated inflammatory cytokines. In conclusion, the present study indicated that anti‑inflammatory effects of VD3 in inflammatory immune responses in T2DM and DN uremia are associated with the TLR4/NF-κB p65 signaling pathway.

  3. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-06

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  4. Drosophila laminin: sequence of B2 subunit and expression of all three subunits during embryogenesis

    PubMed Central

    1989-01-01

    In a previous study, we described the cloning of the genes encoding the three subunits of Drosophila laminin, a substrate adhesion molecule, and the cDNA sequence of the B1 subunit (Montell and Goodman, 1988). This analysis revealed the similarity of Drosophila laminin with the mouse and human complexes in subunit composition, domain structure, and amino acid sequence. In this paper, we report the deduced amino acid sequence of the B2 subunit. We then describe the expression and tissue distribution of the three subunits of laminin during Drosophila embryogenesis using both in situ hybridization and immunolocalization techniques, with particular emphasis on its expression in and around the developing nervous system. PMID:2808533

  5. Diversity of insect nicotinic acetylcholine receptor subunits.

    PubMed

    Jones, Andrew K; Sattelle, David B

    2010-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. They consist of five subunits arranged around a central ion channeL Since the subunit composition determines the functional and pharmacological properties of the receptor the presence of nAChR families comprising several subunit-encodinggenes provides a molecular basis for broad functional diversity. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their nematode andvertebrate counterparts. Thus, the fruit fly (Drosophila melanogaster), malaria mosquito (Anopheles gambiae), honey bee (Apis mellifera), silk worm (Bombyx mon) and the red flour beetle (Tribolium castaneum) possess 10-12 nAChR genes while human and the nematode Caenorhabditis elegans have 16 and 29 respectively. Although insect nAChRgene families are amongst the smallest known, receptor diversity can be considerably increased by the posttranscriptional processes alternative splicing and mRNA A-to-I editingwhich can potentially generate protein products which far outnumber the nAChR genes. These two processes can also generate species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit which may perform species-specific functions. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that target specific pest insects while sparing beneficial species.

  6. Targeting Activation of Specific NF-κB Subunits Prevents Stress-Dependent Atherothrombotic Gene Expression

    PubMed Central

    Djuric, Zdenka; Kashif, Muhammed; Fleming, Thomas; Muhammad, Sajjad; Piel, David; von Bauer, Rüdiger; Bea, Florian; Herzig, Stephan; Zeier, Martin; Pizzi, Marina; Isermann, Berend; Hecker, Markus; Schwaninger, Markus; Bierhaus, Angelika; Nawroth, Peter P

    2012-01-01

    Psychosocial stress has been shown to be a contributing factor in the development of atherosclerosis. Although the underlying mechanisms have not been elucidated entirely, it has been shown previously that the transcription factor nuclear factor-κB (NF-κB) is an important component of stress-activated signaling pathway. In this study, we aimed to decipher the mechanisms of stress-induced NF-κB-mediated gene expression, using an in vitro and in vivo model of psychosocial stress. Induction of stress led to NF-κB-dependent expression of proinflammatory (tissue factor, intracellular adhesive molecule 1 [ICAM-1]) and protective genes (manganese superoxide dismutase [MnSOD]) via p50, p65 or cRel. Selective inhibition of the different subunits and the respective kinases showed that inhibition of cRel leads to the reduction of atherosclerotic lesions in apolipoprotein−/− (ApoE−/−) mice via suppression of proinflammatory gene expression. This observation may therefore provide a possible explanation for ineffectiveness of antioxidant therapies and suggests that selective targeting of cRel activation may provide a novel approach for the treatment of stress-related inflammatory vascular disease. PMID:23114885

  7. DNMT3L interacts with transcription factors to target DNMT3L/DNMT3B to specific DNA sequences: role of the DNMT3L/DNMT3B/p65-NFκB complex in the (de-)methylation of TRAF1.

    PubMed

    Pacaud, Romain; Sery, Quentin; Oliver, Lisa; Vallette, François M; Tost, Jörg; Cartron, Pierre-François

    2014-09-01

    DNMT3L i.e. DNA (cytosine-5)-methyltransferase 3-like protein, is devoid of cytosine methyltransferase activity, despite clear homology to DNMT3A and DNMT3B, due to the mutation of key catalytic residues. However, DNMT3L participates in de novo methylation reactions through its direct interaction with DNMT3A and DNMT3B. In the present study, we investigated if DNMT3L interacts also directly with transcription factors (TFs). Using TF arrays, we identified 73 TFs that interacted with DNMT3L, 13 of which (ASH2L, ATF1, ATF3, BLZF1, CDX2, CERM, E2F3, E2F4, GCNF, GTF2I, GTF3C5, NFkB-p65 and RXRα) interacted only with DNMT3L, but not with DNMT3A/B. By focusing on the interaction with NFkB-p65, we demonstrate that DNMT3L forms a complex with DNMT3B and NFkB-p65 and that this complex is required for the control of DNA methylation at the TRAF1 promoter in the T98G glioma cell line. In addition, our experiments describe the DNA methylation at TRAF1 as being dynamic with a demethylation phase involving TET3. Thus, our data suggests that DNMT3L can address DNMT3A/B to specific sites by directly interacting with TFs that do not directly interact with DNMT3A/B. In summary, our data provide a new avenue for the direction of site-specific de novo DNA methylation catalyzed by DNMT3A/B.

  8. Subunit Recombinant Vaccine Protects Against Monkeypox

    DTIC Science & Technology

    2006-05-27

    Subunit Recombinant Vaccine Protects against Monkeypox1 Jean-Michel Heraud,* Yvette Edghill-Smith,*† Victor Ayala,‡ Irene Kalisz,‡ Janie Parrino ...GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Heraud, JM Edghill-Smith, Y Ayala, V Kalisz, I Parrino , J Kalyanaraman, VS Manischewitz, J King

  9. Subunit-selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections.

    PubMed

    Misas-Villamil, Johana C; van der Burgh, Aranka M; Grosse-Holz, Friederike; Bach-Pages, Marcel; Kovács, Judit; Kaschani, Farnusch; Schilasky, Sören; Emon, Asif Emran Khan; Ruben, Mark; Kaiser, Markus; Overkleeft, Hermen S; van der Hoorn, Renier A L

    2017-01-24

    The proteasome is a nuclear - cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit-selective inhibitors and dual-color fluorescent activity-based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant-microbe interactions. Our data reveals that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1-1 (PtoDC3000(ΔhQ)) whilst the activity profile of the β1 subunit changes. Infection with wild-type PtoDC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species. This article is protected by copyright. All rights reserved.

  10. MUTAGENICITY OF P&#65H-CONTAMINATED SOILS DURING BIOREMEDIATION

    EPA Science Inventory

    Bioremediation of contaminated soils is considered an effective method for reducing potential health hazards. Although it is assumed that (bio)remediation is a detoxifying process, degradation products of compounds such as polycyclic aromatic compounds (PACs) can be more toxic th...

  11. Recent Advances in Subunit Vaccine Carriers

    PubMed Central

    Vartak, Abhishek; Sucheck, Steven J.

    2016-01-01

    The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chemical properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technology, improved analytical methods, and use of computational methodology have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorganic nanoparticles. PMID:27104575

  12. Subunit organization in cytoplasmic dynein subcomplexes

    PubMed Central

    King, Stephen J.; Bonilla, Myriam; Rodgers, Michael E.; Schroer, Trina A.

    2002-01-01

    Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain–binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo. PMID:11967380

  13. DNA sequences, recombinant DNA molecules and processes for producing the A and B subunits of cholera toxin and preparations containing so-obtained subunit or subunits

    SciTech Connect

    Harford, N.; De Wilde, M.

    1987-05-19

    A recombinant DNA molecule is described comprising at least a portion coding for subunits A and B of cholera toxin, or a fragment or derivative of the portion wherein the fragment or derivative codes for a polypeptide have an activity which can induce an immune response to subunit A; can induce an immune response to subunit A and cause epithelial cell penetration and the enzymatic effect leading to net loss of fluid into the gut lumen; can bind to the membrane receptor for the B subunit of cholera toxin; can induce an immune response to subunit B; can induce an immune response to subunit B and bind to the membrane receptor; or has a combination of the activities.

  14. Differential accumulation of ribonucleotide reductase subunits in clam oocytes: the large subunit is stored as a polypeptide, the small subunit as untranslated mRNA

    PubMed Central

    1986-01-01

    Within minutes of fertilization of clam oocytes, translation of a set of maternal mRNAs is activated. One of the most abundant of these stored mRNAs encodes the small subunit of ribonucleotide reductase (Standart, N. M., S. J. Bray, E. L. George, T. Hunt, and J. V. Ruderman, 1985, J. Cell Biol., 100:1968-1976). Unfertilized oocytes do not contain any ribonucleotide reductase activity; such activity begins to appear shortly after fertilization. In virtually all organisms, this enzyme is composed of two dissimilar subunits with molecular masses of approximately 44 and 88 kD, both of which are required for activity. This paper reports the identification of the large subunit of clam ribonucleotide reductase isolated by dATP-Sepharose chromatography as a relatively abundant 86-kD polypeptide which is already present in oocytes, and whose level remains constant during early development. The enzyme activity of this large subunit was established in reconstitution assays using the small subunit isolated from embryos by virtue of its binding to the anti-tubulin antibody YL 1/2. Thus the two components of clam ribonucleotide reductase are differentially stored in the oocyte: the small subunit in the form of untranslated mRNA and the large subunit as protein. When fertilization triggers the activation of translation of the maternal mRNA, the newly synthesized small subunit combines with the preformed large subunit to generate active ribonucleotide reductase. PMID:3536960

  15. Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

    PubMed Central

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2011-01-01

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

  16. Prefoldin subunits are protected from ubiquitin-proteasome system-mediated degradation by forming complex with other constituent subunits.

    PubMed

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2011-06-03

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

  17. Inherent conformational flexibility of F1-ATPase α-subunit.

    PubMed

    Hahn-Herrera, Otto; Salcedo, Guillermo; Barril, Xavier; García-Hernández, Enrique

    2016-09-01

    The core of F1-ATPase consists of three catalytic (β) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in β-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3β3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and β-subunits show on Mg(II) for recognizing ATP.

  18. [Nose surgical anatomy in six aesthetic subunits].

    PubMed

    Chaput, B; Lauwers, F; Lopez, R; Saboye, J; André, A; Grolleau, J-L; Chavoin, J-P

    2013-04-01

    The nose is a complex entity, combining aesthetic and functional roles. Descriptive anatomy is a fundamental science that it can be difficult to relate directly to our daily surgical activity. Reasoning in terms of aesthetic subunits to decide on his actions appeared to us so obvious. The aim of this paper is to resume the anatomical bases relevant to our daily practice in order to fully apprehend the restorative or cosmetic procedures. We discuss the limits of the systematization of these principles in nasal oncology.

  19. Amino-terminal truncations of the ribulose-bisphosphate carboxylase small subunit influence catalysis and subunit interactions.

    PubMed Central

    Paul, K; Morell, M K; Andrews, T J

    1993-01-01

    The first 20 residues at the amino terminus of the small subunit of spinach ribulose-1,5-bisphosphate carboxylase form an irregular arm that makes extensive contacts with the large subunit and also with another small subunit (S. Knight, I. Andersson, and C.-I. Brändén [1990] J Mol Biol 215: 113-160). The influence of these contacts on subunit binding and, indirectly, on catalysis was investigated by constructing truncations from the amino terminus of the small subunit of the highly homologous enzyme from Synechococcus PCC 6301 expressed in Escherichia coli. Removal of the first six residues (and thus the region of contact with a neighboring small subunit) affected neither the affinity with which the small subunits bound to the large subunits nor the catalytic properties of the assembled holoenzyme. Extending the truncation to include the first 12 residues (which encroaches into a highly conserved region that interacts with the large subunit) also did not weaken intersubunit binding appreciably, but it reduced the catalytic activity of the holoenzyme nearly 5-fold. Removal of an additional single residue (i.e. removal of a total of 13 residues) weakened intersubunit binding approximately 80-fold. Paradoxically, this partially restored catalytic activity to approximately 40% of that of the wild-type holoenzyme. None of these truncations materially affected the Km values for ribulose-1,5-bisphosphate or CO2. Removal of all 20 residues of the irregular arm (thereby deleting the conserved region of contact with large subunits) totally abolished the small subunit's ability to bind to large subunits to form a stable holoenzyme. However, this truncated small subunit was still synthesized by the E. coli cells. These data are interpreted in terms of the role of the amino-terminal arm of the small subunit in maintaining the structure of the holoenzyme. PMID:8278544

  20. Complementation of subunits from different bacterial luciferases. Evidence for the role of the. beta. subunit in the bioluminescent mechanism

    SciTech Connect

    Meighen, E.A.; Bartlet, I.

    1980-12-10

    Complementation of the nonidentical subunits (..cap alpha.. and ..beta..) of luciferases isolated from two different bioluminescent strains, Beneckea harveyi and Photobacterium phosphoreum, has resulted in the formation of a functional hybrid luciferase (..cap alpha../sub h/..beta../sub p/) containing the ..cap alpha.. subunit from B. harveyi luciferase (..cap alpha../sub h/) and the ..beta.. subunit from P. phosphoreum luciferase (..beta../sub p/). The complementation was unidirectional; activity could not be restored by complementing the ..cap alpha.. subunit of P. phosphoreum luciferase with the ..beta.. subunit of B. harveyi luciferase, showing that the subunits from these luciferases were not identical. Kinetic parameters of the hybrid luciferase reflecting the intermediate and later steps of the bioluminescent reaction as well as the overall activity and specificity were essentially identical to the same kinetic parameters for B. harveyi luciferase, the source of the ..cap alpha.. subunit, and quite distinct from those of P. phosphoreum luciferase. However, kinetic parameters that reflected the initial step in the reaction involving interaction of FMNH/sub 2/ and luciferase were altered in the hybrid luciferase compared to both the parental luciferases, the K/sub d/ for FMNH/sub 2/ actually being closer to that observed for the P. phosphoreum luciferase (the source of the ..beta.. subunit). These results provide direct evidence that modification or alteration of the ..beta.. subunit in a dimeric luciferase molecule can affect the kinetic properties and indicates that the ..beta.. subunit plays a functional role in the bioluminescent mechanism. It is proposed that both the ..cap alpha.. and ..beta.. subunits are involved with the initial interaction with FMNH/sub 2/, whereas subsequent steps in the mechanism are dictated exclusively by the ..cap alpha.. subunit and are unaffected by alterations in the ..beta.. subunit.

  1. Formation of active bacterial luciferase between interspecific subunits in vivo.

    PubMed

    Almashanu, S; Tuby, A; Hadar, R; Einy, R; Kuhn, J

    1995-01-01

    Interspecific complementation between luxAs and luxBs from Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi and Xenorhabdus luminescens was examined in vivo. The individual genes from these species were cloned on different compatible plasmids or amplified by PCR and brought together to yield cis combinations without extraneous DNA. The beta subunits from V. harveyi and X. luminescens form active enzyme only with alpha subunits from one of these species. All other combinations yield active enzymes. The lack of activity of the V. harveyi and X. luminescens beta subunits with the alpha subunits from V. fischeri and P. leiognathi results from a lack of association. This was shown by in vivo competition in which these beta subunits were overproduced in comparison with the beta and alpha of V. fischeri. No reduction in light was found. Overall, the in vivo results parallel those found in vitro using isolated denatured subunits and renaturation by removal of the denaturant.

  2. Sodium channel β subunits: emerging targets in channelopathies

    PubMed Central

    O’Malley, Heather A.; Isom, Lori L.

    2016-01-01

    Voltage-gated sodium channels (VGSCs) are responsible for initiation and propagation of action potentials in excitable cells. VGSCs in mammalian brain are heterotrimeric complexes of α and β subunits. Originally called “auxiliary,” we now know that β subunit proteins are multifunctional signaling molecules that play roles in both excitable and non-excitable cell types, and with or without the pore-forming α subunit present. β subunits function in VGSC and potassium channel modulation, cell adhesion, and gene regulation, with particularly important roles in brain development. Mutations in the genes encoding β subunits are linked to a number of diseases, including epilepsy, sudden death syndromes like SUDEP and SIDS, and cardiac arrhythmia. While VGSC β subunit-specific drugs have not yet been developed, this protein family is an emerging therapeutic target. PMID:25668026

  3. Quantifying the cooperative subunit action in a multimeric membrane receptor

    PubMed Central

    Wongsamitkul, Nisa; Nache, Vasilica; Eick, Thomas; Hummert, Sabine; Schulz, Eckhard; Schmauder, Ralf; Schirmeyer, Jana; Zimmer, Thomas; Benndorf, Klaus

    2016-01-01

    In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels. PMID:26858151

  4. Genetic analysis of neuronal ionotropic glutamate receptor subunits.

    PubMed

    Granger, Adam J; Gray, John A; Lu, Wei; Nicoll, Roger A

    2011-09-01

    In the brain, fast, excitatory synaptic transmission occurs primarily through AMPA- and NMDA-type ionotropic glutamate receptors. These receptors are composed of subunit proteins that determine their biophysical properties and trafficking behaviour. Therefore, determining the function of these subunits and receptor subunit composition is essential for understanding the physiological properties of synaptic transmission. Here, we discuss and evaluate various genetic approaches that have been used to study AMPA and NMDA receptor subunits. These approaches have demonstrated that the GluA1 AMPA receptor subunit is required for activity-dependent trafficking and contributes to basal synaptic transmission, while the GluA2 subunit regulates Ca(2+) permeability, homeostasis and trafficking to the synapse under basal conditions. In contrast, the GluN2A and GluN2B NMDA receptor subunits regulate synaptic AMPA receptor content, both during synaptic development and plasticity. Ongoing research in this field is focusing on the molecular interactions and mechanisms that control these functions. To accomplish this, molecular replacement techniques are being used, where native subunits are replaced with receptors containing targeted mutations. In this review, we discuss a single-cell molecular replacement approach which should arguably advance our physiological understanding of ionotropic glutamate receptor subunits, but is generally applicable to study of any neuronal protein.

  5. Expression of GABA receptor rho subunits in rat brain.

    PubMed

    Boue-Grabot, E; Roudbaraki, M; Bascles, L; Tramu, G; Bloch, B; Garret, M

    1998-03-01

    The GABA receptor rho1, rho2, and rho3 subunits are expressed in the retina where they form bicuculline-insensitive GABA(C) receptors. We used northern blot, in situ hybridization, and RT-PCR analysis to study the expression of rho subunits in rat brains. In situ hybridization allowed us to detect rho-subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT-PCR experiments indicated that (a) in retina and in domains that may contain functional GABA(C) receptors, rho2 and rho1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABA(C) receptors, rho2 mRNA is enriched relative to rho1 mRNA. These results suggest that both rho1 and rho2 subunits are necessary to form a functional GABA(C) receptor. The use of RT-PCR also showed that, except in the superior colliculus, rho3 is expressed along with rho1 and rho2 subunits. We also raised an antibody against a peptide sequence unique to the rho1 subunit. The use of this antibody on cerebellum revealed the rat rho1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABA(C) receptor subunits to identified neurons paves the way for future electrophysiological studies.

  6. Compilation of small ribosomal subunit RNA structures.

    PubMed Central

    Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

    1993-01-01

    The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

  7. Rubisco small subunit gene family in cassava.

    PubMed

    Yeo, T W; Mak, Y M; Ho, K K

    1999-01-01

    Cassava leaves of two different cultivars, Brazil and Buloh, were used to isolate mRNA. The mRNA isolated was successfully used in the construction of cDNA libraries for each of the cultivars. The cDNA libraries were screened for members of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene family and positive clones were sequenced. A total of seven different SSU genes, of which five were from cultivar Brazil and two were from cultivar Buloh, were isolated. Comparison results show that even though all the sequences are highly similar, they can be classified into three subfamilies. Homology between members of the same subfamily is higher than homology between members from the same cultivar.

  8. Subunit vaccine efficacy against Botulinum neurotoxin subtypes.

    PubMed

    Henkel, James S; Tepp, William H; Przedpelski, Amanda; Fritz, Robert B; Johnson, Eric A; Barbieri, Joseph T

    2011-10-13

    Botulinum neurotoxins (BoNT) are classified into 7 serotypes (A-G) based upon neutralization by serotype-specific anti-sera. Several recombinant serotype-specific subunit BoNT vaccines have been developed, including a subunit vaccine comprising the receptor binding domain (HCR) of the BoNTs. Sequencing of the genes encoding BoNTs has identified variants (subtypes) that possess up to 32% primary amino acid variation among different BoNT serotypes. Studies were conducted to characterize the ability of the HCR of BoNT/A to protect against challenge by heterologous BoNT/A subtypes (A1-A3). High dose vaccination with HCR/A subtypes A1-A4 protected mice from challenge by heterologous BoNT/A subtype A1-A3, while low dose HCR vaccination yielded partial protection to heterologous BoNT/A subtype challenge. Absolute IgG titers to HCRs correlated to the dose of HCR used for vaccination, where HCR/A1 elicited an A1 subtype-specific IgG response, which was not observed with HCR/A2 vaccination. Survival of mice challenged to heterologous BoNT/A2 following low dose HCR/A1 vaccination correlated with elevated IgG titers directed to the denatured C-terminal sub-domain of HCR/A2, while survival of mice to heterologous BoNT/A1 following low dose HCR/A2 vaccination correlated to elevated IgG titers directed to native HCRc/A1. This implies that low dose vaccinations with HCR/A subtypes elicit unique IgG responses, and provides a basis to define how the host develops a neutralizing immune response to BoNT intoxication. These results may provide a reference for the development of pan-BoNT vaccines.

  9. Dynamic regulation of β1 subunit trafficking controls vascular contractility.

    PubMed

    Leo, M Dennis; Bannister, John P; Narayanan, Damodaran; Nair, Anitha; Grubbs, Jordan E; Gabrick, Kyle S; Boop, Frederick A; Jaggar, Jonathan H

    2014-02-11

    Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total β1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular β1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of β1 subunit-containing recycling endosomes, which increased surface β1 almost threefold. These β1 subunits associated with surface-resident BKα proteins, elevating channel Ca(2+) sensitivity and activity. Our data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid β1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types.

  10. Structure of the archaeal Cascade subunit Csa5: relating the small subunits of CRISPR effector complexes.

    PubMed

    Reeks, Judith; Graham, Shirley; Anderson, Linzi; Liu, Huanting; White, Malcolm F; Naismith, James H

    2013-05-01

    The Cascade complex for CRISPR-mediated antiviral immunity uses CRISPR RNA (crRNA) to target invading DNA species from mobile elements such as viruses, leading to their destruction. The core of the Cascade effector complex consists of the Cas5 and Cas7 subunits, which are widely conserved in prokaryotes. Cas7 binds crRNA and forms the helical backbone of Cascade. Many archaea encode a version of the Cascade complex (denoted Type I-A) that includes a Csa5 (or small) subunit, which interacts weakly with the core proteins. Here, we report the crystal structure of the Csa5 protein from Sulfolobus solfataricus. Csa5 comprises a conserved α-helical domain with a small insertion consisting of a weakly conserved β-strand domain. In the crystal, the Csa5 monomers have multimerized into infinite helical threads. At each interface is a strictly conserved intersubunit salt bridge, deletion of which disrupts multimerization. Structural analysis indicates a shared evolutionary history among the small subunits of the CRISPR effector complexes. The same α-helical domain is found in the C-terminal domain of Cse2 (from Type I-E Cascade), while the N-terminal domain of Cse2 is found in Cmr5 of the CMR (Type III-B) effector complex. As Cmr5 shares no match with Csa5, two possibilities present themselves: selective domain loss from an ancestral Cse2 to create two new subfamilies or domain fusion of two separate families to create a new Cse2 family. A definitive answer awaits structural studies of further small subunits from other CRISPR effector complexes.

  11. Liposome-Based Adjuvants for Subunit Vaccines: Formulation Strategies for Subunit Antigens and Immunostimulators

    PubMed Central

    Tandrup Schmidt, Signe; Foged, Camilla; Smith Korsholm, Karen; Rades, Thomas; Christensen, Dennis

    2016-01-01

    The development of subunit vaccines has become very attractive in recent years due to their superior safety profiles as compared to traditional vaccines based on live attenuated or whole inactivated pathogens, and there is an unmet medical need for improved vaccines and vaccines against pathogens for which no effective vaccines exist. The subunit vaccine technology exploits pathogen subunits as antigens, e.g., recombinant proteins or synthetic peptides, allowing for highly specific immune responses against the pathogens. However, such antigens are usually not sufficiently immunogenic to induce protective immunity, and they are often combined with adjuvants to ensure robust immune responses. Adjuvants are capable of enhancing and/or modulating immune responses by exposing antigens to antigen-presenting cells (APCs) concomitantly with conferring immune activation signals. Few adjuvant systems have been licensed for use in human vaccines, and they mainly stimulate humoral immunity. Thus, there is an unmet demand for the development of safe and efficient adjuvant systems that can also stimulate cell-mediated immunity (CMI). Adjuvants constitute a heterogeneous group of compounds, which can broadly be classified into delivery systems or immunostimulators. Liposomes are versatile delivery systems for antigens, and they can carefully be customized towards desired immune profiles by combining them with immunostimulators and optimizing their composition, physicochemical properties and antigen-loading mode. Immunostimulators represent highly diverse classes of molecules, e.g., lipids, nucleic acids, proteins and peptides, and they are ligands for pattern-recognition receptors (PRRs), which are differentially expressed on APC subsets. Different formulation strategies might thus be required for incorporation of immunostimulators and antigens, respectively, into liposomes, and the choice of immunostimulator should ideally be based on knowledge regarding the specific PRR

  12. Epitopes from two soybean glycinin subunits antigenic in pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of a basic and acidic portion joined by disulfide bridges. There are 5 glycinin subunits designated Gy1-Gy5. Results: Twenty seven out of 30 pi...

  13. Specific Roles of NMDA Receptor Subunits in Mental Disorders

    PubMed Central

    Yamamoto, H.; Hagino, Y.; Kasai, S.; Ikeda, K.

    2015-01-01

    N-methyl-D-aspartate (NMDA) receptor plays important roles in learning and memory. NMDA receptors are a tetramer that consists of two glycine-binding subunits GluN1, two glutamate-binding subunits (i.e., GluN2A, GluN2B, GluN2C, and GluN2D), a combination of a GluN2 subunit and glycine-binding GluN3 subunit (i.e., GluN3A or GluN3B), or two GluN3 subunits. Recent studies revealed that the specific expression and distribution of each subunit are deeply involved in neural excitability, plasticity, and synaptic deficits. The present article summarizes reports on the dysfunction of NMDA receptors and responsible subunits in various neurological and psychiatric disorders, including schizophrenia, autoimmune-induced glutamatergic receptor dysfunction, mood disorders, and autism. A key role for the GluN2D subunit in NMDA receptor antagonist-induced psychosis has been recently revealed. PMID:25817860

  14. Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

    ERIC Educational Resources Information Center

    Decatur, Wayne A.

    2010-01-01

    This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

  15. Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly

    PubMed Central

    1990-01-01

    The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin- treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits

  16. Geranyl diphosphate synthase large subunit, and methods of use

    DOEpatents

    Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

    2001-10-16

    A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

  17. A revised model for AMP-activated protein kinase structure: The alpha-subunit binds to both the beta- and gamma-subunits although there is no direct binding between the beta- and gamma-subunits.

    PubMed

    Wong, Kelly A; Lodish, Harvey F

    2006-11-24

    The 5'-AMP-activated protein kinase (AMPK) is a master sensor for cellular metabolic energy state. It is activated by a high AMP/ATP ratio and leads to metabolic changes that conserve energy and utilize alternative cellular fuel sources. The kinase is composed of a heterotrimeric protein complex containing a catalytic alpha-subunit, an AMP-binding gamma-subunit, and a scaffolding beta-subunit thought to bind directly both the alpha- and gamma-subunits. Here, we use coimmunoprecipitation of proteins in transiently transfected cells to show that the alpha2-subunit binds directly not only to the beta-subunit, confirming previous work, but also to the gamma1-subunit. Deletion analysis of the alpha2-subunit reveals that the C-terminal 386-552 residues are sufficient to bind to the beta-subunit. The gamma1-subunit binds directly to the alpha2-subunit at two interaction sites, one within the catalytic domain consisting of alpha2 amino acids 1-312 and a second within residues 386-552. Binding of the alpha2 and the gamma1-subunits was not affected by 400 mum AMP or ATP. Furthermore, we show that the beta-subunit C terminus is essential for binding to the alpha2-subunit but, in contrast to previous work, the beta-subunit does not bind directly to the gamma1-subunit. Taken together, this study presents a new model for AMPK heterotrimer structure where through its C terminus the beta-subunit binds to the alpha-subunit that, in turn, binds to the gamma-subunit. There is no direct interaction between the beta- and gamma-subunits.

  18. The subunit composition and function of mammalian cytochrome c oxidase.

    PubMed

    Kadenbach, Bernhard; Hüttemann, Maik

    2015-09-01

    Cytochrome c oxidase (COX) from mammals and birds is composed of 13 subunits. The three catalytic subunits I-III are encoded by mitochondrial DNA, the ten nuclear-coded subunits (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, VIII) by nuclear DNA. The nuclear-coded subunits are essentially involved in the regulation of oxygen consumption and proton translocation by COX, since their removal or modification changes the activity and their mutation causes mitochondrial diseases. Respiration, the basis for ATP synthesis in mitochondria, is differently regulated in organs and species by expression of tissue-, developmental-, and species-specific isoforms for COX subunits IV, VIa, VIb, VIIa, VIIb, and VIII, but the holoenzyme in mammals is always composed of 13 subunits. Various proteins and enzymes were shown, e.g., by co-immunoprecipitation, to bind to specific COX subunits and modify its activity, but these interactions are reversible, in contrast to the tightly bound 13 subunits. In addition, the formation of supercomplexes with other oxidative phosphorylation complexes has been shown to be largely variable. The regulatory complexity of COX is increased by protein phosphorylation. Up to now 18 phosphorylation sites have been identified under in vivo conditions in mammals. However, only for a few phosphorylation sites and four nuclear-coded subunits could a specific function be identified. Research on the signaling pathways leading to specific COX phosphorylations remains a great challenge for understanding the regulation of respiration and ATP synthesis in mammalian organisms. This article reviews the function of the individual COX subunits and their isoforms, as well as proteins and small molecules interacting and regulating the enzyme.

  19. Subunit structure of the follitropin receptor

    SciTech Connect

    Shin, J.

    1985-01-01

    Both of the ..cap alpha.. and ..beta.. subunits of intact human follitropin (FSH) were radioiodinated with /sup 125/I-FSH-sodium iodide and chloramine-T, and could be resolved on polyacrylamide gels (SDS-PAGE). The electrophoretic mobility of radioiodinated FSH ..cap alpha.. and ..beta.. subunits as well as the ..cap alpha beta.. dimer changed markedly depending on the concentration of reducing agents. /sup 125/I-FSH (Ka = 1.4 x 10/sup 10/ M/sup -1/), complexes to the receptor on procine granulosa cells or in Triton X-100 extracts, was affinity-crosslinked with a cleavable (nondisulfide) homobifunctional reagent, bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone, solubilized in sodium dodecyl sulfate with or without reducing agents, and electrophoresed. Crosslinked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65 (unreduced 62), 83 (unreduced 76) and 117 (unreduced 110)kDa, in addition to hormone bands. Formation of the three bands requires the /sup 125/I-FSH hormone to bind specifically to the receptor with subsequent cross-linking. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22, 18, and 34 kDa components to the FSH ..cap alpha beta.. dimer. The results of reduction of cross-linked complexes demonstrated the existence of disulfide linkage between the three components. FSH was photoactively derivatized with N-hydroxysuccinimide ester of 4-azidobenzolyl-glycine and radioiodinated for photoaffinity labeling. When derivatized /sup 125/I-FSH (Ka = 1.12 10/sup 10/ M/sup -1/) bound to the cell was photolyzed for cross-linking and resolved on the SDS-PAGE, two new bands (106 and 61 kDa) under reducing condition appeared in addition to the hormone bands. Upon reduction with dithiotheitol and second-dimensional electrophoresis, the unreduced 104 kDa (reduced 106 kDa) band released two small components 31 and 14 kDa.

  20. Na, K ATPase beta3 subunit (CD298): association with alpha subunit and expression on peripheral blood cells.

    PubMed

    Chiampanichayakul, S; Khunkaewla, P; Pata, S; Kasinrerk, W

    2006-12-01

    Beta3 subunit is described as one of the Na, K ATPase subunits. Recently, we generated a monoclonal antibody (mAb), termed P-3E10. This mAb was shown to react with the Na, K ATPase beta3 subunit or CD298. By immunofluorescence analysis using mAb P-3E10, it was found that all peripheral blood leukocytes express Na, K ATPase beta3. The presence of beta3 subunit on leukocytes is not in a quantitative polymorphic manner. Upon phytohemagglutinin or phorbol myristate acetate activation, the expression level of the Na, K ATPase beta3 subunit on activated peripheral blood mononuclear cells was not altered in comparison with those of unstimulated cells. Red blood cells (RBCs) of healthy donors showed negative reactivity with mAb P-3E10. However, more than 80% of thalassemic RBCs showed positive reactivity. By immunoprecipitation, moreover, a protein band of 55-65 kDa was precipitated from normal RBC membrane using mAb P-3E10. These results evidenced that the beta3 subunit of Na, K ATPase is expressed on RBC membrane but the epitope recognized by mAb P-3E10 is hidden in normal RBCs. Furthermore, we showed the association of beta3 subunit and alpha subunit of Na, K ATPase. This information is important for further understanding of the functional roles of this molecule.

  1. [A promoter responsible for over-expression of cholera toxin B subunit in cholera toxin A subunit structure gene].

    PubMed

    Cao, C; Shi, C; Li, P; Ma, Q

    1997-01-01

    A promoter sequence, which promotes the transcription of cholera toxin B subunit gene, was found in cholera toxin A subunit structure gene. The transcription starts at the adenine Located at +833, that is 456bp upstream to the A of the initiation codon ATG of cholera toxin B gene. Under the control of the promoter, cholera toxin B subunit was over-expressed as high as 200 mg/L at an optimized culture condition. The chloramphenicol acetyl transferase gene and beta-galactosidase could also be efficiently expressed under the direction of the promoter. This promoter may be responsible for the 6 fold and 7 fold higher expression level of cholera toxin B subunit than cholera toxin A subunit in V. cholerae and Escheria coli respectively. The over-expression of CTB may be useful in preparing vaccine against cholera and facilitating the construction of peptide-bearing immunogenic hybrid proteins.

  2. Altered 40 S ribosomal subunits in omnipotent suppressors of yeast.

    PubMed

    Eustice, D C; Wakem, L P; Wilhelm, J M; Sherman, F

    1986-03-20

    The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

  3. Interactions between the human RNA polymerase II subunits.

    PubMed

    Acker, J; de Graaff, M; Cheynel, I; Khazak, V; Kedinger, C; Vigneron, M

    1997-07-04

    As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.

  4. Echinococcus granulosus Antigen B Structure: Subunit Composition and Oligomeric States

    PubMed Central

    Monteiro, Karina M.; Cardoso, Mateus B.; Follmer, Cristian; da Silveira, Nádya P.; Vargas, Daiani M.; Kitajima, Elliot W.; Zaha, Arnaldo; Ferreira, Henrique B.

    2012-01-01

    Background Antigen B (AgB) is the major protein secreted by the Echinococcus granulosus metacestode and is involved in key host-parasite interactions during infection. The full comprehension of AgB functions depends on the elucidation of several structural aspects that remain unknown, such as its subunit composition and oligomeric states. Methodology/Principal Findings The subunit composition of E. granulosus AgB oligomers from individual bovine and human cysts was assessed by mass spectrometry associated with electrophoretic analysis. AgB8/1, AgB8/2, AgB8/3 and AgB8/4 subunits were identified in all samples analyzed, and an AgB8/2 variant (AgB8/2v8) was found in one bovine sample. The exponentially modified protein abundance index (emPAI) was used to estimate the relative abundance of the AgB subunits, revealing that AgB8/1 subunit was relatively overrepresented in all samples. The abundance of AgB8/3 subunit varied between bovine and human cysts. The oligomeric states formed by E. granulosus AgB and recombinant subunits available, rAgB8/1, rAgB8/2 and rAgB8/3, were characterized by native PAGE, light scattering and microscopy. Recombinant subunits showed markedly distinct oligomerization behaviors, forming oligomers with a maximum size relation of rAgB8/3>rAgB8/2>rAgB8/1. Moreover, the oligomeric states formed by rAgB8/3 subunit were more similar to those observed for AgB purified from hydatid fluid. Pressure-induced dissociation experiments demonstrated that the molecular assemblies formed by the more aggregative subunits, rAgB8/2 and rAgB8/3, also display higher structural stability. Conclusions/Significance For the first time, AgB subunit composition was analyzed in samples from single hydatid cysts, revealing qualitative and quantitative differences between samples. We showed that AgB oligomers are formed by different subunits, which have distinct abundances and oligomerization properties. Overall, our findings have significantly contributed to increase the

  5. NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequences compared for members of the genus Taenia (Cestoda).

    PubMed

    Gasser, R B; Zhu, X; McManus, D P

    1999-12-01

    Nine members of the genus Taenia (Taenia taeniaeformis, Taenia hydatigena, Taenia pisiformis, Taenia ovis, Taenia multiceps, Taenia serialis, Taenia saginata, Taenia solium and the Asian Taenia) were characterised by their mitochondrial NADH dehydrogenase subunit 1 gene sequences and their genetic relationships were compared with those derived from the cytochrome c oxidase subunit 1 sequence data. The extent of inter-taxon sequence difference in NADH dehydrogenase subunit 1 (approximately 5.9-30.8%) was usually greater than in cytochrome c oxidase subunit 1 (approximately 2.5-18%). Although topology of the phenograms derived from NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit 1 sequence data differed, there was concordance in that T. multiceps, T. serialis (of canids), T. saginata and the Asian Taenia (of humans) were genetically most similar, and those four members were genetically more similar to T. ovis and T. solium than they were to T. hydatigena and T. pisiformis (of canids) or T. taeniaeformis (of cats). The NADH dehydrogenase subunit 1 sequence data may prove useful in studies of the systematics and population genetic structure of the Taeniidae.

  6. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; Chapelle, S; De Wachter, R

    1994-01-01

    A database on large ribosomal subunit RNA is made available. It contains 258 sequences. It provides sequence, alignment and secondary structure information in computer-readable formats. Files can be obtained using ftp. PMID:7524023

  7. The eukaryotic RNA exosome: same scaffold but variable catalytic subunits.

    PubMed

    Lykke-Andersen, Søren; Tomecki, Rafal; Jensen, Torben Heick; Dziembowski, Andrzej

    2011-01-01

    The RNA exosome is a versatile ribonucleolytic protein complex that participates in a multitude of cellular RNA processing and degradation events. It consists of an invariable nine-subunit core that associates with a variety of enzymatically active subunits and co-factors. These contribute to or even provide the catalytic activity and substrate specificity of the complex. The S. cerevisiae exosome has been intensively studied since its discovery in 1997 and thus serves as the archetype of eukaryotic exosomes. Notably, its catalytic potential, derived exclusively from associated subunits, differs between the nuclear and cytoplasmic versions of the complex. The same holds true for other eukaryotes, however, recent discoveries from various laboratories including our own have revealed that there are variations on this theme. Here, we review the latest findings concerning catalytic subunits of eukaryotic exosomes, and we discuss the apparent need for differential composition and subcellular distribution of exosome variants.

  8. Genetic Analysis of the Cytoplasmic Dynein Subunit Families

    PubMed Central

    Pfister, K. Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M. C

    2006-01-01

    Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles. PMID:16440056

  9. A process yields large quantities of pure ribosome subunits

    NASA Technical Reports Server (NTRS)

    Friedman, M.; Lu, P.; Rich, A.

    1972-01-01

    Development of process for in-vitro protein synthesis from living cells followed by dissociation of ribosomes into subunits is discussed. Process depends on dialysis or use of chelating agents. Operation of process and advantages over previous methods are outlined.

  10. Subunit-Specific Trafficking of GABAA Receptors during Status Epilepticus

    PubMed Central

    Goodkin, Howard P.; Joshi, Suchitra; Mtchedlishvili, Zakaria; Brar, Jasmit; Kapur, Jaideep

    2010-01-01

    It is proposed that a reduced surface expression of GABAA receptors (GABARs) contributes to the pathogenesis of status epilepticus (SE), a condition characterized by prolonged seizures. This hypothesis was based on the finding that prolonged epileptiform bursting (repetitive bursts of prolonged depolarizations with superimposed action potentials) in cultures of dissociated hippocampal pyramidal neurons (dissociated cultures) results in the increased intracellular accumulation of GABARs. However, it is not known whether this rapid modification in the surface-expressed GABAR pool results from selective, subunit-dependent or nonselective, subunit-independent internalization of GABARs. In hippocampal slices obtained from animals undergoing prolonged SE (SE-treated slices), we found that the surface expression of the GABARβ2/3 and γ2 subunits was reduced, whereas that of the δ subunit was not. Complementary electrophysiological recordings from dentate granule cells in SE-treated slices demonstrated a reduction in GABAR-mediated synaptic inhibition, but not tonic inhibition. A reduction in the surface expression of the γ2 subunit, but not the δ subunit was also observed in dissociated cultures and organotypic hippocampal slice cultures when incubated in an elevated KCl external medium or an elevated KCl external medium supplemented with NMDA, respectively. Additional studies demonstrated that the reduction in the surface expression of the γ2 subunit was independent of direct ligand binding of the GABAR. These findings demonstrate that the regulation of surface-expressed GABAR pool during SE is subunit-specific and occurs independent of ligand binding. The differential modulation of the surface expression of GABARs during SE has potential implications for the treatment of this neurological emergency. PMID:18322097

  11. F-subunit reinforces torque generation in V-ATPase.

    PubMed

    Kishikawa, Jun-ichi; Seino, Akihiko; Nakanishi, Atsuko; Tirtom, Naciye Esma; Noji, Hiroyuki; Yokoyama, Ken; Hayashi, Kumiko

    2014-09-01

    Vacuolar-type H(+)-pumping ATPases (V-ATPases) perform remarkably diverse functions in eukaryotic organisms. They are present in the membranes of many organelles and regulate the pH of several intracellular compartments. A family of V-ATPases is also present in the plasma membranes of some bacteria. Such V-ATPases function as ATP-synthases. Each V-ATPase is composed of a water-soluble domain (V1) and a membrane-embedded domain (Vo). The ATP-driven rotary unit, V[Formula: see text], is composed of A, B, D, and F subunits. The rotary shaft (the DF subcomplex) rotates in the central cavity of the A3B3-ring (the catalytic hexamer ring). The D-subunit, which has a coiled-coil domain, penetrates into the ring, while the F-subunit is a globular-shaped domain protruding from the ring. The minimal ATP-driven rotary unit of V[Formula: see text] is comprised of the A3B3D subunits, and we therefore investigated how the absence of the globular-shaped F-subunit affects the rotary torque generation of V[Formula: see text]. Using a single-molecule technique, we observed the motion of the rotary motors. To obtain the torque values, we then analyzed the measured motion trajectories based on the fluctuation theorem, which states that the law of entropy production in non-equilibrium conditions and has been suggested as a novel and effective method for measuring torque. The measured torque of A3B3D was half that of the wild-type V1, and full torque was recovered in the mutant V1, in which the F-subunit was genetically fused with the D-subunit, indicating that the globular-shaped F-subunit reinforces torque generation in V1.

  12. Transcription Activator Interactions with Multiple SWI/SNF Subunits

    PubMed Central

    Neely, Kristen E.; Hassan, Ahmed H.; Brown, Christine E.; Howe, LeAnn; Workman, Jerry L.

    2002-01-01

    We have previously shown that the yeast SWI/SNF complex stimulates in vitro transcription from chromatin templates in an ATP-dependent manner. SWI/SNF function in this regard requires the presence of an activator with which it can interact directly, linking activator recruitment of SWI/SNF to transcriptional stimulation. In this study, we determine the SWI/SNF subunits that mediate its interaction with activators. Using a photo-cross-linking label transfer strategy, we show that the Snf5, Swi1, and Swi2/Snf2 subunits are contacted by the yeast acidic activators, Gcn4 and Hap4, in the context of the intact native SWI/SNF complex. In addition, we show that the same three subunits can interact individually with acidic activation domains, indicating that each subunit contributes to binding activators. Furthermore, mutations that reduce the activation potential of these activators also diminish its interaction with each of these SWI/SNF subunits. Thus, three distinct subunits of the SWI/SNF complex contribute to its interactions with activation domains. PMID:11865042

  13. Transcriptional regulators of Na,K-ATPase subunits

    PubMed Central

    Li, Zhiqin; Langhans, Sigrid A.

    2015-01-01

    The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic α-subunit, the β-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids, and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits has been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease. PMID:26579519

  14. Hybrid Rubisco of tomato large subunits and tobacco small subunits is functional in tobacco plants.

    PubMed

    Zhang, Xing-Hai; Webb, James; Huang, Yi-Hong; Lin, Li; Tang, Ri-Sheng; Liu, Aimin

    2011-03-01

    Biogenesis of functional ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants requires specific assembly in the chloroplast of the imported, cytosol-synthesized small subunits (SS) with the chloroplast-made large subunits (LS). Accumulating evidence indicates that chloroplasts (plastids) generally have a low tolerance for assembling foreign or modified Rubisco. To explore Rubisco engineering, we created two lines of transplastomic tobacco plants whose rbcL gene was replaced by tomato-derived rbcL: plant LLS2 with Rubisco composed of tobacco SS and Q437R LS and plant LLS4 with a hybrid Rubisco of tobacco SS and tomato LS (representing four substitutions of Y226F, A230T, S279T and Q437R from tobacco LS). Plant LLS2 exhibited similar phenotypes as the wild type. Plant LLS4 showed lower chlorophyll and Rubisco levels particularly in young emerging leaves, lower photosynthesis rates and biomass during early stages of development, but was able to reach reproductive maturity and somewhat wild type-like phenotype under ambient CO₂ condition. In vitro assays detected similar carboxylase activity and RuBP affinity in LLS2 and LLS4 plants as in wild type. Our studies demonstrated that tomato LS was sufficiently assembled with tobacco SS into functional Rubisco. The hybrid Rubisco of tomato LS and tobacco SS can drive photosynthesis that supports photoautotrophic growth and reproduction of tobacco plants under ambient CO₂ and light conditions. We discuss the effect of these residue substitutions on Rubisco activity and the possible attribution of chlorophyll deficiency to the in planta photosynthesis performance in the hybrid Rubisco plants.

  15. NOX Activation by Subunit Interaction and Underlying Mechanisms in Disease

    PubMed Central

    Rastogi, Radhika; Geng, Xiaokun; Li, Fengwu; Ding, Yuchuan

    2017-01-01

    Nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX) is an enzyme complex with the sole function of producing superoxide anion and reactive oxygen species (ROS) at the expense of NADPH. Vital to the immune system as well as cellular signaling, NOX is also involved in the pathologies of a wide variety of disease states. Particularly, it is an integral player in many neurological diseases, including stroke, TBI, and neurodegenerative diseases. Pathologically, NOX produces an excessive amount of ROS that exceed the body’s antioxidant ability to neutralize them, leading to oxidative stress and aberrant signaling. This prevalence makes it an attractive therapeutic target and as such, NOX inhibitors have been studied and developed to counter NOX’s deleterious effects. However, recent studies of NOX have created a better understanding of the NOX complex. Comprised of independent cytosolic subunits, p47-phox, p67-phox, p40-phox and Rac, and membrane subunits, gp91-phox and p22-phox, the NOX complex requires a unique activation process through subunit interaction. Of these subunits, p47-phox plays the most important role in activation, binding and translocating the cytosolic subunits to the membrane and anchoring to p22-phox to organize the complex for NOX activation and function. Moreover, these interactions, particularly that between p47-phox and p22-phox, are dependent on phosphorylation initiated by upstream processes involving protein kinase C (PKC). This review will look at these interactions between subunits and with PKC. It will focus on the interaction involving p47-phox with p22-phox, key in bringing the cytosolic subunits to the membrane. Furthermore, the implication of these interactions as a target for NOX inhibitors such as apocynin will be discussed as a potential avenue for further investigation, in order to develop more specific NOX inhibitors based on the inhibition of NOX assembly and activation. PMID:28119569

  16. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia

    PubMed Central

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-01-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  17. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    PubMed Central

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  18. A distinct holoenzyme organization for two-subunit pyruvate carboxylase

    PubMed Central

    Choi, Philip H.; Jo, Jeanyoung; Lin, Yu-Cheng; Lin, Min-Han; Chou, Chi-Yuan; Dietrich, Lars E. P.; Tong, Liang

    2016-01-01

    Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the β subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4β4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2β4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis. PMID:27708276

  19. Separation and characterization of alpha-chain subunits from tilapia (Tilapia zillii) skin gelatin using ultrafiltration.

    PubMed

    Chen, Shulin; Tang, Lanlan; Su, Wenjin; Weng, Wuyin; Osako, Kazufumi; Tanaka, Munehiko

    2015-12-01

    Alpha-chain subunits were separated from tilapia skin gelatin using ultrafiltration, and the physicochemical properties of obtained subunits were investigated. As a result, α1-subunit and α2-subunit could be successfully separated by 100 kDa MWCO regenerated cellulose membranes and 150 kDa MWCO polyethersulfone membranes, respectively. Glycine was the most dominant amino acid in both α1-subunit and α2-subunit. However, the tyrosine content was higher in α2-subunit than in α1-subunit, resulting in strong absorption near 280 nm observed in the UV absorption spectrum. Based on the DSC analysis, it was found that the glass transition temperatures of gelatin, α1-subunit and α2-subunit were 136.48 °C, 126.77 °C and 119.43 °C, respectively. Moreover, the reduced viscosity and denaturation temperature of α1-subunit were higher than those of α2-subunit, and the reduced viscosity reached the highest when α-subunits were mixed with α1/α2 ratio of approximately 2, suggesting that α1-subunit plays a more important role in the thermostability of gelatin than α2-subunit.

  20. Dengue vaccine: an update on recombinant subunit strategies.

    PubMed

    Martin, J; Hermida, L

    2016-03-01

    Dengue is an increasing public health problem worldwide, with the four serotypes of the virus infecting over 390 million people annually. There is no specific treatment or antiviral drug for dengue, and prevention is largely limited to controlling the mosquito vectors or disrupting the human-vector contact. Despite the considerable progress made in recent years, an effective vaccine against the virus is not yet available. The development of a dengue vaccine has been hampered by many unique challenges, including the need to ensure the absence of vaccine-induced enhanced severity of disease. Recombinant protein subunit vaccines offer a safer alternative to other vaccine approaches. Several subunit vaccine candidates are presently under development, based on different structural and non-structural proteins of the virus. Novel adjuvants or immunopotentiating strategies are also being tested to improve their immunogenicity. This review summarizes the current status and development trends of subunit dengue vaccines.

  1. Cholera Toxin B: One Subunit with Many Pharmaceutical Applications

    PubMed Central

    Baldauf, Keegan J.; Royal, Joshua M.; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

    2015-01-01

    Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction. PMID:25802972

  2. Cholera toxin B: one subunit with many pharmaceutical applications.

    PubMed

    Baldauf, Keegan J; Royal, Joshua M; Hamorsky, Krystal Teasley; Matoba, Nobuyuki

    2015-03-20

    Cholera, a waterborne acute diarrheal disease caused by Vibrio cholerae, remains prevalent in underdeveloped countries and is a serious health threat to those living in unsanitary conditions. The major virulence factor is cholera toxin (CT), which consists of two subunits: the A subunit (CTA) and the B subunit (CTB). CTB is a 55 kD homopentameric, non-toxic protein binding to the GM1 ganglioside on mammalian cells with high affinity. Currently, recombinantly produced CTB is used as a component of an internationally licensed oral cholera vaccine, as the protein induces potent humoral immunity that can neutralize CT in the gut. Additionally, recent studies have revealed that CTB administration leads to the induction of anti-inflammatory mechanisms in vivo. This review will cover the potential of CTB as an immunomodulatory and anti-inflammatory agent. We will also summarize various recombinant expression systems available for recombinant CTB bioproduction.

  3. Antigenic breadth: a missing ingredient in HSV-2 subunit vaccines?

    PubMed

    Halford, William P

    2014-06-01

    The successful human papillomavirus and hepatitis B virus subunit vaccines contain single viral proteins that represent 22 and 12%, respectively, of the antigens encoded by these tiny viruses. The herpes simplex virus 2 (HSV-2) genome is >20 times larger. Thus, a single protein subunit represents 1% of HSV-2's total antigenic breadth. Antigenic breadth may explain why HSV-2 glycoprotein subunit vaccines have failed in clinical trials, and why live HSV-2 vaccines that express 99% of HSV-2's proteome may be more effective. I review the mounting evidence that live HSV-2 vaccines offer a greater opportunity to stop the spread of genital herpes, and I consider the unfounded 'safety concerns' that have kept live HSV-2 vaccines out of U.S. clinical trials for 25 years.

  4. G alpha 12 and G alpha 13 subunits define a fourth class of G protein alpha subunits.

    PubMed Central

    Strathmann, M P; Simon, M I

    1991-01-01

    Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are central to the signaling processes of multicellular organisms. We have explored the diversity of the G protein subunits in mammals and found evidence for a large family of genes that encode the alpha subunits. Amino acid sequence comparisons show that the different alpha subunits fall into at least three classes. These classes have been conserved in animals separated by considerable evolutionary distances; they are present in mammals, Drosophila, and nematodes. We have now obtained cDNA clones encoding two murine alpha subunits, G alpha 12 and G alpha 13, that define a fourth class. The translation products are predicted to have molecular masses of 44 kDa and to be insensitive to ADP-ribosylation by pertussis toxin. They share 67% amino acid sequence identity with each other and less than 45% identity with other alpha subunits. Their transcripts can be detected in every tissue examined, although the relative levels of the G alpha 13 message appear somewhat variable. Images PMID:1905812

  5. Subunits of the Schizosaccharomyces pombe RNA polymerase II: enzyme purification and structure of the subunit 3 gene.

    PubMed Central

    Azuma, Y; Yamagishi, M; Ishihama, A

    1993-01-01

    To improve our understanding of the structure and function of eukaryotic RNA polymerase II, we purified the enzyme from the fission yeast Schizosaccharomyces pombe. The highly purified RNA polymerase II contained more than eleven polypeptides. The sizes of the largest the second-, and the third-largest polypeptides as measured by SDS-polyacrylamide gel electrophoresis were about 210, 150, and 40 kilodaltons (kDa), respectively, and are similar to those of RPB1, 2, and 3 subunits of Saccharomyces cerevisiae RNA polymerase II. Using the degenerated primers designed after amino acid micro-sequencing of the 40 kDa third-largest polypeptide (subunit 3), we cloned the subunit 3 gene (rpb3) and determined its DNA sequence. Taken together with the sequence of parts of PCR-amplified cDNA, the predicted coding sequence of rpb3, interrupted by two introns, was found to encode a polypeptide of 297 amino acid residues in length with a molecular weight of 34 kDa. The S. pombe subunit 3 contains four structural domains conserved for the alpha-subunit family of RNA polymerase from both eukaryotes and prokaryotes. A putative leucine zipper motif was found to exist in the C-terminal proximal conserved region (domain D). Possible functions of the conserved domains are discussed. Images PMID:8367291

  6. Stiffness of γ subunit of F(1)-ATPase.

    PubMed

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2010-11-01

    F(1)-ATPase is a molecular motor in which the γ subunit rotates inside the α(3)β(3) ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F(1)-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F(1) and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F(1) from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the α(3)β(3) ring, and the complex of the external part of the γ subunit and the α(3)β(3) ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F(o) and F(1)-ATPase.

  7. Localisation of AMPK γ subunits in cardiac and skeletal muscles.

    PubMed

    Pinter, Katalin; Grignani, Robert T; Watkins, Hugh; Redwood, Charles

    2013-12-01

    The trimeric protein AMP-activated protein kinase (AMPK) is an important sensor of energetic status and cellular stress, and mutations in genes encoding two of the regulatory γ subunits cause inherited disorders of either cardiac or skeletal muscle. AMPKγ2 mutations cause hypertrophic cardiomyopathy with glycogen deposition and conduction abnormalities; mutations in AMPKγ3 result in increased skeletal muscle glycogen. In order to gain further insight into the roles of the different γ subunits in muscle and into possible disease mechanisms, we localised the γ2 and γ3 subunits, along with the more abundant γ1 subunit, by immunofluorescence in cardiomyocytes and skeletal muscle fibres. The predominant cardiac γ2 variant, γ2-3B, gave a striated pattern in cardiomyocytes, aligning with the Z-disk but with punctate staining similar to T-tubule (L-type Ca(2+) channel) and sarcoplasmic reticulum (SERCA2) markers. In skeletal muscle fibres AMPKγ3 localises to the I band, presenting a uniform staining that flanks the Z-disk, also coinciding with the position of Ca(2+) influx in these muscles. The localisation of γ2-3B- and γ3-containing AMPK suggests that these trimers may have similar functions in the different muscles. AMPK containing γ2-3B was detected in oxidative skeletal muscles which had low expression of γ3, confirming that these two regulatory subunits may be co-ordinately regulated in response to metabolic requirements. Compartmentalisation of AMPK complexes is most likely dependent on the regulatory γ subunit and this differential localisation may direct substrate selection and specify particular functional roles.

  8. Micelle-Based Adjuvants for Subunit Vaccine Delivery

    PubMed Central

    Trimaille, Thomas; Verrier, Bernard

    2015-01-01

    In the development of subunit vaccines with purified or recombinant antigens for cancer and infectious diseases, the design of improved and safe adjuvants able to efficiently target the antigen presenting cells, such as dendritic cells, represents a crucial challenge. Nanoparticle-based antigen delivery systems have been identified as an innovative strategy to improve the efficacy of subunit vaccines. Among them, self-assembled micellar nanoparticles from amphiphilic (macro)molecules have recently emerged as promising candidates. In this short review, we report on the recent research findings highlighting the versatility and potential of such systems in vaccine delivery. PMID:26426060

  9. Advancements in the development of subunit influenza vaccines

    PubMed Central

    Zhang, Naru; Zheng, Bo-Jian; Lu, Lu; Zhou, Yusen; Jiang, Shibo; Du, Lanying

    2014-01-01

    The ongoing threat of influenza epidemics and pandemics has emphasized the importance of developing safe and effective vaccines against infections from divergent influenza viruses. In this review, we first introduce the structure and life cycle of influenza A viruses, describing major influenza A virus-caused pandemics. We then compare different types of influenza vaccines and discuss current advancements in the development of subunit influenza vaccines, particularly those based on nucleoprotein (NP), extracellular domain of matrix protein 2 (M2e) and hemagglutinin (HA) proteins. We also illustrate potential strategies for improving the efficacy of subunit influenza vaccines. PMID:25529753

  10. Carrier subunit of plasma membrane transporter is required for oxidative folding of its helper subunit.

    PubMed

    Rius, Mònica; Chillarón, Josep

    2012-05-25

    We study the amino acid transport system b(0,+) as a model for folding, assembly, and early traffic of membrane protein complexes. System b(0,+) is made of two disulfide-linked membrane subunits: the carrier, b(0,+) amino acid transporter (b(0,+)AT), a polytopic protein, and the helper, related to b(0,+) amino acid transporter (rBAT), a type II glycoprotein. rBAT ectodomain mutants display folding/trafficking defects that lead to type I cystinuria. Here we show that, in the presence of b(0,+)AT, three disulfides were formed in the rBAT ectodomain. Disulfides Cys-242-Cys-273 and Cys-571-Cys-666 were essential for biogenesis. Cys-673-Cys-685 was dispensable, but the single mutants C673S, and C685S showed compromised stability and trafficking. Cys-242-Cys-273 likely was the first disulfide to form, and unpaired Cys-242 or Cys-273 disrupted oxidative folding. Strikingly, unassembled rBAT was found as an ensemble of different redox species, mainly monomeric. The ensemble did not change upon inhibition of rBAT degradation. Overall, these results indicated a b(0,+)AT-dependent oxidative folding of the rBAT ectodomain, with the initial and probably cotranslational formation of Cys-242-Cys-273, followed by the oxidation of Cys-571-Cys-666 and Cys-673-Cys-685, that was completed posttranslationally.

  11. Effects of cations and cosolvents on eukaryotic ribosomal subunit conformation

    SciTech Connect

    Moore, M.N.; Spremulli, L.L.

    1985-01-01

    The effects of various cations and cosolvents on the conformation of wheat germ ribosomes and ribosomal subunits have been investigated by using the techniques of circular dichroism and differential hydrogen exchange. A class of hydrogens on both the 40S and 60S subunits exchange out more rapidly as the Mg/sup 2 +/ concentration is raised, indicating that Mg/sup 2 +/ causes a change in subunit conformation. Ca/sup 2 +/ and the polyamines produce a similar increase in the rate of hydrogen exchange. These results suggest that increases in cation concentrations permit a tightening of ribosome structure and a greater degree of internalization of the rRNA. The cosolvent glycerol causes an alteration in the CD spectrum of 80S ribosomes in both the nucleic acid and protein portions of the spectrum. Glycerol also causes a decrease in the rate of exchange of a number of hydrogens on both the 40S and 60S subunits. These results are interpreted to mean that glycerol favors a more native, less denatured structure in the ribosome.

  12. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2015-01-01

    The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.

  13. The multifaceted subunit interfaces of ionotropic glutamate receptors.

    PubMed

    Green, Tim; Nayeem, Naushaba

    2014-06-06

    The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.

  14. Spectroscopic properties of Carcinus aestuarii hemocyanin and its structural subunits

    NASA Astrophysics Data System (ADS)

    Dolashka-Angelova, Pavlina; Hristova, Rumiyana; Stoeva, Stanka; Voelter, Wolfgang

    1999-12-01

    Hemocyanin (Hc) of Carcinus aestuarii contains three major and one minor electrophoretically separable polypeptide chains which were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography. N-terminal amino acid sequences of four structural subunits (SSs) from C. aestuarii were compared with known N-terminal sequences from other arthropodan hemocyanins. The conformational changes, induced by various treatments, were monitored by far UV, CD and fluorescence spectroscopy. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 52-59°C and coincide with the melting temperatures, Tm (49-55°C), determined by CD spectroscopy. The free energy of stabilization in water, Δ GDH 2O , toward guanidinium hydrochloride is about 1.3 times higher for the dodecameric Hc as compared to the isolated subunits and about one time higher for Ca1, comparing with other SSs. The studies reveal that the conformational stability of the native dodecamer towards various denaturants (temperature and guanidinium hydrochloride) indicate that the quaternary structure is stabilized by oligomerization between structural subunits, and the possibility of a structural role of the sugar mojeties cannot be excluded.

  15. Emergence of ion channel modal gating from independent subunit kinetics

    PubMed Central

    Bicknell, Brendan A.

    2016-01-01

    Many ion channels exhibit a slow stochastic switching between distinct modes of gating activity. This feature of channel behavior has pronounced implications for the dynamics of ionic currents and the signaling pathways that they regulate. A canonical example is the inositol 1,4,5-trisphosphate receptor (IP3R) channel, whose regulation of intracellular Ca2+ concentration is essential for numerous cellular processes. However, the underlying biophysical mechanisms that give rise to modal gating in this and most other channels remain unknown. Although ion channels are composed of protein subunits, previous mathematical models of modal gating are coarse grained at the level of whole-channel states, limiting further dialogue between theory and experiment. Here we propose an origin for modal gating, by modeling the kinetics of ligand binding and conformational change in the IP3R at the subunit level. We find good agreement with experimental data over a wide range of ligand concentrations, accounting for equilibrium channel properties, transient responses to changing ligand conditions, and modal gating statistics. We show how this can be understood within a simple analytical framework and confirm our results with stochastic simulations. The model assumes that channel subunits are independent, demonstrating that cooperative binding or concerted conformational changes are not required for modal gating. Moreover, the model embodies a generally applicable principle: If a timescale separation exists in the kinetics of individual subunits, then modal gating can arise as an emergent property of channel behavior. PMID:27551100

  16. Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

    PubMed Central

    Römling, Ute; Galperin, Michael Y.

    2015-01-01

    Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

  17. ATP-induced helicase slippage reveals highly coordinated subunits

    NASA Astrophysics Data System (ADS)

    Wang, Michelle D.

    2012-02-01

    Helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication, repair and recombination. T7 helicase, a model hexameric motor, has been observed to use dTTP, but not ATP, to unwind dsDNA as it translocates along ssDNA. Whether and how different subunits of the helicase coordinate their chemo-mechanical activities and DNA binding during translocation is still under debate. Here we address this question using a single-molecule approach to monitor helicase unwinding. We found that T7 helicase does in fact unwind dsDNA in the presence of ATP and that the unwinding rate is even faster than that with dTTP. However, unwinding was repeatedly interrupted by sudden slippage events, ultimately preventing unwinding over a substantial distance. This behaviour was greatly reduced with the supplement of a small amount of dTTP. These findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination. Our results support a model where nearly all subunits coordinate their chemo-mechanical activities and DNA binding. Such subunit coordination may be general to many ring-shaped helicases and reveals a potential mechanism for regulation of DNA unwinding during replication.

  18. A World History Sub-Unit: Teaching about Turkey.

    ERIC Educational Resources Information Center

    Lynn, Karen

    This document is a sub-unit teaching plan for world history teachers who want to use multicultural concepts in the world history curriculum. The objective explored includes a student response to the Turkish question of "Who are we"? Teacher preparation involves defining social and cultural roots and outlining periods of Turkish history.…

  19. Glycoprotein hormone assembly in the endoplasmic reticulum: I. The glycosylated end of human alpha-subunit loop 2 is threaded through a beta-subunit hole.

    PubMed

    Xing, Yongna; Myers, Rebecca V; Cao, Donghui; Lin, Win; Jiang, Mei; Bernard, Michael P; Moyle, William R

    2004-08-20

    Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the alpha-subunit straddles a hole in the beta-subunit. This hole is formed when a cysteine at the end of a beta-subunit strand known as the "seatbelt" becomes "latched" by a disulfide to a cysteine in the beta-subunit core. The heterodimer is stabilized in part by the difficulty of threading the glycosylated end of the alpha-subunit loop 2 through this hole, a phenomenon required for subunit dissociation. Subunit combination in vitro, which occurs by the reverse process, can be accelerated by removing the alpha-subunit oligosaccharide. In cells, heterodimer assembly was thought to occur primarily by a mechanism in which the seatbelt is wrapped around the alpha-subunit after the subunits dock. Here we show that this "wraparound" process can be used to assemble disulfide cross-linked human choriogonadotropin analogs that contain an additional alpha-subunit cysteine, but only if the normal beta-subunit latch site has been removed. Normally, the seatbelt is latched before the subunits dock and assembly is completed when the glycosylated end of alpha-subunit loop 2 is threaded beneath the seatbelt. The unexpected finding that most assembly of human choriogonadotropin, human follitropin, and human thyrotropin heterodimers occurs in this fashion, indicates that threading may be an important phenomenon during protein folding and macromolecule assembly in the endoplasmic reticulum. We suggest that the unusual structures of the glycoprotein hormones makes them useful for identifying factors that influence this process in living cells.

  20. Binding interactions with the complementary subunit of nicotinic receptors.

    PubMed

    Blum, Angela P; Van Arnam, Ethan B; German, Laurel A; Lester, Henry A; Dougherty, Dennis A

    2013-03-08

    The agonist-binding site of nicotinic acetylcholine receptors (nAChRs) spans an interface between two subunits of the pentameric receptor. The principal component of this binding site is contributed by an α subunit, and it binds the cationic moiety of the nicotinic pharmacophore. The other part of the pharmacophore, a hydrogen bond acceptor, has recently been shown to bind to the complementary non-α subunit via the backbone NH of a conserved Leu. This interaction was predicted by studies of ACh-binding proteins and confirmed by functional studies of the neuronal (CNS) nAChR, α4β2. The ACh-binding protein structures further suggested that the hydrogen bond to the backbone NH is mediated by a water molecule and that a second hydrogen bonding interaction occurs between the water molecule and the backbone CO of a conserved Asn, also on the non-α subunit. Here, we provide new insights into the nature of the interactions between the hydrogen bond acceptor of nicotinic agonists and the complementary subunit backbone. We studied both the nAChR of the neuromuscular junction (muscle-type) and a neuronal subtype, (α4)2(β4)3. In the muscle-type receptor, both ACh and nicotine showed a strong interaction with the Leu NH, but the potent nicotine analog epibatidine did not. This interaction was much attenuated in the α4β4 receptor. Surprisingly, we found no evidence for a functionally significant interaction with the backbone carbonyl of the relevant Asn in either receptor with an array of agonists.

  1. The centromere-kinetochore complex: a repeat subunit model

    PubMed Central

    1991-01-01

    The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species. PMID:1828250

  2. Expression of glutamate receptor subunits in human cancers.

    PubMed

    Stepulak, Andrzej; Luksch, Hella; Gebhardt, Christine; Uckermann, Ortrud; Marzahn, Jenny; Sifringer, Marco; Rzeski, Wojciech; Staufner, Christian; Brocke, Katja S; Turski, Lechoslaw; Ikonomidou, Chrysanthy

    2009-10-01

    Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.

  3. Targeting signals and subunit interactions in coated vesicle adaptor complexes

    PubMed Central

    1995-01-01

    There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN. The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19. To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains. We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting. Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19. These observations suggest that these other subunits may play an important role in targeting. In contrast, beta- and beta'-adaptins are clearly not involved in this event. Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal. We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system. Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma- adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19. These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane. PMID:7593184

  4. Heterotrimeric G protein subunit Gγ13 is critical to olfaction

    PubMed Central

    Li, Feng; Ponissery-Saidu, Samsudeen; Yee, Karen; Wang, Hong; Chen, Meng-Ling; Iguchi, Naoko; Zhang, Genhua; Jiang, Ping; Reisert, Johannes; Huang, Liquan

    2013-01-01

    The activation of G-protein-coupled olfactory receptors on the olfactory sensory neurons (OSNs) triggers a signaling cascade, which is mediated by a heterotrimeric G protein consisting of α, β and γ subunits. Although its α subunit, Gαolf, has been identified and well characterized, the identities of its β and γ subunits and their function in olfactory signal transduction, however, have not been well established yet. We and others have found the expression of Gγ13 in the olfactory epithelium, particularly in the cilia of the OSNs. In this study, we generated a conditional gene knockout mouse line to specifically nullify Gγ13 expression in the olfactory marker protein-expressing OSNs. Immunohistochemical and Western blot results showed that Gγ13 subunit was indeed eliminated in the mutant mice’s olfactory epithelium. Intriguingly, Gαolf, β1 subunits, Ric-8B and CEP290 proteins were also absent in the epithelium whereas the presence of the effector enzyme adenylyl cyclase III remained largely unaltered. Electro-olfactogram studies showed that the mutant animals had greatly reduced responses to a battery of odorants including three presumable pheromones. Behavioral tests indicated that the mutant mice had a remarkably reduced ability to perform an odor-guided search task although their motivation and agility seemed normal. Our results indicate that Gαolf exclusively forms a functional heterotrimeric G protein with Gβ1 and Gγ13 in OSNs, mediating olfactory signal transduction. The identification of the olfactory G protein’s βγ moiety has provided a novel approach to understanding the feedback regulation of olfactory signal transduction pathways as well as the control of subcellular structures of OSNs. PMID:23637188

  5. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

    PubMed Central

    Beier, Anna; Krisp, Christoph; Wolters, Dirk A.

    2016-01-01

    ABSTRACT The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. PMID:27329756

  6. Arrangement of Kv1 alpha subunits dictates sensitivity to tetraethylammonium.

    PubMed

    Al-Sabi, Ahmed; Shamotienko, Oleg; Dhochartaigh, Sorcha Ni; Muniyappa, Nagesh; Le Berre, Marie; Shaban, Hamdy; Wang, Jiafu; Sack, Jon T; Dolly, J Oliver

    2010-09-01

    Shaker-related Kv1 channels contain four channel-forming alpha subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 alpha subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels' properties. Kv1.1 and 1.2 alpha genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K(+) currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2-1.1-1.2-1.1). Pore-blocking petidergic toxins, alpha dendrotoxin, agitoxin-1, tityustoxin-Kalpha, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of alpha subunits can be directed by this optimized concatenation, and that subunit

  7. Mechanism of β4 Subunit Modulation of BK Channels

    PubMed Central

    Wang, Bin; Rothberg, Brad S.; Brenner, Robert

    2006-01-01

    Large-conductance (BK-type) Ca2+-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca2+. BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (β1–β4). Biophysical characterization has shown that the β4 subunit confers properties of the so-called “type II” BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the β4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca2+ sensitivity. Specifically, channel activity at low Ca2+ is inhibited, while at high Ca2+, activity is enhanced. The goal of this study is to understand the mechanism underlying β4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that β4's most profound effect is a decrease in Po (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, β4 promotes channel opening by increasing voltage dependence of Po-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of β4 on BK channels. β4 reduces channel opening by decreasing the intrinsic gating equilibrium (L0), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, β4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vho) to more negative membrane potentials. The consequence is that β4 causes a net positive shift of the G-V relationship (relative to α subunit alone) at low calcium. At higher calcium, the contribution by Vho and an increase in allosteric coupling to Ca2+ binding (C

  8. Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

    PubMed Central

    Saha, Debarchana; Koli, Swanand; Patgaonkar, Mandar; Reddy, Kudumula Venkata Rami

    2017-01-01

    Hemoglobin (Hb) is a major protein involved in transport of oxygen (O2). It consists of Hb-α and Hb-β subunits, which are normally expressed by cells of erythroid lineage. However, till recently, it was not known whether non-erythroid cells like vaginal cells synthesize Hb and whether it has any functional significance. Therefore, we designed the following objectives: (1) to establish in-vitro culture system of human primary vaginal epithelial cells (hPVECs), (2) to determine whether Hb-α and Hb-β proteins are truly synthesized by hPVECs, (3) to evaluate the effect of LPS (lipopolysaccharide) on the expression of Hb-α and Hb-β proteins (4) to decipher the significance of the Hb-α and Hb-β expression in hPVECs and (5) to determine the molecular mechanism regulating the expression of Hb-α in hPVECs. To accomplish these studies, we applied a battery of assays such as RT-PCR, qRT-PCR, Flow cytometry, western blot, and immunofluorescence, Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The results revealed the expression of Hb-α and Hb-β at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10μg/ml for 6 hrs) and these results are comparable with the expression induced by LPS in human vaginal epithelial cell line (VK2/E6E7). These cells constitutively produced low levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines. Also, the response of phosphorylated (p65)-NF-κB to LPS was upregulated with increased expression of IL-6, Toll-like receptor-4 (TLR4) and human beta defensin-1 (hBD-1) in hPVECs and VK2/E6E7 cells. However, Bay 11–7082 treatment (5μM for 24 hrs) could neutralize the effect of LPS-induced p65-NF-κB activity and represses the production`of Hb-α and Hb-β. The results of EMSA revealed the presence of putative binding sites of NF-κB in the human Hb-α promoter region (nt-115 to -106). ChIP analysis confirmed the binding of NF

  9. Bedaquiline Targets the ε Subunit of Mycobacterial F-ATP Synthase.

    PubMed

    Kundu, Subhashri; Biukovic, Goran; Grüber, Gerhard; Dick, Thomas

    2016-11-01

    The tuberculosis drug bedaquiline inhibits mycobacterial F-ATP synthase by binding to its c subunit. Using the purified ε subunit of the synthase and spectroscopy, we previously demonstrated that the drug interacts with this protein near its unique tryptophan residue. Here, we show that replacement of ε's tryptophan with alanine resulted in bedaquiline hypersusceptibility of the bacteria. Overexpression of the wild-type ε subunit caused resistance. These results suggest that the drug also targets the ε subunit.

  10. Bedaquiline Targets the ε Subunit of Mycobacterial F-ATP Synthase

    PubMed Central

    Kundu, Subhashri; Biukovic, Goran; Grüber, Gerhard

    2016-01-01

    The tuberculosis drug bedaquiline inhibits mycobacterial F-ATP synthase by binding to its c subunit. Using the purified ε subunit of the synthase and spectroscopy, we previously demonstrated that the drug interacts with this protein near its unique tryptophan residue. Here, we show that replacement of ε's tryptophan with alanine resulted in bedaquiline hypersusceptibility of the bacteria. Overexpression of the wild-type ε subunit caused resistance. These results suggest that the drug also targets the ε subunit. PMID:27620476

  11. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function.

    PubMed

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V; Craig, Patricio O; Millar, Neil S; Bouzat, Cecilia; Elgoyhen, Ana Belén

    2017-03-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.

  12. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function

    PubMed Central

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K.; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V.; Craig, Patricio O.; Millar, Neil S.; Bouzat, Cecilia

    2017-01-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits. PMID:28069778

  13. Regulation of expression of a soybean storage protein subunit gene. Progress report

    SciTech Connect

    Thompson, J.F.; Madison, J.T.

    1984-04-23

    We have found that the methionine repression of the ..beta..-subunit gene expression is not due to degradation of the ..beta..-subunit but is due to an effect on synthesis of the ..beta..-subunit. The effect of methionine on the synthesis of the ..beta..-is due to an inhibition of ..beta..-subunit mRNA synthesis. 3 references, 1 figure.

  14. On the specificity of antibiotics targeting the large ribosomal subunit.

    PubMed

    Wilson, Daniel N

    2011-12-01

    The peptidyltransferase center of the large ribosomal subunit is responsible for catalyzing peptide bonds. This active site is the target of a variety of diverse antibiotics, many of which are used clinically. The past decade has seen a plethora of structures of antibiotics in complex with the large ribosomal subunit, providing unprecedented insight into the mechanism of action of these inhibitors. Ten distinct antibiotics (chloramphenicol, clindamycin, linezolid, tiamulin, sparsomycin, and five macrolides) have been crystallized in complex with four distinct ribosomal species, three bacterial, and one archaeal. This review aims to compare these structures in order to provide insight into the conserved and species-specific modes of interaction for particular members of each class of antibiotics. Coupled with the wealth of biochemical data, a picture is emerging defining the specific functional states of the ribosome that antibiotics preferentially target. Such mechanistic insight into antibiotic inhibition will be important for the development of the next generation of antimicrobial agents.

  15. AMPK beta subunits display isoform specific affinities for carbohydrates.

    PubMed

    Koay, Ann; Woodcroft, Ben; Petrie, Emma J; Yue, Helen; Emanuelle, Shane; Bieri, Michael; Bailey, Michael F; Hargreaves, Mark; Park, Jong-Tae; Park, Kwan-Hwa; Ralph, Stuart; Neumann, Dietbert; Stapleton, David; Gooley, Paul R

    2010-08-04

    AMP-activated protein kinase (AMPK) is a heterotrimer of catalytic (alpha) and regulatory (beta and gamma) subunits with at least two isoforms for each subunit. AMPK beta1 is widely expressed whilst AMPK beta2 is highly expressed in muscle and both beta isoforms contain a mid-molecule carbohydrate-binding module (beta-CBM). Here we show that beta2-CBM has evolved to contain a Thr insertion and increased affinity for glycogen mimetics with a preference for oligosaccharides containing a single alpha-1,6 branched residue. Deletion of Thr-101 reduces affinity for single alpha-1,6 branched oligosaccharides by 3-fold, while insertion of this residue into the equivalent position in the beta1-CBM sequence increases affinity by 3-fold, confirming the functional importance of this residue.

  16. The role of auxiliary subunits for the functional diversity of voltage-gated calcium channels.

    PubMed

    Campiglio, Marta; Flucher, Bernhard E

    2015-09-01

    Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α(1) subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein-protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein-protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity.

  17. Electrophysiology and beyond: multiple roles of Na+ channel β subunits in development and disease.

    PubMed

    Patino, Gustavo A; Isom, Lori L

    2010-12-10

    Voltage-gated Na+ channel (VGSC) β Subunits are not "auxiliary." These multi-functional molecules not only modulate Na+ current (I(Na)), but also function as cell adhesion molecules (CAMs)-playing roles in aggregation, migration, invasion, neurite outgrowth, and axonal fasciculation. β subunits are integral members of VGSC signaling complexes at nodes of Ranvier, axon initial segments, and cardiac intercalated disks, regulating action potential propagation through critical intermolecular and cell-cell communication events. At least in vitro, many β subunit cell adhesive functions occur both in the presence and absence of pore-forming VGSC α subunits, and in vivo β subunits are expressed in excitable as well as non-excitable cells, thus β subunits may play important functional roles on their own, in the absence of α subunits. VGSC β1 subunits are essential for life and appear to be especially important during brain development. Mutations in β subunit genes result in a variety of human neurological and cardiovascular diseases. Moreover, some cancer cells exhibit alterations in β subunit expression during metastasis. In short, these proteins, originally thought of as merely accessory to α subunits, are critical players in their own right in human health and disease. Here we discuss the role of VGSC β subunits in the nervous system.

  18. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Caers, A; Van de Peer, Y; De Wachter, R

    1998-01-01

    The rRNA WWW Server at URL http://rrna.uia.ac.be/ now provides a database of 496 large subunit ribosomal RNA sequences. All these sequences are aligned, incorporate secondary structure information, and can be obtained in a number of formats. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available and searchable. If necessary, the data on the server can also be obtained by anonymous ftp. PMID:9399830

  19. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1996-01-01

    Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can easily be obtained through the World Wide Web. The files in the database are also available via anonymous ftp. PMID:8594610

  20. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Caers, A; De Rijk, P; De Wachter, R

    1998-01-01

    About 8600 complete or nearly complete sequences are now available from the Antwerp database on small ribosomal subunit RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and detailed taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/ PMID:9399829

  1. Mcm subunits can assemble into two different active unwinding complexes.

    PubMed

    Kanter, Diane M; Bruck, Irina; Kaplan, Daniel L

    2008-11-07

    The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2-7, Cdc45, and GINS. The Mcm2-7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3' to 5' polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding complexes.

  2. Screening for AMPA receptor auxiliary subunit specific modulators

    PubMed Central

    Azumaya, Caleigh M.; Days, Emily L.; Vinson, Paige N.; Stauffer, Shaun; Sulikowski, Gary; Weaver, C. David; Nakagawa, Terunaga

    2017-01-01

    AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes. PMID:28358902

  3. Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

    PubMed Central

    Hutcheon, Marcus L.; Duncan, Thomas M.; Ngai, Helen; Cross, Richard L.

    2001-01-01

    Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a. PMID:11438702

  4. Na(+), K(+)-ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos.

    PubMed

    Hu, Yau-Chung; Chu, Keng-Fu; Yang, Wen-Kai; Lee, Tsung-Han

    2017-03-10

    The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na(+), K(+)-ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and β-subunits. Among the NKA genes, α1-β1 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α1-β1 isozyme. Based on the sequenced milkfish transcriptome, an NKA β1 subunit gene was identified that had the highest amino acid homology to β233, a NKA β1 subunit paralog originally identified in the eel. Despite this high level of homology to β233, phylogenetic analysis and the fact that only a single NKA β1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA β1 subunit gene. The results of accurate domain prediction of the β1 subunit, co-localization of α1 and β1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and β1 subunits, indicated the formation of a α1-β1 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and β1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA β1 subunit and provided the first evidence for an NKA α1-β1 association in gill ionocytes of euryhaline teleosts.

  5. Cornichon proteins determine the subunit composition of synaptic AMPA receptors.

    PubMed

    Herring, Bruce E; Shi, Yun; Suh, Young Ho; Zheng, Chan-Ying; Blankenship, Sabine M; Roche, Katherine W; Nicoll, Roger A

    2013-03-20

    Cornichon-2 and cornichon-3 (CNIH-2/-3) are AMPA receptor (AMPAR) binding proteins that promote receptor trafficking and markedly slow AMPAR deactivation in heterologous cells, but their role in neurons is unclear. Using CNIH-2 and CNIH-3 conditional knockout mice, we find a profound reduction of AMPAR synaptic transmission in the hippocampus. This deficit is due to the selective loss of surface GluA1-containing AMPARs (GluA1A2 heteromers), leaving a small residual pool of synaptic GluA2A3 heteromers. The kinetics of AMPARs in neurons lacking CNIH-2/-3 are faster than those in WT neurons due to the fast kinetics of GluA2A3 heteromers. The remarkably selective effect of CNIHs on the GluA1 subunit is probably mediated by TARP γ-8, which prevents a functional association of CNIHs with non-GluA1 subunits. These results point to a sophisticated interplay between CNIHs and γ-8 that dictates subunit-specific AMPAR trafficking and the strength and kinetics of synaptic AMPAR-mediated transmission.

  6. Rescue of lethal subunits into functional K+ channels.

    PubMed Central

    Taglialatela, M; Payne, J P; Drewe, J A; Brown, A M

    1994-01-01

    In a chimeric, voltage-dependent K+ channel (CHM), the valine at position 369 and the leucine at position 374 interact within the pore or P-region to regulate ion permeation and block. Here we show that the point mutation, CHM V369L, abolished channel function whereas previous experiments showed that CHM V369 and CHM V369I are functional. Coinjection of "lethal" CHM V369L cRNA with CHM L374V cRNA but not CHM cRNA generated functional heteromultimers. The whole-cell Rb+/K+ conductance ratio was 2.98 +/- 0.43 for CHM L374V and was reduced to 0.87 +/- 0.04 for the coexpressed CHM V369L and CHM L374V subunits. When single-channel currents were recorded, a single class of CHM V369L/CHM L374V heteromultimers was identified. This class was readily distinguishable from CHM L374V homomultimers by K+ conductance, gating, and blockade by internal tetraethylammonium. Coinjection experiments at various RNA ratios suggest that the CHM V369L/CHM L374V heteromultime, assuming it to be a tetramer, was composed of three CHM L374V subunits and one CHM V369L subunit. It appears that in the critical P-region of CHM position 369 may tolerate only one leucine. Images FIGURE 7 PMID:8130337

  7. Differential Localization of G Protein βγ Subunits

    PubMed Central

    2015-01-01

    G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system. PMID:24568373

  8. Molecular basis of AKAP specificity for PKA regulatory subunits.

    PubMed

    Gold, Matthew G; Lygren, Birgitte; Dokurno, Pawel; Hoshi, Naoto; McConnachie, George; Taskén, Kjetil; Carlson, Cathrine R; Scott, John D; Barford, David

    2006-11-03

    Localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) by A kinase-anchoring proteins (AKAPs) restricts the action of this broad specificity kinase. The high-resolution crystal structures of the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA both in the apo state and in complex with the high-affinity anchoring peptide AKAP-IS explain the molecular basis for AKAP-regulatory subunit recognition. AKAP-IS folds into an amphipathic alpha helix that engages an essentially preformed shallow groove on the surface of the RII dimer D/D domains. Conserved AKAP aliphatic residues dominate interactions to RII at the predominantly hydrophobic interface, whereas polar residues are important in conferring R subunit isoform specificity. Using a peptide screening approach, we have developed SuperAKAP-IS, a peptide that is 10,000-fold more selective for the RII isoform relative to RI and can be used to assess the impact of PKA isoform-selective anchoring on cAMP-responsive events inside cells.

  9. Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.

    PubMed Central

    García-Colunga, J; Miledi, R

    1996-01-01

    Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds. Images Fig. 3 PMID:8633003

  10. Molecular Modeling of the Misfolded Insulin Subunit and Amyloid Fibril

    PubMed Central

    Choi, Jay H.; May, Barnaby C.H.; Wille, Holger; Cohen, Fred E.

    2009-01-01

    Abstract Insulin, a small hormone protein comprising 51 residues in two disulfide-linked polypeptide chains, adopts a predominantly α-helical conformation in its native state. It readily undergoes protein misfolding and aggregates into amyloid fibrils under a variety of conditions. Insulin is a unique model system in which to study protein fibrillization, since its three disulfide bridges are retained in the fibrillar state and thus limit the conformational space available to the polypeptide chains during misfolding and fibrillization. Taking into account this unique conformational restriction, we modeled possible monomeric subunits of the insulin amyloid fibrils using β-solenoid folds, namely, the β-helix and β-roll. Both models agreed with currently available biophysical data. We performed molecular dynamics simulations, which allowed some limited insights into the relative structural stability, suggesting that the β-roll subunit model may be more stable than the β-helix subunit model. We also constructed β-solenoid-based insulin fibril models and conducted fiber diffraction simulation to identify plausible fibril architectures of insulin amyloid. A comparison of simulated fiber diffraction patterns of the fibril models to the experimental insulin x-ray fiber diffraction data suggests that the model fibers composed of six twisted β-roll protofilaments provide the most reasonable fit to available experimental diffraction patterns and previous biophysical studies. PMID:20006956

  11. Ribosomal small subunit domains radiate from a central core

    PubMed Central

    Gulen, Burak; Petrov, Anton S.; Okafor, C. Denise; Vander Wood, Drew; O’Neill, Eric B.; Hud, Nicholas V.; Williams, Loren Dean

    2016-01-01

    The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2′OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit. PMID:26876483

  12. Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase*

    PubMed Central

    Brandt, Karsten; Maiwald, Sarah; Herkenhoff-Hesselmann, Brigitte; Gnirß, Kerstin; Greie, Jörg-Christian; Dunn, Stanley D.; Deckers-Hebestreit, Gabriele

    2013-01-01

    FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. bI and bII). The results show the b dimer to be located at a non-catalytic α/β cleft, with bI close to subunit α, whereas bII is proximal to subunit β. Furthermore, bI can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-bI-α, bII-β, α-bI-bII-β, and a-bI-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that bI is functionally related to the single b subunit present in mitochondrial ATP synthase. PMID:23846684

  13. Two-subunit DNA escort mechanism and inactive subunit bypass in an ultra-fast ring ATPase

    DOE PAGES

    Liu, Ninning; Chistol, Gheorghe; Bustamante, Carlos

    2015-10-09

    SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in Bacillus subtilis . Here, we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base pair, we deduce that SpoIIIE’s step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism thatmore » is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such a flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell.« less

  14. Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus.

    PubMed

    Didelon, F; Mladinic', M; Cherubini, E; Bradbury, A

    2000-12-01

    The cDNA library screening strategy was used to identify the genes encoding for GABA(A) receptor subunits in the rat hippocampus during development. With this technique, genes encoding eleven GABA(A) receptor subunits were identified. The alpha5 subunit was by far the most highly expressed, followed by the gamma2, alpha2 and alpha4 subunits respectively. The expression of the beta2, alpha1, gamma1, beta1 and beta3 subunits was moderate, although that of the alpha3 and delta subunits was weak. In situ hybridization experiments, using digoxigenin-labeled cRNA probes, confirmed that the delta subunit was expressed in the neonatal as well as in the adult hippocampus, and is likely to form functional receptors in association with other subunits of the GABA(A) receptor. When the more sensitive RT-PCR approach was used, the gamma3 subunit was also detected, suggesting that this subunit is present in the hippocampus during development but at low levels of expression. The insertion of the delta subunit into functional GABA(A) receptors may enhance the efficacy of GABA in the immediate postnatal period when this amino acid is still exerting a depolarizing and excitatory action.

  15. Modulation of BK Channel Function by Auxiliary Beta and Gamma Subunits

    PubMed Central

    Li, Q.; Yan, J.

    2016-01-01

    The large-conductance, Ca2+- and voltage-activated K+ (BK) channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological characteristics. This diversity is in part conferred by channel modulation with different regulatory auxiliary subunits. To date, two distinct classes of BK channel auxiliary subunits have been identified: β subunits and γ subunits. Modulation of BK channels by the four auxiliary β (β1–β4) subunits has been well established and intensively investigated over the past two decades. The auxiliary γ subunits, however, were identified only very recently, which adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. This chapter will review the current understanding of BK channel modulation by auxiliary β and γ subunits, especially the latest findings. PMID:27238261

  16. Functional Diversification of Maize RNA Polymerase IV and V subtypes via Alternative Catalytic Subunits

    SciTech Connect

    Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; Sidorenko, Lyudmila; Nicora, Carrie D.; Norbeck, Angela D.; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; Mcginnis, Karen A.; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

    2014-10-01

    Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic ana- lyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two sub- types of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  17. Ribosome Subunit Stapling for Orthogonal Translation in E.  coli.

    PubMed

    Fried, Stephen D; Schmied, Wolfgang H; Uttamapinant, Chayasith; Chin, Jason W

    2015-10-19

    The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl-transferase center, for unnatural polymer synthesis in cells.

  18. Subunit regulation of the neuronal alpha 1A Ca2+ channel expressed in Xenopus oocytes.

    PubMed Central

    De Waard, M; Campbell, K P

    1995-01-01

    1. Voltage-dependent Ca2+ channels are multi-protein complexes composed of at least three subunits: alpha 1, alpha 2 delta and beta. Ba2+ currents were recorded in Xenopus oocytes expressing the neuronal alpha 1A Ca2+ channel, using the two-electrode voltage-clamp technique. Various subunit combinations were studied: alpha 1A, alpha 1A alpha 2 delta b, alpha 1A beta or alpha 1A alpha 2 delta b beta. 2. The alpha 1A subunit alone directs the expression of functional Ca2+ channels. It carries all the properties of the channel: gating, permeability, voltage dependence of activation and inactivation, and pharmacology. The alpha 1A channel is activated by low voltages when physiological concentrations of the permeant cation are used. Both ancillary subunits alpha 2 delta and beta induced considerable changes in the biophysical properties of the alpha 1A current. The subunit specificity of the changes in current properties was analysed for all four beta gene products by coexpressing beta 1b, beta 2a, beta 3 and beta 4. 3. All beta subunits induce a stimulation in the current amplitude, a change in inactivation kinetics, and two hyperpolarizing shifts--one in the voltage dependence of activation and a second in the voltage dependence of steady-state inactivation. The most significant difference in regulation among beta subunits is the induction of variable rate constants of current inactivation. Rates of inactivation were induced in the following order (fastest to slowest): beta 3 > beta 1b = beta 4 > beta 2a. 4. The alpha 2 delta b subunit does not modify the properties of alpha 1A Ca2+ channels in the absence of beta subunits. However, this subunit increases the beta-induced stimulation in current amplitude and also regulates the beta-induced change in inactivation kinetics. 5. Of all the subunit combinations tested, Ca2+ channels that included a beta subunit were the most prone to decrease in activity. It is concluded that beta subunits are the primary target for the

  19. Characterization of the interface between gamma and epsilon subunits of Escherichia coli F1-ATPase.

    PubMed

    Tang, C; Capaldi, R A

    1996-02-09

    The interaction faces of the gamma and epsilon subunits in the Escherichia coli F1-ATPase have been explored by a combination of cross-linking and chemical modification experiments using several mutant epsilon subunits as follows: epsilonS10C, epsilonH38C, epsilonT43C, epsilonS65C, epsilonS108C, and epsilonM138C, along with a mutant of the gamma subunit, gammaT106C. The replacement of Ser-10 by a Cys or Met-138 by a Cys reduced the inhibition of ECF1 by the epsilon subunit, while the mutation S65C increased this inhibitory effect. Modification of the Cys at position 10 with N-ethylmaleimide or fluoroscein maleimide further reduced the binding affinity of, and the maximal inhibition by, the epsilon subunit. Similar chemical modification of the Cys at position 43 of the epsilon subunit (in the mutant epsilonT43C) and a Cys at position 106 of the gamma subunit (gammaT106C) also affected the inhibition of ECF1 by the epsilon subunit. The various epsilon subunit mutants were reacted with TFPAM3, and the site(s) of cross-linking within the ECF1 complex was determined. Previous studies have shown cross-linking from the Cys at positions 10 and 38 with the gamma subunit and from a Cys at position 108 to an alpha subunit (Aggeler, R., Chicas-Cruz, K., Cai, S. X., Keana, J. F. W., and Capaldi, R. A. (1992) Biochemistry 31, 2956-2961; Aggeler, R., Weinreich, F., and Capaldi, R. A. (1995) Biochim. Biophys. Acta 1230, 62-68). Here, cross-linking was found from a Cys at position 43 to the gamma subunit and from the Cys at position 138 to a beta subunit. The site of cross-linking from Cys-10 of epsilon to the gamma subunit was localized by peptide mapping to a region of the gamma subunit between residues 222 and 242. Cross-linking from a Cys at position 38 and at position 43 was with the C-terminal part of the gamma subunit, between residues 202 and 286. ECF1 treated with trypsin at pH 7.0 still binds purified epsilon subunit, while enzyme treated with the protease at pH 8.0 does

  20. Properties and subunit structure of pig heart pyruvate dehydrogenase.

    PubMed

    Hamada, M; Hiraoka, T; Koike, K; Ogasahara, K; Kanzaki, T

    1976-06-01

    Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.

  1. Spinal NMDA NR1 Subunit Expression Following Transient TNBS Colitis

    PubMed Central

    Zhou, QiQi; Price, Donald D.; Caudle, Robert M.; Verne, G. Nicholas

    2009-01-01

    Background: N-methyl-D-aspartic acid (NMDA) receptors play an important role in the development of hypersensitivity to visceral and somatic stimuli following inflammation or tissue injury. Our objective was to investigate the role of NMDA NR1 receptors in the spinal cord (T10-L1; L4-S1) of a subset of rats that remain hypersensitive following histological resolution of TNBS-induced colitis compared to saline treated rats and rats that had recovered both behaviorally and histologically. We hypothesized that NMDA NR1 subunit expression mediates hypersensitivity following transient TNBS colitis. Methods: Male Sprague-Dawley rats (150g-250g) received 20mg/rat intracolonic trinitrobenzene sulfonic acid (TNBS) in 50% ethanol or saline. Animals underwent nociceptive visceral/somatic pain testing 16 weeks after resolution of TNBS colitis. Animals were sacrificed and their spinal cord (T10-L1; L4-S1) was retrieved and 2-dimensional polyacrylamide gel electrophoresis and immunohistocytochemistry techniques were used to investigate spinal-NMDA receptor expression. Results: NR1001 was the only NMDA NR1 receptor subunit that was expressed in recovered and control rats, whereas hypersensitive animals expressed NR1011 and NR1111 as well as NR1001 subunits. Immunohistochemistry analysis demonstrated increased expression of NMDA NR1-N1, C1, and C2-plus expression in lamina I & II of the spinal cord (T10-L1; L4-S1) in hypersensitive rats but not in recovered/control rats. Conclusions: Selective increases in the expression of the NMDA NR1 splice variants occur in hypersensitive rats following resolution of TNBS colitis. This suggests that the NMDA NR1 receptor play an important role in the development of neuronal plasticity and central sensitization. The recombination of NR1 splice variants may serve as a key functional protein that maintains hypersensitivity following resolution of TNBS colitis. PMID:19406112

  2. The β Subunit of Voltage-Gated Ca2+ Channels

    PubMed Central

    Buraei, Zafir; Yang, Jian

    2015-01-01

    Calcium regulates a wide spectrum of physiological processes such as heartbeat, muscle contraction, neuronal communication, hormone release, cell division, and gene transcription. Major entry-ways for Ca2+ in excitable cells are high-voltage activated (HVA) Ca2+channels. These are plasma membrane proteins composed of several subunits, including α1, α2δ, β, and γ. Although the principal α1 subunit (Cavα1) contains the channel pore, gating machinery and most drug binding sites, the cytosolic auxiliary β subunit (Cavβ) plays an essential role in regulating the surface expression and gating properties of HVA Ca2+ channels. Cavβ is also crucial for the modulation of HVA Ca2+ channels by G proteins, kinases, and the Ras-related RGK GTPases. New proteins have emerged in recent years that modulate HVA Ca2+ channels by binding to Cavβ. There are also indications that Cavβ may carry out Ca2+ channel-independent functions, including directly regulating gene transcription. All four subtypes of Cavβ, encoded by different genes, have a modular organization, consisting of three variable regions, a conserved guanylate kinase (GK) domain, and a conserved Src-homology 3 (SH3) domain, placing them into the membrane-associated guanylate kinase (MAGUK) protein family. Crystal structures of Cavβs reveal how they interact with Cavα1, open new research avenues, and prompt new inquiries. In this article, we review the structure and various biological functions of Cavβ, with both a historical perspective as well as an emphasis on recent advances. PMID:20959621

  3. Crystal Structure of the Cytoplasmic N-Terminal Domain of Subunit I, a Homolog of Subunit a, of V-ATPase

    SciTech Connect

    Srinivasan, Sankaranarayanan; Vyas, Nand K.; Baker, Matthew L.; Quiocho, Florante A.

    2012-02-27

    Subunit 'a' is associated with the membrane-bound (VO) complex of eukaryotic vacuolar H{sup +}-ATPase acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit 'I' homolog of subunit a. The structure is composed of a curved long central {alpha}-helix bundle capped on both ends by two lobes with similar {alpha}/{beta} architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstructions from electron microscopy of prokaryotic and eukaryotic vacuolar H{sup +}-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.

  4. Database on the structure of large ribosomal subunit RNA.

    PubMed Central

    De Rijk, P; Van de Peer, Y; De Wachter, R

    1997-01-01

    The latest release of the large ribosomal subunit RNA database contains 429 sequences. All these sequences are aligned, and incorporate secondary structure information. The rRNA WWW Server at URL http://rrna.uia.ac.be/ provides researchers with an easily accessible resource to obtain the data in this database in a number of computer-readable formats. A new query interface has been added to the server. If necessary, the data can also be obtained by anonymous ftp from the same site. PMID:9016517

  5. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Nicolaï, S; De Rijk, P; De Wachter, R

    1996-01-01

    The Antwerp database on small ribosomal subunit RNA offers over 4300 nucleotide sequences (August 1995). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. The complete database is made available to the scientific community through anonymous ftp and World Wide Web(WWW). PMID:8594609

  6. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Van den Broeck, I; De Rijk, P; De Wachter, R

    1994-01-01

    The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library. PMID:7524022

  7. Database on the structure of small ribosomal subunit RNA.

    PubMed Central

    Van de Peer, Y; Jansen, J; De Rijk, P; De Wachter, R

    1997-01-01

    The Antwerp database on small ribosomal subunit RNA now offers more than 6000 nucleotide sequences (August 1996). All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Besides the primary and secondary structure information, literature references, accession numbers and detailed taxonomic information are also compiled. For ease of use, the complete database is made available to the scientific community via World Wide Web at URL http://rrna.uia.ac.be/ssu/ . PMID:9016516

  8. Determination of kainate receptor subunit ratios in mouse brain using novel chimeric protein standards.

    PubMed

    Watanabe-Iida, Izumi; Konno, Kohtarou; Akashi, Kaori; Abe, Manabu; Natsume, Rie; Watanabe, Masahiko; Sakimura, Kenji

    2016-01-01

    Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KAR subunits are predominant in the brain, with distinct subunit combinations between the hippocampus and cerebellum. Kainate receptors, an unconventional member of the iGluR receptor family, have a tetrameric structure assembled from low-affinity (GluK1-3) and high-affinity (GluK4 and GluK5) subunits. We used a simple but novel procedure to measure the relative abundance of both low- and

  9. Crystal structure and molecular imaging of the Nav channel β3 subunit indicates a trimeric assembly.

    PubMed

    Namadurai, Sivakumar; Balasuriya, Dilshan; Rajappa, Rajit; Wiemhöfer, Martin; Stott, Katherine; Klingauf, Jurgen; Edwardson, J Michael; Chirgadze, Dimitri Y; Jackson, Antony P

    2014-04-11

    The vertebrate sodium (Nav) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Nav 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations.

  10. Ca(2+) channel inactivation heterogeneity reveals physiological unbinding of auxiliary beta subunits.

    PubMed Central

    Restituito, S; Cens, T; Rousset, M; Charnet, P

    2001-01-01

    Voltage gated Ca(2+) channel (VGCC) auxiliary beta subunits increase membrane expression of the main pore-forming alpha(1) subunits and finely tune channel activation and inactivation properties. In expression studies, co-expression of beta subunits also reduced neuronal Ca(2+) channel regulation by heterotrimeric G protein. Biochemical studies suggest that VGCC beta subunits and G protein betagamma can compete for overlapping interaction sites on VGCC alpha(1) subunits, suggesting a dynamic association of these subunits with alpha(1). In this work we have analyzed the stability of the alpha(1)/beta association under physiological conditions. Regulation of the alpha(1A) Ca(2+) channel inactivation properties by beta(1b) and beta(2a) subunits had two major effects: a shift in voltage-dependent inactivation (E(in)), and an increase of the non-inactivating current (R(in)). Unexpectedly, large variations in magnitude of the effects were recorded on E(in), when beta(1b) was expressed, and R(in), when beta(2a) was expressed. These variations were not proportional to the current amplitude, and occurred at similar levels of beta subunit expression. beta(2a)-induced variations of R(in) were, however, inversely proportional to the magnitude of G protein block. These data underline the two different mechanisms used by beta(1b) and beta(2a) to regulate channel inactivation, and suggest that the VGCC beta subunit can unbind the alpha1 subunit in physiological situations. PMID:11423397

  11. TWIST1 associates with NF-κB subunit RELA via carboxyl-terminal WR domain to promote cell autonomous invasion through IL8 production

    PubMed Central

    2012-01-01

    Background Metastasis is the primary cause of death for cancer patients. TWIST1, an evolutionarily conserved basic helix-loop-helix (bHLH) transcription factor, is a strong promoter of metastatic spread and its expression is elevated in many advanced human carcinomas. However, the molecular events triggered by TWIST1 to motivate dissemination of cancer cells are largely unknown. Results Here we show that TWIST1 induces the production of interleukin 8 (IL8), which activates matrix metalloproteinases and promotes invasion of breast epithelial and cancer cells. In this novel mechanism, TWIST1-mediated IL8 transcription is induced through the TWIST1 carboxy-terminal WR (Trp-Arg) domain instead of the classic DNA binding bHLH domain. Co-immunoprecipitation analyses revealed that the WR domain mediates the formation of a protein complex comprised of TWIST1 and the nuclear factor-kappaB (NF-κB) subunit RELA (p65/NF-κB3), which synergistically activates the transcriptional activity of NF-κB. This activation leads to increased DNA binding affinity of RELA to the IL8 promoter and thus induces the expression of the cytokine. Blockage of IL8 signaling by IL8 neutralizing antibodies or receptor inhibition reduced the invasiveness of both breast epithelial and cancer cells, indicating that TWIST1 induces autonomous cell invasion by establishing an IL8 antocrine loop. Conclusions Our data demonstrate that the TWIST1 WR domain plays a critical role in TWIST1-induced IL8 expression through interactions with and activation of NF-κB. The produced IL8 signals through an autocrine loop and promotes extracellular matrix degradation to enable cell invasion across the basement membrane. PMID:22891766

  12. Smallpox subunit vaccine produced in planta confers protection in mice

    PubMed Central

    Golovkin, Maxim; Spitsin, Sergei; Andrianov, Vyacheslav; Smirnov, Yuriy; Xiao, Yuhong; Pogrebnyak, Natalia; Markley, Karen; Brodzik, Robert; Gleba, Yuri; Isaacs, Stuart N.; Koprowski, Hilary

    2007-01-01

    We report here the in planta production of the recombinant vaccinia virus B5 antigenic domain (pB5), an attractive component of a subunit vaccine against smallpox. The antigenic domain was expressed by using efficient transient and constitutive plant expression systems and tested by various immunization routes in two animal models. Whereas oral administration in mice or the minipig with collard-derived insoluble pB5 did not generate an anti-B5 immune response, intranasal administration of soluble pB5 led to a rise of B5-specific immunoglobulins, and parenteral immunization led to a strong anti-B5 immune response in both mice and the minipig. Mice immunized i.m. with pB5 generated an antibody response that reduced virus spread in vitro and conferred protection from challenge with a lethal dose of vaccinia virus. These results indicate the feasibility of producing safe and inexpensive subunit vaccines by using plant production systems. PMID:17428917

  13. Binding of ATP by pertussis toxin and isolated toxin subunits

    SciTech Connect

    Hausman, S.Z.; Manclark, C.R.; Burns, D.L. )

    1990-07-03

    The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.

  14. Unraveling Subunit Cooperativity in Homotetrameric HCN2 Channels

    PubMed Central

    Benndorf, Klaus; Thon, Susanne; Schulz, Eckhard

    2012-01-01

    In a multimeric receptor protein, the binding of a ligand can modulate the binding of a succeeding ligand. This phenomenon, called cooperativity, is caused by the interaction of the receptor subunits. By using a complex Markovian model and a set of parameters determined previously, we analyzed how the successive binding of four ligands leads to a complex cooperative interaction of the subunits in homotetrameric HCN2 pacemaker channels. The individual steps in the model were characterized by Gibbs free energies for the equilibria and activation energies, specifying the affinity of the binding sites and the transition rates, respectively. Moreover, cooperative free energies were calculated for each binding step in both the closed and the open channel. We show that the cooperativity sequence positive-negative-positive determined for the binding affinity is generated by the combined effect of very different cooperativity sequences determined for the binding and unbinding rates, which are negative-negative-positive and no-negative-no, respectively. It is concluded that in the ligand-induced activation of HCN2 channels, the sequence of cooperativity based on the binding affinity is caused by two even qualitatively different sequences of cooperativity that are based on the rates of ligand binding and unbinding. PMID:23199914

  15. A small subunit processome protein promotes cancer by altering translation.

    PubMed

    Yang, H W; Kim, T-M; Song, S S; Menon, L; Jiang, X; Huang, W; Black, P M; Park, P J; Carroll, R S; Johnson, M D

    2015-08-20

    Dysregulation of ribosome biogenesis or translation can promote cancer, but the underlying mechanisms remain unclear. UTP18 is a component of the small subunit processome, a nucleolar multi-protein complex whose only known function is to cleave pre-ribosomal RNA to yield the 18S ribosomal RNA component of 40S ribosomal subunits. Here, we show that UTP18 also alters translation to promote stress resistance and growth, and that UTP18 is frequently gained and overexpressed in cancer. We observed that UTP18 localizes to the cytoplasm in a subset of cells, and that serum withdrawal increases cytoplasmic UTP18 localization. Cytoplasmic UTP18 associates with the translation complex and Hsp90 to upregulate the translation of IRES-containing transcripts such as HIF1a, Myc and VEGF, thereby inducing stress resistance. Hsp90 inhibition decreases cytoplasmic UTP18 and UTP18-induced increases in translation. Importantly, elevated UTP18 expression correlates with increased aggressiveness and decreased survival in numerous cancers. Enforced UTP18 overexpression promotes transformation and tumorigenesis, whereas UTP18 knockdown inhibits these processes. This stress adaptation mechanism is thus co-opted for growth by cancers, and its inhibition may represent a promising new therapeutic target.

  16. An assembly landscape for the 30S ribosomal subunit

    PubMed Central

    Talkington, Megan W. T.; Siuzdak, Gary

    2005-01-01

    Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, mRNA processing, translation, DNA replication, and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes. Our understanding of 30S assembly is incomplete, due to the challenges of monitoring the association of many components simultaneously. We have developed a new method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S rRNA during formation of the functional particle. These data represent the first detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we have found that local transformations throughout the assembling subunit have similar but distinct activation energies. This observation shows that the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to a view in which the assembly of the complex traverses a landscape dotted with a variety of local conformational transitions. PMID:16319883

  17. Thermostable cross-protective subunit vaccine against Brucella species.

    PubMed

    Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

    2014-12-01

    A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation.

  18. Palmitoylation of the β4-Subunit Regulates Surface Expression of Large Conductance Calcium-activated Potassium Channel Splice Variants*

    PubMed Central

    Chen, Lie; Bi, Danlei; Tian, Lijun; McClafferty, Heather; Steeb, Franziska; Ruth, Peter; Knaus, Hans Guenther; Shipston, Michael J.

    2013-01-01

    Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels. PMID:23504458

  19. [Chromatographic and spectroscopic characterization of phycocyanin and its subunits purified from Anabaena variabilis CCC421].

    PubMed

    Chakdar, N; Sakha, S; Pabbi, S

    2014-01-01

    Phycocyanin, a high value pigment was purified from diazotrophic cyanobacteria Anabaena variabilis CCC421 using a strategy involving ammonium sulfate precipitation, dialysis and anion exchange chromatography using DEAE-cellulose column. 36% phycocyanin with a purity of 2.75 was recovered finally after anion exchange chromatography. Purified phycocyanin was found to contain 2 subunits of 17 and 18 kDa which were identified as a-and (3 subunits by SDS-PAGE and MALDI-TOE HPLC method using a C5 column coupled with fluorescence or photodiode-based detection was also developed to separate and detect the A. variabilis CCC421 phycocyanin subunits. The fluorescence method was more sensitive than photodiode one. The purified phycocyanin from A. variabilis CCC421 as well as its subunits was characterized with respect to absorption and IR spectra. Spectral characterization of the subunits revealed that alpha and beta subunits contained one and two phycocyanobilin groups as chromophores, respectively.

  20. Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels.

    PubMed

    Zeng, Xu-Hui; Xia, Xiao-Ming; Lingle, Christopher J

    2003-06-01

    An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.

  1. The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

    PubMed

    Asher, O; Jensen, B S; Lupu-Meiri, M; Oron, Y; Fuchs, S

    1998-04-17

    The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

  2. Efficient Expression of Functional (α6β2)2β3 AChRs in Xenopus Oocytes from Free Subunits Using Slightly Modified α6 Subunits

    PubMed Central

    Ley, Carson Kai-Kwong; Kuryatov, Alexander; Wang, Jingyi; Lindstrom, Jon Martin

    2014-01-01

    Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)2β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)2β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells. PMID:25068303

  3. Efficient expression of functional (α6β2)2β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.

    PubMed

    Ley, Carson Kai-Kwong; Kuryatov, Alexander; Wang, Jingyi; Lindstrom, Jon Martin

    2014-01-01

    Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)2β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)2β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.

  4. Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

    PubMed

    Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

    2006-09-08

    The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

  5. Evidence for inhibition mediated by coassembly of GABAA and GABAC receptor subunits in native central neurons.

    PubMed

    Milligan, Carol J; Buckley, Noel J; Garret, Maurice; Deuchars, Jim; Deuchars, Susan A

    2004-08-18

    Fast inhibition in the nervous system is commonly mediated by GABA(A) receptors comprised of 2alpha/2beta/1gamma subunits. In contrast, GABA(C) receptors containing only rho subunits (rho1-rho3) have been predominantly detected in the retina. However, here using reverse transcription-PCR and in situ hybridization we show that mRNA encoding the rho1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the rho1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABA(C) receptor agonist cis-4-aminocrotonic acid (100-800 microM) requires the rho1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABA(A) (bicuculline, 10 microM) and GABA(C) [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA); 40-160 microM] receptors. Responses to GABA(C) agonists were also enhanced by the GABA(A) receptor modulator pentobarbitone (300 microM). Spontaneous and evoked IPSPs were reduced in amplitude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABA(A) and GABA(C) subunits formed a heteromeric receptor. Immunohistochemistry indicated that rho1 and alpha1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABA(C) receptor agonists were enhanced by the GABA(A) receptor modulator zolpidem (500 nm), which acts on the alpha1 subunit when the gamma2 subunit is also present. Finally, coimmunoprecipitation indicated that the rho1 subunit formed complexes that also containedalpha1 and gamma2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABA(A) and GABA(C) receptor subunits.

  6. Accessory subunits are integral for assembly and function of human mitochondrial complex I.

    PubMed

    Stroud, David A; Surgenor, Elliot E; Formosa, Luke E; Reljic, Boris; Frazier, Ann E; Dibley, Marris G; Osellame, Laura D; Stait, Tegan; Beilharz, Traude H; Thorburn, David R; Salim, Agus; Ryan, Michael T

    2016-10-06

    Complex I (NADH:ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chain and is composed of 45 subunits in humans, making it one of the largest known multi-subunit membrane protein complexes. Complex I exists in supercomplex forms with respiratory chain complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP. Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with mitochondrial disease, Parkinson's disease and ageing. Bacterial and human complex I share 14 core subunits that are essential for enzymatic function; however, the role and necessity of the remaining 31 human accessory subunits is unclear. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. Here we use gene editing to generate human knockout cell lines for each accessory subunit. We show that 25 subunits are strictly required for assembly of a functional complex and 1 subunit is essential for cell viability. Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes after the loss of specific modules revealed that ATP5SL and DMAC1 are required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enables the study of complex dysfunction at a cellular level.

  7. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

    SciTech Connect

    Watanabe, Kanako; Kanno, Takeshi; Oshima, Tadayuki; Miwa, Hiroto; Tashiro, Chikara; Nishizaki, Tomoyuki

    2008-03-07

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G{sub 1} phase of cell cycling and decreased the proportion in the S/G{sub 2} phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G{sub 1} phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.

  8. Interspecific luciferase beta subunit hybrids between Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi.

    PubMed

    Almashanu, S; Gendler, I; Hadar, R; Kuhn, J

    1996-09-01

    Bacterial luciferase (EC 1.14.14.3) is a heterodimer composed of alpha- and beta-chains encoded by luxA and luxB, respectively. Although some interspecific combinations of these subunits lead to active enzyme, others do not. The beta subunits of Vibrio fischeri and Photobacterium leiognathi form active enzyme with the alpha subunits of V.fischeri, P.leiognathi and Vibrio harveyi, while the beta subunit from V.harveyi only complements the alpha subunit of V.harveyi. Inactivity is caused by a lack of dimerization of the beta subunit of V.harveyi with the alpha subunits of V.fischeri and P.leiognathi. These observations served as the basis for a search to discover which segment of the beta polypeptide confers the ability to dimerize with the alpha subunits of V.fischeri and P.leiognathi. Intragenic beta subunit hybrids were made between V.harveyi, V.fischeri and P.leiognathi. Unique restriction sites were introduced into the respective luxB genes to divide them into four roughly equal segments. In all, 78 hybrids were constructed by in vitro techniques. The N-terminal segment of the peptide contains the signals that differentiate between the beta subunits of V.fischeri and P. leiognathi and the beta subunit of V. harveyi, and allow the former to dimerize with their alpha subunits. The second segment has no major effect on enzyme activity but does exhibit some context effects. Important interactions were found between the third and fourth segments of the polypeptide with respect to enzymatic activity.

  9. The Role of Auxiliary Subunits for the Functional Diversity of Voltage-Gated Calcium Channels

    PubMed Central

    Campiglio, Marta; Flucher, Bernhard E

    2015-01-01

    Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α1 subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2019–2031, 2015. © 2015 Wiley Periodicals, Inc. PMID:25820299

  10. The NH2-terminal php domain of the alpha subunit of the Escherichia coli replicase binds the epsilon proofreading subunit.

    PubMed

    Wieczorek, Anna; McHenry, Charles S

    2006-05-05

    The alpha subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746-3752). Deletion of 60 residues from the NH2 terminus of the alpha php domain destroys epsilon binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for epsilon binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds epsilon with the same affinity as the 1160-residue full-length alpha subunit. A subset of mutations of a conserved acidic residue (Asp43 in Escherichia coli alpha) present in the php domain of all bacterial replicases resulted in defects in epsilon binding. Using sequence alignments, we show that the prototypical gram+ Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an epsilon-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.

  11. Structure–Function Relationships in Fungal Large-Subunit Catalases

    SciTech Connect

    Diaz, A.; Valdez, V; Rudino-Pinera, E; Horjales, E; Hansberg, W

    2009-01-01

    Neurospora crassa has two large-subunit catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and accumulates particularly in asexual spores; CAT-3 is associated with growing cells and is induced under different stress conditions. It is our interest to elucidate the structure-function relationships in large-subunit catalases. Here we have determined the CAT-3 crystal structure and compared it with the previously determined CAT-1 structure. Similar to CAT-1, CAT-3 hydrogen peroxide (H{sub 2}O{sub 2}) saturation kinetics exhibited two components, consistent with the existence of two active sites: one saturated in the millimolar range and the other in the molar range. In the CAT-1 structure, we found three interesting features related to its unusual kinetics: (a) a constriction in the channel that conveys H{sub 2}O{sub 2} to the active site; (b) a covalent bond between the tyrosine, which forms the fifth coordination bound to the iron of the heme, and a vicinal cysteine; (c) oxidation of the pyrrole ring III to form a cis-hydroxyl group in C5 and a cis-{gamma}-spirolactone in C6. The site of heme oxidation marks the starts of the central channel that communicates to the central cavity and the shortest way products can exit the active site. CAT-3 has a similar constriction in its major channel, which could function as a gating system regulated by the H{sub 2}O{sub 2} concentration before the gate. CAT-3 functional tyrosine is not covalently bonded, but has instead the electron relay mechanism described for the human catalase to divert electrons from it. Pyrrole ring III in CAT-3 is not oxidized as it is in other large-subunit catalases whose structure has been determined. Different in CAT-3 from these enzymes is an occupied central cavity. Results presented here indicate that CAT-3 and CAT-1 enzymes represent a functional group of catalases with distinctive structural characteristics that determine similar kinetics.

  12. Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy.

    PubMed Central

    Klinger, C; Huet, J; Song, D; Petersen, G; Riva, M; Bautz, E K; Sentenac, A; Oudet, P; Schultz, P

    1996-01-01

    Immunoelectron microscopy was used to determine the spatial organization of the yeast RNA polymerase I core subunits on a three-dimensional model of the enzyme. Images of antibody-labeled enzymes were compared with the native enzyme to determine the localization of the antibody binding site on the surface of the model. Monoclonal antibodies were used as probes to identify the two largest subunits homologous to the bacterial beta and beta' subunits. The epitopes for the two monoclonal antibodies were mapped using subunit-specific phage display libraries, thus allowing a direct correlation of the structural data with functional information on conserved sequence elements. An epitope close to conserved region C of the beta-like subunit is located at the base of the finger-like domain, whereas a sequence between conserved regions C and D of the beta'-like subunit is located in the apical region of the enzyme. Polyclonal antibodies outlined the alpha-like subunit AC40 and subunit AC19 which were found co-localized also in the apical region of the enzyme. The spatial location of the subunits is correlated with their biological activity and the inhibitory effect of the antibodies. Images PMID:8887555

  13. Stage and Tissue Specific Expression of Four TCR Subunits in Olive Flounder (Paralichthys olivaceus).

    PubMed

    Lee, Young Mee; Lee, Jeong-Ho; Noh, Jae Koo; Kim, Hyun Chul; Park, Choul-Ji; Park, Jong-Won; Hwang, In Joon; Kim, Sung Yeon

    2013-12-01

    TCR subunits are members of membrane-bound receptors which allow the fast and efficient elimination of the specific fish pathogens have regulated function in adaptive immunity. Sequence structure of TCR subunits have been reported for various teleosts, but the information of each TCR subunit functional characterization through expression analysis in fish was unknown. In this study, we examined the gene expression of TCR subunits in the early developmental stages and observed transcript levels in various tissues from healthy adult olive flounder by RT-PCR. The mRNA expression of alpha subunit was already detected in the previous hatching step. But the transcripts of another TCR subunit were not observed during embryo development and increased after hatching and maintained until metamorphosis at the same level. It was found that all TCR subunits mRNAs are commonly expressed in the immune-related organ such as spleen, kidney and gill, also weak expressed in fin and eye. TCR alpha and beta subunit were expressed in brain, whereas gamma and delta were not expressed same tissue. The sequence alignment analysis shows that there are more than 80% sequence homology between TCR subunits. Because it has a high similarity of amino acid sequence to expect similar in function, but expression analysis show that will have may functional diversity due to different time and place of expression.

  14. Enrichment of GABAA Receptor α-Subunits on the Axonal Initial Segment Shows Regional Differences

    PubMed Central

    Gao, Yudong; Heldt, Scott A.

    2016-01-01

    Although it is generally recognized that certain α-subunits of γ-aminobutyric acid type A receptors (GABAARs) form enriched clusters on the axonal initial segment (AIS), the degree to which these clusters vary in different brain areas is not well known. In the current study, we quantified the density, size, and enrichment ratio of fluorescently labeled α1-, α2-, or α3-subunits aggregates co-localized with the AIS-marker ankyrin G and compared them to aggregates in non-AIS locations among different brain areas including hippocampal subfields, basal lateral amygdala (BLA), prefrontal cortex (PFC), and sensory cortex (CTX). We found regional differences in the enrichment of GABAAR α-subunits on the AIS. Significant enrichment was identified in the CA3 of hippocampus for α1-subunits, in the CA1, CA3, and BLA for α2-subunits, and in the BLA for α3-subunits. Using α-subunit knock-out (KO) mice, we found that BLA enrichment of α2- and α3-subunits were physiologically independent of each other, as the enrichment of one subunit was unaffected by the genomic deletion of the other. To further investigate the unique pattern of α-subunit enrichment in the BLA, we examined the association of α2- and α3-subunits with the presynaptic vesicular GABA transporter (vGAT) and the anchoring protein gephyrin (Geph). As expected, both α2- and α3-subunits on the AIS within the BLA received prominent GABAergic innervation from vGAT-positive terminals. Further, we found that the association of α2- and α3-subunits with Geph was weaker in AIS versus non-AIS locations, suggesting that Geph might be playing a lesser role in the enrichment of α2- and α3-subunits on the AIS. Overall, these observations suggest that GABAARs on the AIS differ in subunit composition across brain regions. As with somatodendritic GABAARs, the distinctive expression pattern of AIS-located GABAAR α-subunits in the BLA, and other brain areas, likely contribute to unique forms of GABAergic inhibitory

  15. The α2δ subunits of voltage-gated calcium channels.

    PubMed

    Dolphin, Annette C

    2013-07-01

    Voltage-gated calcium channels consist of the main pore-forming α1 subunit, together, except in the case of the T-type channels, with β and α2δ and sometimes γ subunits, which are collectively termed auxiliary or accessory subunits. This review will concentrate on the properties and role of the α2δ subunits of these channels. These proteins are largely extracellular, membrane-associated proteins which influence the trafficking, localization, and biophysical properties of the channels. This article is part of a Special Issue entitled: Calcium channels.

  16. Differential expression of the protein kinase A subunits in normal adrenal glands and adrenocortical adenomas.

    PubMed

    Weigand, Isabel; Ronchi, Cristina L; Rizk-Rabin, Marthe; Dalmazi, Guido Di; Wild, Vanessa; Bathon, Kerstin; Rubin, Beatrice; Calebiro, Davide; Beuschlein, Felix; Bertherat, Jérôme; Fassnacht, Martin; Sbiera, Silviu

    2017-12-01

    Somatic mutations in protein kinase A catalytic α subunit (PRKACA) were found to be causative for 30-40% of cortisol-producing adenomas (CPA) of the adrenal gland, rendering PKA signalling constitutively active. In its resting state, PKA is a stable and inactive heterotetramer, consisting of two catalytic and two regulatory subunits with the latter inhibiting PKA activity. The human genome encodes three different PKA catalytic subunits and four different regulatory subunits that are preferentially expressed in different organs. In normal adrenal glands all regulatory subunits are expressed, while CPA exhibit reduced protein levels of the regulatory subunit IIβ. In this study, we linked for the first time the loss of RIIβ protein levels to the PRKACA mutation status and found the down-regulation of RIIβ to arise post-transcriptionally. We further found the PKA subunit expression pattern of different tumours is also present in the zones of the normal adrenal cortex and demonstrate that the different PKA subunits have a differential expression pattern in each zone of the normal adrenal gland, indicating potential specific roles of these subunits in the regulation of different hormones secretion.

  17. beta-subunits of Snf1 kinase are required for kinase function and substrate definition.

    PubMed

    Schmidt, M C; McCartney, R R

    2000-09-15

    The Snf1 kinase and its mammalian homolog, the AMP-activated protein kinase, are heterotrimeric enzymes composed of a catalytic alpha-subunit, a regulatory gamma-subunit and a beta-subunit that mediates heterotrimer formation. Saccharomyces cerevisiae encodes three beta-subunit genes, SIP1, SIP2 and GAL83. Earlier studies suggested that these subunits may not be required for Snf1 kinase function. We show here that complete and precise deletion of all three beta-subunit genes inactivates the Snf1 kinase. The sip1Delta sip2Delta gal83Delta strain is unable to derepress invertase, grows poorly on alternative carbon sources and fails to direct the phosphorylation of the Mig1 and Sip4 proteins in vivo. The SIP1 sip2Delta gal83Delta strain manifests a subset of Snf phenotypes (Raf(+), Gly(-)) observed in the snf1Delta 10 strain (Raf(-), Gly(-)), suggesting that individual beta-subunits direct the Snf1 kinase to a subset of its targets in vivo. Indeed, deletion of individual beta-subunit genes causes distinct differences in the induction and phosphorylation of Sip4, strongly suggesting that the beta-subunits play an important role in substrate definition.

  18. Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase.

    PubMed

    Ishmukhametov, Robert R; DeLeon-Rangel, Jessica; Zhu, Shaotong; Vik, Steven B

    2017-04-01

    Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.

  19. Structure of the large ribosomal subunit from human mitochondria

    PubMed Central

    Bai, Xiao-chen; Sugimoto, Yoichiro; Edwards, Patricia C.; Murshudov, Garib; Scheres, Sjors H. W.; Ramakrishnan, V.

    2014-01-01

    Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases, and are often the unintended targets of various clinically useful antibiotics. Using single-particle electron cryo-microscopy we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance including recruitment of mitochondrial tRNAVal to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs. PMID:25278503

  20. Expression and secretion of cholera toxin B subunit in lactobacilli.

    PubMed

    Okuno, Takahiro; Kashige, Nobuhiro; Satho, Tomomitsu; Irie, Keiichi; Hiramatsu, Yukihiro; Sharmin, Tanjina; Fukumitsu, Yuki; Uyeda, Saori; Yamada, Seitaro; Harakuni, Tetsuya; Miyata, Takeshi; Arakawa, Takeshi; Imoto, Masumi; Toda, Akihisa; Nakashima, Yukihiko; Miake, Fumio

    2013-01-01

    Lactic acid bacteria (LAB) are used in various fields, including in food and medical supplies. There has been a great deal of research into vaccine development using LAB as carriers due to their "generally recognized as safe" status. Cholera is an infectious disease that causes diarrhea due to cholera toxin (CT) produced by Vibrio cholerae. The pentameric cholera toxin B (CTB) subunit has no toxicity, and is used as an antigen in cholera vaccines and as a delivery molecule in vaccines to various diseases. In this study, we generated recombinant LAB expressing and secreting CTB. Here, we first report that CTB expressed and secreted from LAB bound to GM1 ganglioside. The secreted CTB was purified, and its immunogenicity was determined by intranasal administration into mice. The results of the present study suggested that it may be useful as the basis of a new oral cholera vaccine combining LAB and CTB.

  1. Structure of the large ribosomal subunit from human mitochondria.

    PubMed

    Brown, Alan; Amunts, Alexey; Bai, Xiao-chen; Sugimoto, Yoichiro; Edwards, Patricia C; Murshudov, Garib; Scheres, Sjors H W; Ramakrishnan, V

    2014-11-07

    Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases and are often the unintended targets of various clinically useful antibiotics. Using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance, including recruitment of mitochondrial valine transfer RNA (tRNA(Val)) to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs.

  2. The subunit interfaces of weakly associated homodimeric proteins.

    PubMed

    Dey, Sucharita; Pal, Arumay; Chakrabarti, Pinak; Janin, Joël

    2010-04-23

    We analyzed subunit interfaces in 315 homodimers with an X-ray structure in the Protein Data Bank, validated by checking the literature for data that indicate that the proteins are dimeric in solution and that, in the case of the "weak" dimers, the homodimer is in equilibrium with the monomer. The interfaces of the 42 weak dimers, which are smaller by a factor of 2.4 on average than in the remainder of the set, are comparable in size with antibody-antigen or protease-inhibitor interfaces. Nevertheless, they are more hydrophobic than in the average transient protein-protein complex and similar in amino acid composition to the other homodimer interfaces. The mean numbers of interface hydrogen bonds and hydration water molecules per unit area are also similar in homodimers and transient complexes. Parameters related to the atomic packing suggest that many of the weak dimer interfaces are loosely packed, and we suggest that this contributes to their low stability. To evaluate the evolutionary selection pressure on interface residues, we calculated the Shannon entropy of homologous amino acid sequences at 60% sequence identity. In 93% of the homodimers, the interface residues are better conserved than the residues on the protein surface. The weak dimers display the same high degree of interface conservation as other homodimers, but their homologs may be heterodimers as well as homodimers. Their interfaces may be good models in terms of their size, composition, and evolutionary conservation for the labile subunit contacts that allow protein assemblies to share and exchange components, allosteric proteins to undergo quaternary structure transitions, and molecular machines to operate in the cell.

  3. Effective polymer adjuvants for sustained delivery of protein subunit vaccines.

    PubMed

    Adams, Justin R; Haughney, Shannon L; Mallapragada, Surya K

    2015-03-01

    We have synthesized thermogelling cationic amphiphilic pentablock copolymers that have the potential to act as injectable vaccine carriers and adjuvants that can simultaneously provide sustained delivery and enhance the immunogenicity of released antigen. While these pentablock copolymers have shown efficacy in DNA delivery in past studies, the ability to deliver both DNA and protein for subunit vaccines using the same polymeric carrier can provide greater flexibility and efficacy. We demonstrate the ability of these pentablock copolymers, and the parent triblock Pluronic copolymers to slowly release structurally intact and antigenically stable protein antigens in vitro, create an antigen depot through long-term injection-site persistence and enhance the in vivo immune response to these antigens. We show release of the model protein antigen ovalbumin in vitro from the thermogelling block copolymers with the primary, secondary and tertiary structures of the released protein unchanged compared to the native protein, and its antigenicity preserved upon release. The block copolymers form a gel at physiological temperatures that serves as an antigenic depot and persists in vivo at the site of injection for over 50days. The pentablock copolymers show a significant fivefold enhancement in the immune response compared to soluble protein alone, even 6weeks after the administration, based on measurement of antibody titers. These results demonstrate the potential of these block copolymers hydrogels to persist for several weeks and sustain the release of antigen with minimal effects on protein stability and antigenicity; and their ability to be used simultaneously as a sustained delivery device as well as a subunit vaccine adjuvant platform.

  4. Subunit interface mutants of rabbit muscle aldolase form active dimers.

    PubMed Central

    Beernink, P. T.; Tolan, D. R.

    1994-01-01

    We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability. PMID:7833800

  5. Molecular cloning and functional analysis of three subunits of yeast proteasome.

    PubMed Central

    Emori, Y; Tsukahara, T; Kawasaki, H; Ishiura, S; Sugita, H; Suzuki, K

    1991-01-01

    The genes encoding three subunits of Saccharomyces cerevisiae proteasome were cloned and sequenced. The deduced amino acid sequences were homologous not only to each other (30 to 40% identity) but also to those of rat and Drosophila proteasomes (25 to 65% identity). However, none of these sequences showed any similarity to any other known sequences, including various proteases, suggesting that these proteasome subunits may constitute a unique gene family. Gene disruption analyses revealed that two of the three subunits (subunits Y7 and Y8) are essential for growth, indicating that the proteasome and its individual subunits play an indispensable role in fundamental biological processes. On the other hand, subunit Y13 is not essential; haploid cells with a disrupted Y13 gene can proliferate, although the doubling time is longer than that of cells with nondisrupted genes. In addition, biochemical analysis revealed that proteasome prepared from the Y13 disrupted cells contains tryptic and chymotryptic activities equivalent to those of nondisrupted cells, indicating that the Y13 subunit is not essential for tryptic or chymotryptic activity. However, the chymotryptic activity of the Y13 disrupted cells is not dependent on sodium dodecyl sulfate (SDS), an activator of proteasome, since nearly full activity was observed in the absence of SDS. Thus, the activity in proteasome of the Y13 disrupted cells might result in unregulated intracellular proteolysis, thus leading to the prolonged cell cycle. These results indicate that cloned proteasome subunits having similar sequences to the yeast Y13 subunit are structural, but not catalytic, components of proteasome. It is also suggested that two subunits (Y7 and Y8) might occupy positions essential to proteasome structure or activity, whereas subunit Y13 is in a nonessential but important position. Images PMID:1898763

  6. Trends in Nasal Subunit Reconstruction by Facial Plastic and Reconstructive Surgeons.

    PubMed

    Larrabee, Yuna C; Phillips, David J; Sclafani, Anthony P

    2017-02-01

    To determine if facial plastic and reconstructive surgeons still adhere to the classic nasal subunit principle as described by Burget and Menick. Observational survey. A Weill Cornell Medicine institutional review board approved electronic survey that was sent via e-mail to active members of the American Academy of Facial Plastic and Reconstructive Surgery (AAFPRS). The survey consisted of 32 multiple-choice questions pertaining to the operative management of small (22-30%), medium (50-58%), and large (75-81%) defects of each subunit of the nose, as well as demographic, provider, and practice characteristics. There were 111 responses to the survey (10.1% response rate). Ninety-eight percent of respondents reported familiarity with the subunit principle, and 59.6% considered the subunit principle in greater than 90% of cases. Almost three-quarters (70.4%) of respondents felt the subunit principle should be applied but could be modified based on the particular nasal defect, whereas 28.7% felt it was only sometimes helpful and was not mandatory for successful nasal reconstruction. Large defects of the tip and ala are generally treated by excision of the remaining subunit (79.4 and 80.6%, respectively). Fewer surgeons would excise the remaining subunit for large defects of the dorsum (39.8%), sidewall (38.8%), and soft tissue facet (18.4%). Simple repair without additional excision was the treatment of choice for small defects of the tip (58.2%), ala (59.2%), sidewall (65%), dorsum (68%), and soft tissue facet (71.8%). However, in many small- (up to 32%) and medium- (up to 51%) sized defects of the tip, ala, sidewall, and dorsum, respondents reported partial subunit excision. The majority of AAFPRS members abide to the classical subunit principle by completely excising the remaining subunit for large defects of the tip and ala. Many surgeons modify the subunit principle in small and medium defects.

  7. Differential regulation of thyrotropin subunit apoprotein and carbohydrate biosynthesis by thyroid hormone

    SciTech Connect

    Taylor, T.; Weintraub, B.D.

    1985-04-01

    The regulation of TSH apoprotein and carbohydrate biosynthesis by thyroid hormone was studied by incubating pituitaries from normal and hypothyroid (3 weeks post-thyroidectomy) rats in medium containing (/sup 14/C)alanine and (/sup 3/H) glucosamine. After 6 h, samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to clear the sample of LH and free LH beta, then anti-LH alpha to precipitate free alpha-subunit. Total proteins were acid precipitated. All precipitates were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, which were then sliced and assayed by scintillation spectrometry. In hypothyroid pituitaries plus medium, (/sup 14/C)alanine incorporation in combined and free beta-subunits was 26 times normal and considerably greater than the 3.4-fold increase seen in total protein; combined and free alpha-subunits showed no specific increase in apoprotein synthesis. (/sup 3/H)Glucosamine incorporation in combined alpha- and beta-subunits in hypothyroid samples was 13 and 21 times normal, respectively, and was greater than the 1.9-fold increase in total protein; free alpha-subunit showed no specific increase in carbohydrate synthesis. The glucosamine to alanine ratio, reflecting relative glycosylation of newly synthesized molecules, was increased in hypothyroidism for combined alpha-subunits, but not for combined beta-subunits, free alpha-subunits, or total proteins. In summary, short term hypothyroidism selectively stimulated TSH beta apoprotein synthesis and carbohydrate synthesis of combined alpha- and beta-subunits. Hypothyroidism also increased the relative glycosylation of combined alpha-subunit. Thus, thyroid hormone deficiency appears to alter the rate-limiting step in TSH assembly (i.e. beta-subunit synthesis) as well as the carbohydrate structure of TSH, which may play important roles in its biological function.

  8. Reduction of tomato polygalacturonase beta subunit expression affects pectin solubilization and degradation during fruit ripening.

    PubMed Central

    Watson, C F; Zheng, L; DellaPenna, D

    1994-01-01

    The developmental changes that accompany tomato fruit ripening include increased solubilization and depolymerization of pectins due to the action of polygalacturonase (PG). Two PG isoenzymes can be extracted from ripe fruit: PG2, which is a single catalytic PG polypeptide, and PG1, which is composed of PG2 tightly associated with a second noncatalytic protein, the beta subunit. Previous studies have correlated ripening-associated increases in pectin solubilization and depolymerization with the presence of extractable PG1 activity, prior to the appearance of PG2, suggesting a functional role for the beta subunit and PG1 in pectin metabolism. To assess the function of the beta subunit, we produced and characterized transgenic tomatoes constitutively expressing a beta subunit antisense gene. Fruit from antisense lines had greatly reduced levels of beta subunit mRNA and protein and accumulated < 1% of their total extractable PG activity in ripe fruit as PG1, as compared with 25% for wild type. Inhibition of beta subunit expression resulted in significantly elevated levels of EDTA-soluble polyuronides at all stages of fruit ripening and a significantly higher degree of depolymerization at later ripening stages. Decreased beta subunit protein and extractable PG1 enzyme activity and increased pectin solubility and depolymerization all cosegregated with the beta subunit antisense transgene in T2 progeny. These results indicate (1) that PG2 is responsible for pectin solubilization and depolymerization in vivo and (2) that the beta subunit protein is not required for PG2 activity in vivo but (3) does play a significant role in regulating pectin metabolism in wild-type fruit by limiting the extent of pectin solubilization and depolymerization that can occur during ripening. Whether this occurs by direct interaction of the beta subunit with PG2 or indirectly by interaction of the beta subunit with the pectic substrate remains to be determined. PMID:7827495

  9. Cytochrome oxidase subunit V gene of Neurospora crassa: DNA sequences, chromosomal mapping, and evidence that the cya-4 locus specifies the structural gene for subunit V.

    PubMed Central

    Sachs, M S; Bertrand, H; Metzenberg, R L; RajBhandary, U L

    1989-01-01

    The sequences of cDNA and genomic DNA clones for Neurospora cytochrome oxidase subunit V show that the protein is synthesized as a 171-amino-acid precursor containing a 27-amino-acid N-terminal extension. The subunit V protein sequence is 34% identical to that of Saccharomyces cerevisiae subunit V; these proteins, as well as the corresponding bovine subunit, subunit IV, contain a single hydrophobic domain which most likely spans the inner mitochondrial membrane. The Neurospora crassa subunit V gene (cox5) contains two introns, 398 and 68 nucleotides long, which share the conserved intron boundaries 5'GTRNGT...CAG3' and the internal consensus sequence ACTRACA. Two short sequences, YGCCAG and YCCGTTY, are repeated four times each in the cox5 gene upstream of the mRNA 5' termini. The cox5 mRNA 5' ends are heterogeneous, with the major mRNA 5' end located 144 to 147 nucleotides upstream from the translational start site. The mRNA contains a 3'-untranslated region of 186 to 187 nucleotides. Using restriction-fragment-length polymorphism, we mapped the cox5 gene to linkage group IIR, close to the arg-5 locus. Since one of the mutations causing cytochrome oxidase deficiency in N. crassa, cya-4-23, also maps there, we transformed the cya-4-23 strain with the wild-type cox5 gene. In contrast to cya-4-23 cells, which grow slowly, cox5 transformants grew quickly, contained cytochrome oxidase, and had 8- to 11-fold-higher levels of subunit V in their mitochondria. These data suggest (i) that the cya-4 locus in N. crassa specifies structural information for cytochrome oxidase subunit V and (ii) that, in N. crassa, as in S. cerevisiae, deficiencies in the production of nuclearly encoded cytochrome oxidase subunits result in deficiency in cytochrome oxidase activity. Finally, we show that the lower levels of subunit V in cya-4-23 cells are most likely due to substantially reduced levels of translatable subunit V mRNA. Images PMID:2540423

  10. Species-specific Differences among KCNMB3 BK β3 Auxiliary Subunits: Some β3 N-terminal Variants May Be Primate-specific Subunits

    PubMed Central

    Zeng, Xuhui; Xia, Xiao-Ming; Lingle, Christopher J.

    2008-01-01

    The KCNMB3 gene encodes one of a family of four auxiliary β subunits found in the mammalian genome that associate with Slo1 α subunits and regulate BK channel function. In humans, the KCNMB3 gene contains four N-terminal alternative exons that produce four functionally distinct β3 subunits, β3a–d. Three variants, β3a–c, exhibit kinetically distinct inactivation behaviors. Since investigation of the physiological roles of BK auxiliary subunits will depend on studies in rodents, here we have determined the identity and functional properties of mouse β3 variants. Whereas β1, β2, and β4 subunits exhibit 83.2%, 95.3%, and 93.8% identity between mouse and human, the mouse β3 subunit, excluding N-terminal splice variants, shares only 62.8% amino acid identity with its human counterpart. Based on an examination of the mouse genome and screening of mouse cDNA libraries, here we have identified only two N-terminal candidates, β3a and β3b, of the four found in humans. Both human and mouse β3a subunits produce a characteristic use-dependent inactivation. Surprisingly, whereas the hβ3b exhibits rapid inactivation, the putative mβ3b does not inactivate. Furthermore, unlike hβ3, the mβ3 subunit, irrespective of the N terminus, mediates a shift in gating to more negative potentials at a given Ca2+ concentration. The shift in gating gradually is lost following patch excision, suggesting that the gating shift involves some regulatory process dependent on the cytosolic milieu. Examination of additional genomes to assess conservation among splice variants suggests that the putative mβ3b N terminus may not be a true orthologue of the hβ3b N terminus and that both β3c and β3d appear likely to be primate-specific N-terminal variants. These results have three key implications: first, functional properties of homologous β3 subunits may differ among mammalian species; second, the specific physiological roles of homologous β3 subunits may differ among mammalian

  11. Copolymer semiconductors comprising thiazolothiazole or benzobisthiazole, or benzobisoxazole electron acceptor subunits, and electron donor subunits, and their uses in transistors and solar cells

    DOEpatents

    Jenekhe, Samson A; Subramaniyan, Selvam; Ahmed, Eilaf; Xin, Hao; Kim, Felix Sunjoo

    2014-10-28

    The inventions disclosed, described, and/or claimed herein relate to copolymers comprising copolymers comprising electron accepting A subunits that comprise thiazolothiazole, benzobisthiazole, or benzobisoxazoles rings, and electron donating subunits that comprise certain heterocyclic groups. The copolymers are useful for manufacturing organic electronic devices, including transistors and solar cells. The invention also relates to certain synthetic precursors of the copolymers. Methods for making the copolymers and the derivative electronic devices are also described.

  12. Functional hybrid rubisco enzymes with plant small subunits and algal large subunits: engineered rbcS cDNA for expression in chlamydomonas.

    PubMed

    Genkov, Todor; Meyer, Moritz; Griffiths, Howard; Spreitzer, Robert J

    2010-06-25

    There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO(2) fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO(2)/O(2) specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO(2)/O(2) specificity but a lower carboxylation V(max) than Chlamydomonas Rubisco, the hybrid enzymes have 3-11% increases in CO(2)/O(2) specificity and retain near normal V(max) values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO(2) is concentrated for optimal photosynthesis.

  13. Rescue of gamma2 subunit-deficient mice by transgenic overexpression of the GABAA receptor gamma2S or gamma2L subunit isoforms.

    PubMed

    Baer, K; Essrich, C; Balsiger, S; Wick, M J; Harris, R A; Fritschy, J M; Lüscher, B

    2000-07-01

    The gamma2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high-affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the gamma2 subunit, gamma2 short (gamma2S) and gamma2 long (gamma2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the gamma2S or gamma2L subunit on a gamma2 subunit-deficient background are phenotypically indistinguishable from wild-type. They express nearly normal levels of gamma2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild-type in both juvenile and adult mice. Our results indicate that the two gamma2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform-specific roles in phosphorylation-dependent regulatory mechanisms.

  14. Interaction between G-protein beta and gamma subunit types is selective.

    PubMed Central

    Pronin, A N; Gautam, N

    1992-01-01

    Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, alpha, beta, and gamma. Each of these subunits comprises a family of proteins. The rules for association between members of one family with members of another to form a multimer are not known; it is not clear whether associations are specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified G proteins contain more than one subtype of beta or gamma subunits. The Gt alpha subunit is associated only with beta 1 and gamma 1. It is not known whether this specificity is due to the differential expression of these subunit types in a cell type or due to intrinsically different affinities between different beta and gamma subunit types. We have used a transfected cell assay system to examine the association of the beta 1, beta 2, and beta 3 proteins with the gamma 1 and gamma 2 proteins. Results show that gamma 1 does not associate with beta 2 and that beta 3 does not associate with gamma 1 or gamma 2. Differences in affinities between types of G protein subunits will impose restrictions on the formation of certain heterotrimers and determine which G protein will be active in a cell. A chimeric molecule of beta 1 and beta 2 was used to broadly map the regions on these subunits that determine specificity of association. Images PMID:1631113

  15. Isolation and characterization of the subunits of Phaseolus vulgaris alpha-amylase inhibitor.

    PubMed

    Yamaguchi, H

    1991-11-01

    An alpha-amylase inhibitor (PHA-I) of the white kidney bean (Phaseolus vulgaris) was found to be composed of two kinds of subunits and they were isolated on a size-exclusion column by HPLC under denaturing conditions. The alpha-subunit was free from tryptophan and cysteine and the beta-subunit contained no methionine or cysteine. There was no marked resemblance in tryptic peptide map between these subunit polypeptides. The alpha-subunit contained 28% by weight of carbohydrate, mainly made up of high mannose-type oligosacharides, whereas the sugar moiety of the beta-subunit amounted to 7% by weight and seemed to be predominantly composed of xylomannose-type oligosaccharides. By SDS-PAGE following deglycosylation, the molecular weights of the polypeptides of alpha- and beta-subunits were shown to be 7,800 and 14,000, respectively. These values were consistent with molecular sizes obtained for alpha- and beta-subunits by gel permeation HPLC in 6 M guanidine hydrochloride. The molecular weight of the native PHA-I, 28,800, obtained by gel permeation HPLC under non-denaturing conditions, suggested a heterodimeric structure for PHA-I.

  16. Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis.

    PubMed

    Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan; Urano, Daisuke; Trusov, Yuri; Sheahan, Michael B; McCurdy, David W; Assmann, Sarah M; Jones, Alan M; Botella, José R

    2015-02-01

    Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex comprises one Gα, one Gβ, and one Gγ subunit. However, in addition to the canonical Gγ subunits (class A), plants also possess two unusual, plant-specific classes of Gγ subunits (classes B and C) that have not yet been found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (class B), which is important for membrane anchoring of the protein; the presence of such subunits gives rise to a flexible sub-population of Gβ/γ heterodimers that are not necessarily restricted to the plasma membrane. Plants also contain class C Gγ subunits, which are twice the size of canonical Gγ subunits, with a predicted transmembrane domain and a large cysteine-rich extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology have been unequivocally demonstrated. Here, we provide compelling evidence that AGG3, a class C Gγ subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.

  17. Regulation of Voltage-Activated K(+) Channel Gating by Transmembrane β Subunits.

    PubMed

    Sun, Xiaohui; Zaydman, Mark A; Cui, Jianmin

    2012-01-01

    Voltage-activated K(+) (K(V)) channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. K(V) channels contain a central pore-gate domain (PGD) surrounded by four voltage-sensing domains (VSDs). The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many K(V) channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the K(V) β subunits that contain transmembrane (TM) segments including the KCNE family and the β subunits of large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels. These TM β subunits affect the voltage-dependent activation of K(V) α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening, and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into K(V) channel modulation by TM β subunits.

  18. Incorporation of high-molecular-weight glutenin subunits into doughs using 2 gram mixograph and extensigraphs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To study the contributions of high-molecular-weight glutenin subunits (HMW-GS) to the gluten macropolymer and dough properties, wheat HMW-GS (x- and y-types) are synthesized in a bacterial expression system. These subunits are then purified and used to supplement dough mixing and extensigraph exper...

  19. Conformational Transitions of Subunit ɛ in ATP Synthase from Thermophilic Bacillus PS3

    PubMed Central

    Feniouk, Boris A.; Kato-Yamada, Yasuyuki; Yoshida, Masasuke; Suzuki, Toshiharu

    2010-01-01

    Abstract Subunit ɛ of bacterial and chloroplast FOF1-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit ɛ can adopt two conformations. In the “extended”, inhibitory conformation, its two C-terminal α-helices are stretched along subunit γ. In the “contracted”, noninhibitory conformation, these helices form a hairpin. The transition of subunit ɛ from an extended to a contracted state was studied in ATP synthase incorporated in Bacillus PS3 membranes at 59°C. Fluorescence energy resonance transfer between fluorophores introduced in the C-terminus of subunit ɛ and in the N-terminus of subunit γ was used to follow the conformational transition in real time. It was found that ATP induced the conformational transition from the extended to the contracted state (half-maximum transition extent at 140 μM ATP). ADP could neither prevent nor reverse the ATP-induced conformational change, but it did slow it down. Acid residues in the DELSEED region of subunit β were found to stabilize the extended conformation of ɛ. Binding of ATP directly to ɛ was not essential for the ATP-induced conformational change. The ATP concentration necessary for the half-maximal transition (140 μM) suggests that subunit ɛ probably adopts the extended state and strongly inhibits ATP hydrolysis only when the intracellular ATP level drops significantly below the normal value. PMID:20141757

  20. All three subunits of soybean beta-conglycinin are potential food allergens.

    PubMed

    Krishnan, Hari B; Kim, Won-Seok; Jang, Sungchan; Kerley, Monty S

    2009-02-11

    Soybeans are recognized as one of the "big 8" food allergens. IgE antibodies from soybean-sensitive patients recognize more than 15 soybean proteins. Among these proteins only the alpha-subunit of beta-conglycinin, but not the highly homologous alpha'- and beta-subunits, has been shown to be a major allergenic protein. The objective of this study was to examine if the alpha'- and beta-subunits of beta-conglycinin can also serve as potential allergens. Immunoblot analysis using sera collected from soybean-allergic patients revealed the presence of IgE antibodies that recognized several soy proteins including 72, 70, 52, 34, and 21 kDa proteins. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analysis of trypsin-digested 72, 70, and 52 kDa proteins indicated that these proteins were the alpha'-, alpha-, and beta-subunits of beta-conglycinin, respectively. Additionally, purified alpha'-, alpha-, and beta-subunits of beta-conglycinin were recognized by IgE antibodies present in the soybean-allergic patients. The IgE reactivity to the beta-subunit of beta-conglycinin was not abolished when this glycoprotein was either deglycosylated using glycosidases or expressed as a recombinant protein in Escherichia coli . The results suggest that in addition to the previously recognized alpha-subunit of beta-conglycinin, the alpha'- and beta-subunits of beta-conglycinin also are potential food allergens.

  1. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells

    PubMed Central

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. DOI: http://dx.doi.org/10.7554/eLife.18426.001 PMID:27537197

  2. Skeletal muscle sodium channel is affected by an epileptogenic beta1 subunit mutation.

    PubMed

    Moran, O; Conti, F

    2001-03-23

    The syndrome of generalized epilepsy with febrile seizures plus type 1 (GEFS+) has been associated to the gene SCN1B coding for the sodium channel beta1 subunit (Wallace, R. H. et al. (1998) Nature Genetics 19, 366-370). In patients, a mutation of the cysteine 121 to trpyptophane (C121W) would cause a lack of modulatory activity of the beta1 subunit on sodium channels expressed in the brain, rendering neurons hyperexcitable. We have confirmed that the normal beta1-modulation of type-IIA adult brain alpha subunits (BIIA) expressed in frog oocytes is defective in C121W. We observed that the mixture of wild-type and mutant beta1 subunits is less effective than wild-type alone, suggesting that the mutant beta1 subunit does bind the alpha subunit. However, we also observed a similar lack of modulation by C121W of the in adult skeletal muscle alpha subunit (SkM1). This finding is in contrast with the simple idea that the mutational effect observed in the oocyte expression system is the principal physiopathological correlate of GEFS+, because no skeletal muscle symptoms have been reported in GEFS+ patients. We conclude that the manifestation of the pathological phenotype is conditioned by the presence of susceptibility genes and/or that the frog oocyte expression system is inadequate for the study of the mutant beta1 subunit physiopathology.

  3. Identification and isolation of three proteasome subunits and their encoding genes from Trypanosoma brucei.

    PubMed

    Huang, L; Shen, M; Chernushevich, I; Burlingame, A L; Wang, C C; Robertson, C D

    1999-08-20

    We have determined peptide sequences of three Trypanosoma brucei proteasome subunit proteins by mass spectrometry of tryptic digests of the proteins purified by two-dimensional (2-D) polyacrylamide gel electrophoresis. Three genes identified by the sequence of their cDNA encode the peptides identified in these three proteins. The three proteins predicted from the gene sequences have significant similarity to other known proteasome subunits and represent an alpha6 type subunit (TbPSA6), and two beta-type subunits belonging to the beta1-type (TbPSB1) and beta2 type (TbPSB2). The sequences of both beta-subunits predict formation of catalytically active subunits through proteolytic processing. The prediction is supported by the presence in each of the two beta-subunits of a tryptic peptide that has the correctly processed N-terminus that creates the threonine nucleophile of the mature protein. This peptide cannot be generated by trypsin because of the required cleavage of a glycine-threonine bond. It is thus likely that there are at least two catalytically active beta-subunits, TbPSB1 and TbPSB2, present in the mature 20S proteasome from T. brucei.

  4. Utility of Respiratory Vaccination With Recombinant Subunit Vaccines for Protection Against Pneumonic Plague

    DTIC Science & Technology

    2002-01-01

    Immunity at mucosal sites can prevent pathogen infection of the host. A) oral poliovirus vaccine B) inhaled influenza vaccine C) kennel cough & Newcastle...Utility of respiratory vaccination with recombinant subunit vaccines for protection against pneumonic plague. Douglas S. Reed & Jennifer Smoll...2. REPORT TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Utility of respiratory vaccination with recombinant subunit vaccines for

  5. Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

    NASA Astrophysics Data System (ADS)

    Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

    1995-02-01

    The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

  6. Identification of Four Distinct Subunit Types in the Unique 6×6 Hemocyanin of the Centipede Scutigera coleoptrata

    NASA Astrophysics Data System (ADS)

    Gebauer, W.; Markl, J.

    We isolated 6×6 hemocyanin, dissociated it into subunits, and examined it by electron microscopy. The subunits were separated by native polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate PAGE, and crossed immunoelectrophoresis. Single subunits were isolated by gel cutting from native PAGE and identified as hemocyanin by measuring their ultraviolet spectrum. A total of four distinct hemocyanin subunits were identified, and the subunit pattern of the three electrophoresis systems assigned to each other. The relative proportion of subunits a:b:c:d were 2 : 2 :>: 1 as determined by densitometry. Presumably, c and d act as linkers between hexamers.

  7. Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit

    SciTech Connect

    Ream, Thomas S.; Haag, Jeremy R.; Pontvianne, Frederic; Nicora, Carrie D.; Norbeck, Angela D.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2015-05-02

    Using affinity purification and mass spectrometry, we identified the subunits of Arabidopsis thaliana multisubunit RNA Polymerases I and III (abbreviated as Pol I and Pol III), providing the first description of their physical compositions in plants. AC40 and AC19 subunits are typically common to Pol I (a.k.a. Pol A) and Pol III (a.k.a. Pol C) and are encoded by single genes whose mutation, in humans, is a cause of the craniofacial disorder, Treacher-Collins Syndrome. Surprisingly, A. thaliana, and related species, express two distinct AC40 paralogs, one of which assembles into Pol I and the other of which assembles into Pol III. Changes at eight amino acid positions correlate with this functional divergence of Pol I and Pol III-specific AC40 paralogs. Two genes encode homologs of the yeast C53 subunit, and either variant can assemble into Pol III. By contrast, only one of two potential C17 variants, and one of two potential C31 variants were detected in Pol III. We introduce a new nomenclature system for plant Pol I and Pol III subunits in which the twelve subunits that are structurally and functionally homologous among Pols I through V are assigned equivalent numbers.

  8. Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit

    DOE PAGES

    Ream, Thomas S.; Haag, Jeremy R.; Pontvianne, Frederic; ...

    2015-05-02

    Using affinity purification and mass spectrometry, we identified the subunits of Arabidopsis thaliana multisubunit RNA Polymerases I and III (abbreviated as Pol I and Pol III), providing the first description of their physical compositions in plants. AC40 and AC19 subunits are typically common to Pol I (a.k.a. Pol A) and Pol III (a.k.a. Pol C) and are encoded by single genes whose mutation, in humans, is a cause of the craniofacial disorder, Treacher-Collins Syndrome. Surprisingly, A. thaliana, and related species, express two distinct AC40 paralogs, one of which assembles into Pol I and the other of which assembles into Polmore » III. Changes at eight amino acid positions correlate with this functional divergence of Pol I and Pol III-specific AC40 paralogs. Two genes encode homologs of the yeast C53 subunit, and either variant can assemble into Pol III. By contrast, only one of two potential C17 variants, and one of two potential C31 variants were detected in Pol III. We introduce a new nomenclature system for plant Pol I and Pol III subunits in which the twelve subunits that are structurally and functionally homologous among Pols I through V are assigned equivalent numbers.« less

  9. "Silent" Amino Acid Residues at Key Subunit Interfaces Regulate the Geometry of Protein Nanocages.

    PubMed

    Zhang, Shengli; Zang, Jiachen; Zhang, Xiaorong; Chen, Hai; Mikami, Bunzo; Zhao, Guanghua

    2016-11-22

    Rendering the geometry of protein-based assemblies controllable remains challenging. Protein shell-like nanocages represent particularly interesting targets for designed assembly. Here, we introduce an engineering strategy-key subunit interface redesign (KSIR)-that alters a natural subunit-subunit interface by selective deletion of a small number of "silent" amino acid residues (no participation in interfacial interactions) into one that triggers the generation of a non-native protein cage. We have applied KSIR to construct a non-native 48-mer nanocage from its native 24-mer recombinant human H-chain ferritin (rHuHF). This protein is a heteropolymer composed of equal numbers of two different subunits which are derived from one polypeptide. This strategy has allowed the study of conversion between protein nanocages with different geometries by re-engineering key subunit interfaces and the demonstration of the important role of the above-mentioned specific residues in providing geometric specificity for protein assembly.

  10. Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli.

    PubMed

    Wagenknecht, T; Grassucci, R; Frank, J

    1988-01-05

    Electron micrographs of frozen-hydrated, large ribosomal subunits from Escherichia coli have been analyzed by computer image processing. Images of subunits in the so-called "crown" orientation were analyzed by correlation alignment procedures developed for negatively stained specimens. Averages of the aligned images showed both similarities and differences to averages determined for negatively stained specimens. The L1 ridge is more dense and stalk-like in frozen-hydrated as compared with negatively stained subunits, possibly because it is associated with ribosomal RNA. The results show that it should be feasible to determine the three-dimensional structure of the large ribosomal subunit from micrographs of individual, frozen-hydrated subunits that have been tilted in the electron microscope.

  11. Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits.

    PubMed

    Piazza, Ilaria; Rutkowska, Anna; Ori, Alessandro; Walczak, Marta; Metz, Jutta; Pelechano, Vicent; Beck, Martin; Haering, Christian H

    2014-06-01

    Condensin complexes have central roles in the three-dimensional organization of chromosomes during cell divisions, but how they interact with chromatin to promote chromosome segregation is largely unknown. Previous work has suggested that condensin, in addition to encircling chromatin fibers topologically within the ring-shaped structure formed by its SMC and kleisin subunits, contacts DNA directly. Here we describe the discovery of a binding domain for double-stranded DNA formed by the two HEAT-repeat subunits of the Saccharomyces cerevisiae condensin complex. From detailed mapping data of the interfaces between the HEAT-repeat and kleisin subunits, we generated condensin complexes that lack one of the HEAT-repeat subunits and consequently fail to associate with chromosomes in yeast and human cells. The finding that DNA binding by condensin's HEAT-repeat subunits stimulates the SMC ATPase activity suggests a multistep mechanism for the loading of condensin onto chromosomes.

  12. Balancing acts of two HEAT subunits of condensin I support dynamic assembly of chromosome axes.

    PubMed

    Kinoshita, Kazuhisa; Kobayashi, Tetsuya J; Hirano, Tatsuya

    2015-04-06

    Condensin I is a five-subunit protein complex that plays a central role in mitotic chromosome assembly and segregation in eukaryotes. To dissect its mechanism of action, we reconstituted wild-type and mutant complexes from recombinant subunits and tested their abilities to assemble chromosomes in Xenopus egg cell-free extracts depleted of endogenous condensins. We find that ATP binding and hydrolysis by SMC subunits have distinct contributions to the action of condensin I and that continuous ATP hydrolysis is required for structural maintenance of chromosomes. Mutant complexes lacking either one of two HEAT subunits produce abnormal chromosomes with highly characteristic defects and have contrasting structural effects on chromosome axes preassembled with the wild-type complex. We propose that balancing acts of the two HEAT subunits support dynamic assembly of chromosome axes under the control of the SMC ATPase cycle, thereby governing construction of rod-shaped chromosomes in eukaryotic cells.

  13. Use of concatemers of ligand-gated ion channel subunits to study mechanisms of steroid potentiation.

    PubMed

    Steinbach, Joe Henry; Akk, Gustav

    2011-12-01

    Synaptic receptors of the nicotinic receptor gene family are pentamers of subunits. This modular structure creates problems in studies of drug actions, related to the number of copies of a subunit that are present and their position. A separate issue concerns the mechanism of action of many anesthetics, which involves potentiation of responses to neurotransmitters. Potentiation requires an interaction between a transmitter and a potentiator, mediated through the target receptor. We have studied the mechanism by which neurosteroids potentiate transmitter responses, using concatemers of covalently linked subunits to control the number and position of subunits in the assembled receptor and to selectively introduce mutations into positionally defined copies of a subunit. We found that the steroid needs to interact with only one site to produce potentiation, that the native sites for steroid interaction have indistinguishable properties, and that steroid potentiation appears to result from a global effect on receptor function.

  14. Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymatic activity.

    PubMed

    Müller, M; Schlapfer, B; Azzi, A

    1988-09-20

    Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.

  15. Lateral mobility and anchoring of recombinant GABAA receptors depend on subunit composition.

    PubMed

    Peran, M; Hicks, B W; Peterson, N L; Hooper, H; Salas, R

    2001-10-01

    The clustering of type A gamma-aminobutyric acid receptors (GABA(A)R) at discrete and functionally significant domains on the nerve cell surface is an important determinant in the integration of synaptic inputs. To discern the role that the subunits of the GABA(A)R play in determining the receptor's cell surface topography and mobility, the alpha1, beta1, beta3, and gamma2s subunits were transfected into COS7, HEK293, and PC12 cells and the distribution and cell surface mobility of these recombinant receptors were examined. Our results show that alpha1 subunits are retained in the endoplasmic reticulum while beta1 and beta3 subunits are sorted to the plasma membrane where they form clusters. Co-expression and co-assembly of alpha1 and beta3 subunits result in the rescue of intracellular alpha1 subunits, which are transported as alphabeta subunit complexes to the cell surface where they formed clusters. Fluorescence photobleach recovery and single particle tracking of recombinant receptors show that, despite clustering, beta3 subunit homooligomers are mobile within a cell surface domain. Inclusion of alpha1 in beta3 or beta3gamma2s complexes, however, dramatically reduces the receptor's lateral mobility in COS 7 and PC12 cells and anchors GABA(A)Rs on the cell surface, suggesting the formation of a direct link to a component of the cytoskeleton. The mobility of recombinant receptors that include the alpha1 subunit mirrors the mobility of GABA(A)Rs on cell bodies and dendrites of cortical and spinal cord neurons. The results suggest that incorporation of alpha1 subunits give rise to a population of GABA(A)Rs that are immobilized on the cell surface.

  16. ASSESSMENT OF SUBUNIT-DEPENDENT DIRECT GATING AND ALLOSTERIC MODULATORY EFFECTS OF CARISOPRODOL AT GABAA RECEPTORS

    PubMed Central

    Kumar, Manoj; González, Lorie A.; Dillon, Glenn H.

    2016-01-01

    Carisoprodol is a widely prescribed muscle relaxant, abuse of which has grown considerably in recent years. It directly activates and allosterically modulates α1β2γ2 GABAARs, although the site(s) of action are unknown. To gain insight into the actions of carisoprodol, subunit-dependent effects of this drug were assessed. Whole-cell patch clamp recordings were obtained from HEK293 cells expressing α1β2, α1β3 or αxβzγ2 (where x = 1–6 and z = 1–3) GABAARs, and in receptors incorporating the δ subunit (modeling extrasynaptic receptors). The ability to directly gate and allosterically potentiate GABA-gated currents was observed for all configurations. Presence or absence of the γ2 subunit did not affect the ability of carisoprodol to directly gate or allosterically modulate the receptor. Presence of the β1 subunit conferred highest efficacy for direct activation relative to maximum GABA currents, while presence of the β2 subunit conferred highest efficacy for allosteric modulation of the GABA response. With regard to α subunits, carisoprodol was most efficacious at enhancing the actions of GABA in receptors incorporating the α1 subunit. The ability to directly gate the receptor was generally comparable regardless of the α subunit isoform, although receptors incorporating the α3 subunit showed significantly reduced direct gating efficacy and affinity. In extrasynaptic (α1β3δ and α4β3δ) receptors, carisoprodol had greater efficacy than GABA as a direct gating agonist. In addition, carisoprodol allosterically potentiated both EC20 and saturating GABA concentrations in these receptors. In assessing voltage-dependence, we found direct gating and inhibitory effects were insensitive to membrane voltage, whereas allosteric modulatory effects were affected by membrane voltage. Our findings demonstrate direct and allosteric effects of carisoprodol at synaptic and extrasynpatic GABAARs and that subunit isoform influences these effects. PMID:25896767

  17. Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

    PubMed Central

    He, Jiuya; Ford, Holly C.; Carroll, Joe; Ding, Shujing; Fearnley, Ian M.

    2017-01-01

    The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme’s rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP. PMID:28289229

  18. Translation initiation factor 3 regulates switching between different modes of ribosomal subunit joining.

    PubMed

    MacDougall, Daniel D; Gonzalez, Ruben L

    2015-05-08

    Ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (IFs) regulate association of the 30S initiation complex (IC) with the 50S subunit to control formation of a 70S IC that can enter into the elongation stage of protein synthesis. The GTP-bound form of IF2 accelerates subunit joining, whereas IF3 antagonizes subunit joining and plays a prominent role in maintaining translation initiation fidelity. The molecular mechanisms through which IF2 and IF3 collaborate to regulate the efficiency of 70S IC formation, including how they affect the dynamics of subunit joining, remain poorly defined. Here, we use single-molecule fluorescence resonance energy transfer to monitor the interactions between IF2 and the GTPase-associated center (GAC) of the 50S subunit during real-time subunit joining reactions in the absence and presence of IF3. In the presence of IF3, IF2-mediated subunit joining becomes reversible, and subunit joining events cluster into two distinct classes corresponding to formation of shorter- and longer-lifetime 70S ICs. Inclusion of IF3 within the 30S IC was also found to alter the conformation of IF2 relative to the GAC, suggesting that IF3's regulatory effects may stem in part from allosteric modulation of IF2-GAC interactions. The results are consistent with a model in which IF3 can exert control over the efficiency of subunit joining by modulating the conformation of the 30S IC, which in turn influences the formation of stabilizing intersubunit contacts and thus the reaction's degree of reversibility.

  19. Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

    PubMed Central

    John, D C; Grant, M E; Bulleid, N J

    1993-01-01

    Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI. Images PMID:8385607

  20. Mechanism of Auxiliary Subunit Modulation of Neuronal α1E Calcium Channels

    PubMed Central

    Jones, Lisa P.; Wei, Shao-kui; Yue, David T.

    1998-01-01

    Voltage-gated calcium channels are composed of a main pore-forming α1 moiety, and one or more auxiliary subunits (β, α2δ) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of α1E calcium channels in combination with a β (β1–β4) and/or the α2δ subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting α2δ with β subunits, of transfecting two different β subunits simultaneously, and of COOH-terminal truncation of α1E to remove a second β binding site. The main results are as follows. (a) The α2δ and β subunits modulate α1E in fundamentally different ways. The sole effect of α2δ is to increase current density by elevating channel density. By contrast, though β subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different β isoforms produce nearly indistinguishable effects on activation. However, β subunits produced clear, isoform-specific effects on inactivation properties. (b) All the β subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different β subunits. (d) The modulatory features found here for α1E do not generalize uniformly to other α1 channel types, as α1C activation gating shows marked β isoform dependence that is absent for α1E. Together, these results help to establish a more

  1. Design of a hyperstable 60-subunit protein icosahedron

    NASA Astrophysics Data System (ADS)

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David

    2016-07-01

    The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

  2. Calmodulin is a subunit of nitric oxide synthase from macrophages

    PubMed Central

    1992-01-01

    A central issue in nitric oxide (NO) research is to understand how NO can act in some settings as a servoregulator and in others as a cytotoxin. To answer this, we have sought a molecular basis for the differential regulation of the two known types of NO synthase (NOS). Constitutive NOS's in endothelium and neurons are activated by agonist- induced elevation of Ca2+ and resultant binding of calmodulin (CaM). In contrast, NOS in macrophages does not require added Ca2+ or CaM, but is regulated instead by transcription. We show here that macrophage NOS contains, as a tightly bound subunit, a molecule with the immunologic reactivity, high performance liquid chromatography retention time, tryptic map, partial amino acid sequence, and exact molecular mass of CaM. In contrast to most CaM-dependent enzymes, macrophage NOS binds CaM tightly without a requirement for elevated Ca2+. This may explain why NOS that is independent of Ca2+ and elevated CaM appears to be activated simply by being synthesized. PMID:1380065

  3. Design of a hyperstable 60-subunit protein icosahedron

    PubMed Central

    Hsia, Yang; Bale, Jacob B.; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K.; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N.; Gonen, Tamir; King, Neil P.; Baker, David

    2016-01-01

    The icosahedron and the dodecahedron are the largest of the Platonic solids, and icosahedral protein structures are widely utilized in biological systems for packaging and transport1,2. There has been considerable interest in repurposing such structures3–5, for example, virus-like particles for the targeted delivery and vaccine design. The ability to design proteins that self assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein 'containers' that could exhibit properties custom-made for various applications. In this manuscript, we describe the computational design of an icosahedral nano-cage that self-assembles from trimeric building blocks. Electron microscopy images of the designed protein expressed in E. coli reveals a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 M guanidine hydrochloride at up to 80 °C, and undergo extremely abrupt, but reversible, disassembly between 2 M and 2.25 M guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of superfolder GFP can be fused to each of the 60 subunits to create highly fluorescent standard candles for light microscopy, and a designed protein pentamer can be placed in the center of each of the twenty pentameric faces to potentially gate macromolecule access to the nanocage interior. Such robust designed nanocages should have considerable utility for targeted drug delivery6, vaccine design7, and synthetic biology8. PMID:27309817

  4. Design of a hyperstable 60-subunit protein dodecahedron. [corrected].

    PubMed

    Hsia, Yang; Bale, Jacob B; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K; Nattermann, Una; Xu, Chunfu; Huang, Po-Ssu; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N; Gonen, Tamir; King, Neil P; Baker, David

    2016-07-07

    The dodecahedron [corrected] is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The dodecahedron [corrected] is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

  5. Immunoproteasome subunit LMP7 Deficiency Improves Obesity and Metabolic Disorders

    PubMed Central

    Kimura, Hiroaki; Usui, Fumitake; Karasawa, Tadayoshi; Kawashima, Akira; Shirasuna, Koumei; Inoue, Yoshiyuki; Komada, Takanori; Kobayashi, Motoi; Mizushina, Yoshiko; Kasahara, Tadashi; Suzuki, Koichi; Iwasaki, Yusaku; Yada, Toshihiko; Caturegli, Patrizio; Takahashi, Masafumi

    2015-01-01

    Inflammation plays an important role in the development of obesity and metabolic disorders; however, it has not been fully understood how inflammation occurs and is regulated in their pathogenesis. Low-molecular mass protein-7 (LMP7) is a proteolytic subunit of the immunoproteasome that shapes the repertoire of antigenic peptides on major histocompatibility complex class I molecule. In this study, we investigated the role of LMP7 in the development of obesity and metabolic disorders using LMP7-deficient mice. LMP7 deficiency conveyed resistant to obesity, and improved glucose intolerance and insulin sensitivity in mice fed with high-fat diet (HFD). LMP7 deficiency decreased pancreatic lipase expression, increased fecal lipid contents, and inhibited the increase of plasma triglyceride levels upon oral oil administration or HFD feeding. Using bone marrow-transferred chimeric mice, we found that LMP7 in both bone marrow- and non-bone marrow-derived cells contributes to the development of HFD-induced obesity. LMP7 deficiency decreased inflammatory responses such as macrophage infiltration and chemokine expression while it increased serum adiponection levels. These findings demonstrate a novel role for LMP7 and provide new insights into the mechanisms underlying inflammation in the pathophysiology of obesity and metabolic disorders. PMID:26515636

  6. The Regulation of NF-κB Subunits by Phosphorylation

    PubMed Central

    Christian, Frank; Smith, Emma L.; Carmody, Ruaidhrí J.

    2016-01-01

    The NF-κB transcription factor is the master regulator of the inflammatory response and is essential for the homeostasis of the immune system. NF-κB regulates the transcription of genes that control inflammation, immune cell development, cell cycle, proliferation, and cell death. The fundamental role that NF-κB plays in key physiological processes makes it an important factor in determining health and disease. The importance of NF-κB in tissue homeostasis and immunity has frustrated therapeutic approaches aimed at inhibiting NF-κB activation. However, significant research efforts have revealed the crucial contribution of NF-κB phosphorylation to controlling NF-κB directed transactivation. Importantly, NF-κB phosphorylation controls transcription in a gene-specific manner, offering new opportunities to selectively target NF-κB for therapeutic benefit. This review will focus on the phosphorylation of the NF-κB subunits and the impact on NF-κB function. PMID:26999213

  7. On the evolution of the single-subunit RNA polymerases.

    PubMed

    Cermakian, N; Ikeda, T M; Miramontes, P; Lang, B F; Gray, M W; Cedergren, R

    1997-12-01

    Many eukaryotic nuclear genomes as well as mitochondrial plasmids contain genes displaying evident sequence similarity to those encoding the single-subunit RNA polymerase (ssRNAP) of bacteriophage T7 and its relatives. We have collected and aligned these ssRNAP sequences and have constructed unrooted phylogenetic trees that demonstrate the separation of ssRNAPs into three well-defined and nonoverlapping clusters (phage-encoded, nucleus-encoded, and plasmid-encoded). Our analyses indicate that these three subfamiles of T7-like RNAPs shared a common ancestor; however, the order in which the groups diverged cannot be inferred from available data. On the basis of structural similarities and mutational data, we suggest that the ancestral ssRNAP gene may have arisen via duplication and divergence of a DNA polymerase or reverse transcriptase gene. Considering the current phylogenetic distribution of ssRNAP sequences, we further suggest that the origin of the ancestral ssRNAP gene closely paralleled in time the introduction of mitochondria into eukaryotic cells through a eubacterial endosymbiosis.

  8. Expansion of transducin subunit gene families in early vertebrate tetraploidizations.

    PubMed

    Lagman, David; Sundström, Görel; Ocampo Daza, Daniel; Abalo, Xesús M; Larhammar, Dan

    2012-10-01

    Hundreds of gene families expanded in the early vertebrate tetraploidizations including many gene families in the phototransduction cascade. We have investigated the evolution of the heterotrimeric G-proteins of photoreceptors, the transducins, in relation to these events using both phylogenetic analyses and synteny comparisons. Three alpha subunit genes were identified in amniotes and the coelacanth, GNAT1-3; two of these were identified in amphibians and teleost fish, GNAT1 and GNAT2. Most tetrapods have four beta genes, GNB1-4, and teleosts have additional duplicates. Finally, three gamma genes were identified in mammals, GNGT1, GNG11 and GNGT2. Of these, GNGT1 and GNGT2 were found in the other vertebrates. In frog and zebrafish additional duplicates of GNGT2 were identified. Our analyses show all three transducin families expanded during the early vertebrate tetraploidizations and the beta and gamma families gained additional copies in the teleost-specific genome duplication. This suggests that the tetraploidizations contributed to visual specialisations.

  9. Extensions to the D-Cam sub-unit architecture

    NASA Astrophysics Data System (ADS)

    Ryan, Padraig; Connell, Joseph

    2005-06-01

    Multispectral imaging produces large amounts of data which extend processing, transmission and storage systems to their upper limits. Although there are several interface standards specific to image data acquisition, such as CameraLink, it is Firewire which provides a high-speed data bus, integrated control capability, without loss of flexibility, and which is commonly available as a low cost solution. The class of multispectral imaging requires a different treatment of the processing principals than standard imaging. The same spatial region is captured multiple times using different optical wavelengths. This technique finds application in such diverse areas as coastal monitoring, fruit sorting and automated agriculture. Modifications and additional features to the camera operating and configuration parameters are therefore required which are not generally present with conventional imaging sensors. This paper describes extensions to the IIDC Digital Camera (D-Cam) specification in the development of a Firewire technology platform for transmitting the data structures described and for providing real-time, online control of spectral information acquisition. Additionally, it describes how a set of registers in the sub-unit architecture of the Firewire protocol is augmented to accommodate the demands of a multispectral system. The extensions are specification conformant and do not alter underlining compliance with the base standard. The paper also describes the implementation of the extended D-Cam in the Firewire subsystem of a smart multispectral camera used in commercial applications.

  10. Phylogenetic Analyses of Meloidogyne Small Subunit rDNA

    PubMed Central

    De Ley, Irma Tandingan; De Ley, Paul; Vierstraete, Andy; Karssen, Gerrit; Moens, Maurice; Vanfleteren, Jacques

    2002-01-01

    Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species. PMID:19265950

  11. Characterization and mutagenesis of the gene encoding the A49 subunit of RNA polymerase A in Saccharomyces cerevisiae.

    PubMed Central

    Liljelund, P; Mariotte, S; Buhler, J M; Sentenac, A

    1992-01-01

    The gene encoding the 49-kDa subunit of RNA polymerase A in Saccharomyces cerevisiae has been identified by formation of a hybrid enzyme between the S. cerevisiae A49 subunit and Saccharomyces douglasii subunits based on a polymorphism existing between the subunits of RNA polymerase A in these two species. The sequence of the gene reveals a basic protein with an unusually high lysine content, which may account for the affinity for DNA shown by the subunit. No appreciable homology with any polymerase subunits, enzymes, or transcription factors is found. Complete deletion of the single-copy RPA49 gene leads to viable but slowly growing colonies. Insertion of the HIS3 gene halfway into the RPA49 coding region results in synthesis of a truncated A49 subunit that is incorporated into the polymerase. The truncated and wild-type subunits compete equally for assembly in the heterozygous diploid, although the wild type is phenotypically dominant. Images PMID:1409638

  12. Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to better u...

  13. Recombinant GABAA receptor desensitization: the role of the gamma 2 subunit and its physiological significance.

    PubMed

    Dominguez-Perrot, C; Feltz, P; Poulter, M O

    1996-11-15

    1. The purpose of these investigations was to examine the role that the gamma 2 subunit plays in human GABAA receptor desensitization. Two different recombinant GABAA receptors (alpha 1 beta 3 and alpha 1 beta 3 gamma 2) were compared by measuring the relaxation of whole-cell currents during the application of GABA, isoguvacine or taurine. 2. At concentrations which trigger a maximum response (100-500 microM GABA) the current relaxation usually fitted the sum of two exponentials. For alpha 1 beta 3 subunit receptors these values were tau 1 = 145 +/- 12 ms and tau 2 = 6.3 +/- 2.1 s (means +/- S.E.M.). Receptors consisting of alpha 1 beta 3 gamma 2 subunits desensitized faster: tau 1 = 41.6 +/- 8.3 ms and tau 2 = 2.4 +/- 0.6 s. 3. The Hill slope, determined for each receptor subunit combination, was the same and greater than 1.0, implying two binding steps in the activation of both receptor subunit combinations. 4. For alpha 1 beta 3 subunit receptors the fast desensitization rates were unaltered by reducing the GABA concentration from the EC100 (100 microM) to the approximate EC50 values (10-20 microM), whereas for alpha 1 beta 3 gamma 2 subunit receptors a significant slowing was observed. The fast desensitization disappeared at agonist concentrations below the EC50 for both subunit combinations. In contrast, the slow desensitization appeared at agonist concentrations near the EC20. This rate was dependent on agonist concentration reaching a maximum near the EC60 value of GABA. 5. The fast desensitization rates were unaltered by changing the holding potential of the cell during agonist application. However, for alpha 1 beta 3 gamma 2 subunit receptors the slow desensitization rate increased by approximately 15- to 20-fold over the range of voltages of -60 to +40 mV. This indicates that the gamma 2 subunit makes GABAA receptor desensitization voltage dependent. 6. Recovery from desensitization was also biphasic. The first recovery phase was faster for alpha 1 beta 3

  14. Molecular cloning of pituitary glycoprotein alpha-subunit and follicle stimulating hormone and chorionic gonadotropin beta-subunits from New World squirrel monkey and owl monkey.

    PubMed

    Scammell, Jonathan G; Funkhouser, Jane D; Moyer, Felricia S; Gibson, Susan V; Willis, Donna L

    2008-02-01

    The goal of this study was to characterize the gonadotropins expressed in pituitary glands of the New World squirrel monkey (Saimiri sp.) and owl monkey (Aotus sp.). The various subunits were amplified from total RNA from squirrel monkey and owl monkey pituitary glands by reverse transcription-polymerase chain reaction and the deduced amino acid sequences compared to those of other species. Mature squirrel monkey and owl monkey glycoprotein hormone alpha-polypeptides (96 amino acids in length) were determined to be 80% homologous to the human sequence. The sequences of mature beta subunits of follicle stimulating hormone (FSHbeta) from squirrel monkey and owl monkey (111 amino acids in length) are 92% homologous to human FSHbeta. New World primate glycoprotein hormone alpha-polypeptides and FSHbeta subunits showed conservation of all cysteine residues and consensus N-linked glycosylation sites. Attempts to amplify the beta-subunit of luteinizing hormone from squirrel monkey and owl monkey pituitary glands were unsuccessful. Rather, the beta-subunit of chorionic gonadotropin (CG) was amplified from pituitaries of both New World primates. Squirrel monkey and owl monkey CGbeta are 143 and 144 amino acids in length and 77% homologous with human CGbeta. The greatest divergence is in the C terminus, where all four sites for O-linked glycosylation in human CGbeta, responsible for delayed metabolic clearance, are predicted to be absent in New World primate CGbetas. It is likely that CG secreted from pituitary of New World primates exhibits a relatively short half-life compared to human CG.

  15. Mapping of a conformational epitope on the cashew allergen Ana o 2: a discontinuous large subunit epitope dependent upon homologous or heterologous small subunit association.

    PubMed

    Xia, Lixin; Willison, LeAnna N; Porter, Lauren; Robotham, Jason M; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2010-05-01

    The 11S globulins are members of the cupin protein superfamily and represent an important class of tree nut allergens for which a number of linear epitopes have been mapped. However, specific conformational epitopes for these allergens have yet to be described. We have recently reported a cashew Ana o 2 conformational epitope defined by murine mAb 2B5 and competitively inhibited by a subset of patient IgE antibodies. The 2B5 epitope appears to reside on the large (acidic) subunit, is dependent upon small (basic) subunit association for expression, and is highly susceptible to denaturation. Here we fine map the epitope using a combination of recombinant chimeric cashew Ana o 2-soybean Gly m 6 chimeras, deletion and point mutations, molecular modeling, and electron microscopy of 2B5-Ana o 2 immune complexes. Key residues appear confined to a 24 amino acid segment near the N-terminus of the large subunit peptide, a portion of which makes direct contact with the small subunit. These data provide an explanation for both the small subunit dependence and the structurally labile nature of the epitope.

  16. Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core

    PubMed Central

    Conte, Emanuele; Vincelli, Gabriele; Schaaper, Roel M.; Bressanin, Daniela; Stefan, Alessandra; Dal Piaz, Fabrizio; Hochkoeppler, Alejandro

    2012-01-01

    Escherichia coli DNA polymerase III holoenzyme (HE) contains a core polymerase consisting of three subunits: α(polymerase), ε(3′-5′ exonuclease), and θ. Genetic experiments suggested that θ subunit stabilizes the intrinsically labile ε subunit and, furthermore, that θ might affect the cellular amounts of Pol III core and HE. Here, we provide biochemical evidence supporting this model by analyzing the amounts of the relevant proteins. First, we show that a ΔholE strain (lacking θ subunit) displays reduced amounts of free ε. We also demonstrate the existence of a dimer of ε, which may be involved in the stabilization of the protein. Second, θ, when overexpressed, dissociates the ε dimer and significantly increases the amount of Pol III core. The stability of ε also depends on cellular chaperones, including DnaK. Here, we report that: (i) temperature shift-up of ΔdnaK strains leads to rapid depletion of ε, and (ii) overproduction of θ overcomes both the depletion of ε and the temperature sensitivity of the strain. Overall, our data suggest that ε is a critical factor in the assembly of Pol III core, and that this is role is strongly influenced by the θ subunit through its prevention of ε degradation. PMID:22546509

  17. Tuning the Biological Activity Profile of Antibacterial Polymers via Subunit Substitution Pattern

    PubMed Central

    2015-01-01

    Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp3 carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications. PMID:24601599

  18. A Chaperonin Subunit with Unique Structures Is Essential for Folding of a Specific Substrate

    PubMed Central

    Peng, Lianwei; Fukao, Yoichiro; Myouga, Fumiyoshi; Motohashi, Reiko; Shinozaki, Kazuo; Shikanai, Toshiharu

    2011-01-01

    Type I chaperonins are large, double-ring complexes present in bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating the folding of newly synthesized, translocated, or stress-denatured proteins. In Escherichia coli, GroEL comprises 14 identical subunits and has been exquisitely optimized to fold its broad range of substrates. However, multiple Cpn60 subunits with different expression profiles have evolved in chloroplasts. Here, we show that, in Arabidopsis thaliana, the minor subunit Cpn60β4 forms a heterooligomeric Cpn60 complex with Cpn60α1 and Cpn60β1–β3 and is specifically required for the folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex (NDH). Other Cpn60β subunits cannot complement the function of Cpn60β4. Furthermore, the unique C-terminus of Cpn60β4 is required for the full activity of the unique Cpn60 complex containing Cpn60β4 for folding of NdhH. Our findings suggest that this unusual kind of subunit enables the Cpn60 complex to assist the folding of some particular substrates, whereas other dominant Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the folding of other obligate substrates. PMID:21483722

  19. Kv5, Kv6, Kv8, and Kv9 subunits: No simple silent bystanders

    PubMed Central

    2016-01-01

    Members of the electrically silent voltage-gated K+ (Kv) subfamilies (Kv5, Kv6, Kv8, and Kv9, collectively identified as electrically silent voltage-gated K+ channel [KvS] subunits) do not form functional homotetrameric channels but assemble with Kv2 subunits into heterotetrameric Kv2/KvS channels with unique biophysical properties. Unlike the ubiquitously expressed Kv2 subunits, KvS subunits show a more restricted expression. This raises the possibility that Kv2/KvS heterotetramers have tissue-specific functions, making them potential targets for the development of novel therapeutic strategies. Here, I provide an overview of the expression of KvS subunits in different tissues and discuss their proposed role in various physiological and pathophysiological processes. This overview demonstrates the importance of KvS subunits and Kv2/KvS heterotetramers in vivo and the importance of considering KvS subunits and Kv2/KvS heterotetramers in the development of novel treatments. PMID:26755771

  20. Tuning the biological activity profile of antibacterial polymers via subunit substitution pattern.

    PubMed

    Liu, Runhui; Chen, Xinyu; Chakraborty, Saswata; Lemke, Justin J; Hayouka, Zvi; Chow, Clara; Welch, Rodney A; Weisblum, Bernard; Masters, Kristyn S; Gellman, Samuel H

    2014-03-19

    Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp(3) carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,β,β-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications.

  1. Assembly and Intracellular Targeting of the βγ Subunits of Heterotrimeric G Proteins

    PubMed Central

    Rehm, Armin; Ploegh, Hidde L.

    1997-01-01

    The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent α, β, and γ subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-β antibodies (ARC5 and ARC9), raised against immunoaffinity purified βγ complexes, recognize β subunits when not associated with γ and can thus be used to monitor assembly of βγ complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of βγ complexes with the plasma membrane fraction starts between 15–30 min of chase. Three pools of β subunits can be distinguished based on their association with γ subunits, their localization, and their detergent solubility. Association of β and α subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of βγ subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane. PMID:9128244

  2. Physiological effects of unassembled chaperonin Cct subunits in the yeast Saccharomyces cerevisiae.

    PubMed

    Kabir, M Anaul; Kaminska, Joanna; Segel, George B; Bethlendy, Gabor; Lin, Paul; Della Seta, Flavio; Blegen, Casey; Swiderek, Kristine M; Zoładek, Teresa; Arndt, Kim T; Sherman, Fred

    2005-02-01

    Eukaryotic chaperonins, the Cct complexes, are assembled into two rings, each of which is composed of a stoichiometric array of eight different subunits, which are denoted Cct1p-Cct8p. Overexpression of a single CCT gene in Saccharomyces cerevisiae causes an increase of the corresponding Cct subunit, but not of the Cct complex. Nevertheless, overexpression of certain Cct subunits, especially CCT6, suppresses a wide range of abnormal phenotypes, including those caused by the diverse types of conditional mutations tor2-21, lst8-2 and rsp5-9 and those caused by the concomitant overexpression of Sit4p and Sap155p. The examination of 73 altered forms of Cct6p revealed that the cct6-24 mutation, containing GDGTT --> AAAAA replacements of the conserved ATP-binding motif, was unable to suppress any of these traits, although the cct6-24 allele was completely functional for growth. These results provide evidence for functional differences among Cct subunits and for physiological properties of unassembled subunits. We suggest that the suppression is due to the competition of specific Cct subunits for activities that normally modify various cellular components. Furthermore, we also suggest that the Cct subunits can act as suppressors only in certain states, such as when associated with ATP.

  3. Structural and spectroscopic studies of the native hemocyanin from Maia squinado and its structural subunits

    NASA Astrophysics Data System (ADS)

    Dolashka-Angelova, Pavlina; Hristova, Rumijana; Schuetz, Juergen; Stoeva, Stanka; Schwarz, Heinz; Voelter, Wolfgang

    2000-09-01

    The dodecameric hemocyanin of the crab Maia squinado contains five major electrophoretically separable polypeptide chains (structural subunits) which have been purified by FPLC ion exchange chromatography. The various proteins have been characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, caesium chloride and potassium iodide as tryptophan quenchers. The results show that the tryptophyl side chains of dodecameric Hc are deeply buried in hydrophobic regions of the hemocyanin aggregates and the quenching efficiency values for the native Hc in comparison with those from the constituent subunits are two to four times less. The conformational stabilities of the native dodecameric aggregate and its isolated structural subunits towards various denaturants (pH, temperature, guanidinium hydrochloride) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby, both, the oxy- and apo-forms of the protein have been considered. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 50-60°C, coinciding with the melting temperatures, Tm, determined by CD spectroscopy. The free energy of stabilization in water, Δ GDH 2O , toward guanidinium hydrochloride is about two times higher for the dodecamer as compared to the isolated subunits. These studies reveal that oligomerization between functional subunits has a stabilizing effect on the whole molecule and differences in the primary structures result in different stabilities of the subunits.

  4. Subunit Conformations and Assembly States of a DNA Translocating Motor: The Terminase of Bacteriophage P22

    PubMed Central

    Němeček, Daniel; Gilcrease, Eddie B.; Kang, Sebyung; Prevelige, Peter E.; Casjens, Sherwood; Thomas, George J.

    2007-01-01

    Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42 kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wildtype gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a ten subunit ring, despite a subunit fold indistinguishable from wildtype. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages. PMID:17945256

  5. Kv2 subunits underlie slowly inactivating potassium current in rat neocortical pyramidal neurons

    PubMed Central

    Guan, D; Tkatch, T; Surmeier, D J; Armstrong, W E; Foehring, R C

    2007-01-01

    We determined the expression of Kv2 channel subunits in rat somatosensory and motor cortex and tested for the contributions of Kv2 subunits to slowly inactivating K+ currents in supragranular pyramidal neurons. Single cell RT-PCR showed that virtually all pyramidal cells expressed Kv2.1 mRNA and ∼80% expressed Kv2.2 mRNA. Immunocytochemistry revealed striking differences in the distribution of Kv2.1 and Kv2.2 subunits. Kv2.1 subunits were clustered and located on somata and proximal dendrites of all pyramidal cells. Kv2.2 subunits were primarily distributed on large apical dendrites of a subset of pyramidal cells from deep layers. We used two methods for isolating currents through Kv2 channels after excluding contributions from Kv1 subunits: intracellular diffusion of Kv2.1 antibodies through the recording pipette and extracellular application of rStromatoxin-1 (ScTx). The Kv2.1 antibody specifically blocked the slowly inactivating K+ current by 25–50% (at 8 min), demonstrating that Kv2.1 subunits underlie much of this current in neocortical pyramidal neurons. ScTx (300 nm) also inhibited ∼40% of the slowly inactivating K+ current. We observed occlusion between the actions of Kv2.1 antibody and ScTx. In addition, Kv2.1 antibody- and ScTx-sensitive currents demonstrated similar recovery from inactivation and voltage dependence and kinetics of activation and inactivation. These data indicate that both agents targeted the same channels. Considering the localization of Kv2.1 and 2.2 subunits, currents from truncated dissociated cells are probably dominated by Kv2.1 subunits. Compared with Kv2.1 currents in expression systems, the Kv2.1 current in neocortical pyramidal cells activated and inactivated at relatively negative potentials and was very sensitive to holding potential. PMID:17379638

  6. Postsynaptic clustering of γ-aminobutyric acid type A receptors by the γ3 subunit in vivo

    PubMed Central

    Baer, Kristin; Essrich, Christian; Benson, Jack A.; Benke, Dietmar; Bluethmann, Horst; Fritschy, Jean-Marc; Lüscher, Bernhard

    1999-01-01

    Synaptic localization of γ-aminobutyric acid type A (GABAA) receptors is a prerequisite for synaptic inhibitory function, but the mechanism by which different receptor subtypes are localized to postsynaptic sites is poorly understood. The γ2 subunit and the postsynaptic clustering protein gephyrin are required for synaptic localization and function of major GABAA receptor subtypes. We now show that transgenic overexpression of the γ3 subunit in γ2 subunit-deficient mice restores benzodiazepine binding sites, benzodiazepine-modulated whole cell currents, and postsynaptic miniature currents, suggesting the formation of functional, postsynaptic receptors. Moreover, the γ3 subunit can substitute for γ2 in the formation of GABAA receptors that are synaptically clustered and colocalized with gephyrin in vivo. These clusters were formed even in brain regions devoid of endogenous γ3 subunit, indicating that the factors present for clustering of γ2 subunit-containing receptors are sufficient to cluster γ3 subunit-containing receptors. The GABAA receptor and gephyrin-clustering properties of the ectopic γ3 subunit were also observed for the endogenous γ3 subunit, but only in the absence of the γ2 subunit, suggesting that the γ3 subunit is at a competitive disadvantage with the γ2 subunit for clustering of postsynaptic GABAA receptors in wild-type mice. PMID:10536013

  7. A genetic analysis of Plasmodium falciparum RNA polymerase II subunits in yeast.

    PubMed

    Hazoume, Adonis; Naderi, Kambiz; Candolfi, Ermanno; Kedinger, Claude; Chatton, Bruno; Vigneron, Marc

    2011-04-01

    RNA polymerase II is an essential nuclear multi subunit enzyme that transcribes nearly the whole genome. Its inhibition by the alpha-amanitin toxin leads to cell death. The enzyme of Plasmodium falciparum remains poorly characterized. Using a complementation assay in yeast as a genetic test, we demonstrate that five Plasmodium putative RNA polymerase subunits are indeed functional in vivo. The active site of this enzyme is built from the two largest subunits. Using site directed mutagenesis we were able to modify the active site of the yeast RNA polymerase II so as to introduce Plasmodium or human structural motifs. The resulting strains allow the screening of chemical libraries for potential specific inhibitors.

  8. Stoichiometry of the Human Glycine Receptor Revealed by Direct Subunit counting

    PubMed Central

    Durisic, Nela; Godin, Antoine G.; Wever, Claudia M.; Heyes, Colin D.; Lakadamyali, Melike; Dent, Joseph A.

    2012-01-01

    The subunit stoichiometry of heteromeric glycine-gated channels (GlyRs) determines fundamental properties of these key inhibitory neurotransmitter receptors; however the ratio of α1 to β-subunits per receptor remains controversial. We used single molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2β. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations. PMID:22973015

  9. Cereblon inhibits proteasome activity by binding to the 20S core proteasome subunit beta type 4.

    PubMed

    Lee, Kwang Min; Lee, Jongwon; Park, Chul-Seung

    2012-10-26

    In humans, mutations in the gene encoding cereblon (CRBN) are associated with mental retardation. Although CRBN has been investigated in several cellular contexts, its function remains unclear. Here, we demonstrate that CRBN plays a role in regulating the ubiquitin-proteasome system (UPS). Heterologous expression of CRBN inhibited proteasome activity in a human neuroblastoma cell line. Furthermore, proteasome subunit beta type 4 (PSMB4), the β7 subunit of the 20S core complex, was identified as a direct binding partner of CRBN. These findings suggest that CRBN may modulate proteasome activity by directly interacting with the β7 subunit.

  10. zeta-COP, a subunit of coatomer, is required for COP-coated vesicle assembly

    PubMed Central

    1993-01-01

    cDNA encoding the 20-kD subunit of coatomer, zeta-COP, predicts a protein of 177-amino acid residues, similar in sequence to AP17 and AP19, subunits of the clathrin adaptor complexes. Polyclonal antibody directed to zeta-COP blocks the binding of coatomer to Golgi membranes and prevents the assembly of COP-coated vesicles on Golgi cisternae. Unlike other coatomer subunits (beta-, beta'-, gamma-, and epsilon- COP), zeta-COP exists in both coatomer bound and free pools. PMID:8276893

  11. Significance of alpha-subunit HCG demonstrated in breast carcinomas by the immunoperoxidase technique.

    PubMed Central

    Walker, R A

    1978-01-01

    Fifty-three breast carcinomas were examined by an indirect immunoperoxidase technique for the presence of the alpha-subunit of human chorionic gonadotrophin. Positive staining occurred in 12 (22.6%). There was no correlation between alpha-subunit production and specific histological features, but production was related to the presence of lymph node metastases and hence a poorer prognosis. The alpha-subunit could be used as a prognostic indicator in the assessment of breast carcinoma. Images Fig. 1 Fig. 2 PMID:346610

  12. CHARACTERIZATION OF NICOTINE ACETYLCHOLINE RECEPTOR SUBUNITS IN THE COCKROACH Periplaneta americana MUSHROOM BODIES REVEALS A STRONG EXPRESSION OF β1 SUBUNIT: INVOLVEMENT IN NICOTINE-INDUCED CURRENTS.

    PubMed

    Taillebois, Emiliane; Thany, Steeve H

    2016-09-01

    Nicotinic acetylcholine receptors are ligand-gated ion channels expressed in many insect structures, such as mushroom bodies, in which they play a central role. We have recently demonstrated using electrophysiological recordings that different native nicotinic receptors are expressed in cockroach mushroom bodies Kenyon cells. In the present study, we demonstrated that eight genes coding for cockroach nicotinic acetylcholine receptor subunits are expressed in the mushroom bodies. Quantitative real-time polymerase chain reaction (PCR) experiments demonstrated that β1 subunit was the most expressed in the mushroom bodies. Moreover, antisense oligonucleotides performed against β1 subunit revealed that inhibition of β1 expression strongly decreases nicotine-induced currents amplitudes. Moreover, co-application with 0.5 μM α-bungarotoxin completely inhibited nicotine currents whereas 10 μM d-tubocurarine had a partial effect demonstrating that β1-containing neuronal nicotinic acetylcholine receptor subtypes could be sensitive to the nicotinic acetylcholine receptor antagonist α-bungarotoxin.

  13. Cholera toxin subunit B detection in microfluidic devices.

    PubMed

    Bunyakul, Natinan; Edwards, Katie A; Promptmas, Chamras; Baeumner, Antje J

    2009-01-01

    Fluorescence and electrochemical microfluidic biosensors were developed for the detection of cholera toxin subunit B (CTB) as a model analyte. The microfluidic devices were made from polydimethylsiloxane (PDMS) using soft lithography from silicon templates. The polymer channels were sealed with a glass plate and packaged in a polymethylmethacrylate housing that provided leakproof sealing and a connection to a syringe pump. In the electrochemical format, an interdigitated ultramicroelectrode array (IDUA) was patterned onto the glass slide using photolithography, gold evaporation and lift-off processes. For CTB recognition, CTB-specific antibodies were immobilized onto superparamagnetic beads and ganglioside GM(1) was incorporated into liposomes. The fluorescence dye sulforhodamine B (SRB) and the electroactive compounds potassium hexacyanoferrate (II)/hexacyanoferrate (III) were used as detection markers that were encapsulated inside the liposomes for the fluorescence and electrochemical detection formats, respectively. Initial optimization experiments were carried out by applying the superparamagnetic beads in microtiter plate assays and SRB liposomes before they were transferred to the microfluidic systems. The limits of detection (LoD) of both assay formats for CTB were found to be 6.6 and 1.0 ng mL(-1) for the fluorescence and electrochemical formats, respectively. Changing the detection system was very easy, requiring only the synthesis of different marker-encapsulating liposomes, as well as the exchange of the detection unit. It was found that, in addition to a lower LoD, the electrochemical format assay showed advantages over the fluorescence format in terms of flexibility and reliability of signal recording.

  14. Early diagnosis of sepsis using serum hemoglobin subunit Beta.

    PubMed

    Yoo, Hayoung; Ku, Sae-Kwang; Kim, Shin-Woo; Bae, Jong-Sup

    2015-02-01

    The development of new sepsis-specific biomarkers is mandatory to improve the detection and monitoring of the disease. Hemoglobin is the main oxygen and carbon dioxide carrier in cells of the erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. Hemoglobin subunit beta (HBβ) is a component of a larger protein called hemoglobin. The aim of this study was to evaluate blood levels of HBβ in septic patients. A prospective study of 82 patients with sepsis was conducted. Furthermore, C57BL/6 mice were subjected to cecal ligation and puncture (CLP) surgery. Alternatively, human umbilical vein endothelial cells (HUVECs) or C57BL/6 mice were exposed to lipopolysaccharide (LPS, 100 ng/ml to HUVECs or 10 mg/kg to mice). The data showed that LPS induced upregulation of the synthesis and secretion of HBβ in LPS-treated HUVECs and in LPS-injected and CLP mice. In patients admitted to the intensive care unit with sepsis, circulating levels of HBβ were significantly high (sepsis, 64.93-114.76 ng/ml, n = 30; severe sepsis, 157.37-268.69 ng/ml, n = 22; septic shock, 309.98-427.03 ng/ml, n = 30) when compared to the levels of control donors (9.76-12.28 ng/ml, n = 21). Patients with septic shock had higher HBβ levels when compared to patients with severe sepsis. Furthermore, the HBβ levels in septic patients were higher than those in healthy volunteers. These results suggest that in septic patients, HBβ blood level is related to the severity of sepsis and may represent a novel endothelial cell dysfunction marker. Moreover, HBβ can be used as a biomarker to determine the severity of sepsis.

  15. P. berghei telomerase subunit TERT is essential for parasite survival.

    PubMed

    Religa, Agnieszka A; Ramesar, Jai; Janse, Chris J; Scherf, Artur; Waters, Andrew P

    2014-01-01

    Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA), though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO) homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR) in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF) analysis. TERT and TR were detected in blood stages and an average telomere length of ∼ 950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert- mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further investigations to

  16. Subunit-dependent effects of nickel on NMDA receptor channels.

    PubMed

    Marchetti, Carla; Gavazzo, Paola

    2003-10-07

    Nickel (Ni2+) is a transition metal that affects different neuronal ionic channels. We investigated its effects on glutamate channels of the NMDA-type in the presence of saturating concentration of glutamate or NMDA (50 microM), in 0 external Mg and in the continuous presence of saturating glycine (30 microM). In neonatal rat cerebellar granule cells, Ni2+ inhibited the current evoked by NMDA at -60 mV with an IC50 close to 40 microM. The inhibition was weakly voltage-dependent and the current at +40 mV was inhibited with IC50=86 microM. Wash out of the metal unmasked a stimulatory effect which persisted for a few seconds. In HEK293 cells transiently transfected with recombinant NR1a-NR2A receptors, Ni2+ inhibited the current elicited by glutamate with an IC50=52 microM at -60 mV and 90 microM at +40 mV. In HEK293 expressing NR1a-NR2B receptors, 0.1-100 microM Ni2+ caused a potentiation of the current, with EC50=4 microM, while with 300 microM, a voltage-dependent block became apparent (IC50=170 microM). As previously reported, the current through both classes of recombinant receptors was steeply dependent on external pH, and in both cases the protonic block had an IC50 close to pH 7.2. Application of Ni2+ showed that stimulation of NR1a-NR2B receptor channels was dependent on external pH, while voltage-independent inhibition of NR1a-NR2A was less sensitive to pH change. These results indicate that Ni2+ has multiple and complex effects on NMDA channels, which are largely dependent on the NR2 subunit.

  17. Elongated polyproline motifs facilitate enamel evolution through matrix subunit compaction.

    PubMed

    Jin, Tianquan; Ito, Yoshihiro; Luan, Xianghong; Dangaria, Smit; Walker, Cameron; Allen, Michael; Kulkarni, Ashok; Gibson, Carolyn; Braatz, Richard; Liao, Xiubei; Diekwisch, Thomas G H

    2009-12-01

    Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i) in a compaction of protein matrix subunit dimensions, (ii) reduced conformational variability, (iii) an increase in polyproline II helices, and (iv) promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem repeat fragment

  18. Preclinical and clinical development of a dengue recombinant subunit vaccine.

    PubMed

    Manoff, Susan B; George, Sarah L; Bett, Andrew J; Yelmene, Michele L; Dhanasekaran, Govindarajan; Eggemeyer, Linda; Sausser, Michele L; Dubey, Sheri A; Casimiro, Danilo R; Clements, David E; Martyak, Timothy; Pai, Vidya; Parks, D Elliot; Coller, Beth-Ann G

    2015-12-10

    This review focuses on a dengue virus (DENV) vaccine candidate based on a recombinant subunit approach which targets the DENV envelope glycoprotein (E). Truncated versions of E consisting of the N-terminal portion of E (DEN-80E) have been expressed recombinantly in the Drosophila S2 expression system and shown to have native-like conformation. Preclinical studies demonstrate that formulations containing tetravalent DEN-80E adjuvanted with ISCOMATRIX™ adjuvant induce high titer virus neutralizing antibodies and IFN-γ producing T cells in flavivirus-naïve non-human primates. The preclinical data further suggest that administration of such formulations on a 0, 1, 6 month schedule may result in higher maximum virus neutralizing antibody titers and better durability of those titers compared to administration on a 0, 1, 2 month schedule. In addition, the virus neutralizing antibody titers induced by adjuvanted tetravalent DEN-80E compare favorably to the titers induced by a tetravalent live virus comparator. Furthermore, DEN-80E was demonstrated to be able to boost virus neutralizing antibody titers in macaques that have had a prior DENV exposure. A monovalent version of the vaccine candidate, DEN1-80E, was formulated with Alhydrogel™ and studied in a proof-of-principle Phase I clinical trial by Hawaii Biotech, Inc. (NCT00936429). The clinical trial results demonstrate that both the 10 μg and 50 μg formulations of DEN1-80E with 1.25 mg of elemental aluminum were immunogenic when administered in a 3-injection series (0, 1, 2 months) to healthy, flavivirus-naïve adults. The vaccine formulations induced DENV-1 neutralizing antibodies in the majority of subjects, although the titers in most subjects were modest and waned over time. Both the 10 μg DEN1-80E and the 50 μg DEN1-80E formulations with Alhydrogel™ were generally well tolerated.

  19. Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction

    PubMed Central

    Luan, Xianghong; Dangaria, Smit; Walker, Cameron; Allen, Michael; Kulkarni, Ashok; Gibson, Carolyn; Braatz, Richard; Liao, Xiubei; Diekwisch, Thomas G. H.

    2009-01-01

    Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i) in a compaction of protein matrix subunit dimensions, (ii) reduced conformational variability, (iii) an increase in polyproline II helices, and (iv) promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem repeat fragment

  20. Use of an α3β4 nicotinic acetylcholine receptor subunit concatamer to characterize ganglionic receptor subtypes with specific subunit composition reveals species-specific pharmacologic properties.

    PubMed

    Stokes, Clare; Papke, Roger L

    2012-09-01

    Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain α4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing α3β4 binding sites and other subunits, including β4, β2, α5, or α3 as a structural subunit in the pentamer. Additional interest in α3 β4 α5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in α5 in particular, to lung cancer and heavy smoking. While α3 and β4 readily form receptors in expression system such as the Xenopus oocyte, since α5 is not required for function, simple co-expression approaches may under-represent α5-containing receptors. We used a concatamer of human α3 and β4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These α3β4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for β4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important α4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the α5 SNP. However, our data validate this approach for further investigations.

  1. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes

    PubMed Central

    Ilca, Serban L.; Kotecha, Abhay; Sun, Xiaoyu; Poranen, Minna M.; Stuart, David I.; Huiskonen, Juha T.

    2015-01-01

    Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems. PMID:26534841

  2. Extensive subunit contacts underpin herpesvirus capsid stability and interior-to-exterior allostery

    PubMed Central

    Huet, Alexis; Makhov, Alexander M.; Huffman, Jamie B.; Vos, Matthijn; Homa, Fred L.; Conway, James F.

    2016-01-01

    The herpesvirus capsid is a complex protein assembly that includes hundreds of copies of four major subunits and lesser numbers of several minor proteins, all essential for infectivity. Cryo-electron microscopy is uniquely suited for studying interactions that govern the assembly and function of such large and functional complexes. Here we report two high quality capsid structures, from human herpes simplex virus type 1 (HSV-1) and the animal pseudorabies virus (PRV), imaged inside intact virions at ~7 Å resolution. From these we developed a complete model of subunit and domainal organization and identified extensive networks of subunit contacts that underpin capsid stability and form a pathway that may signal the completion of DNA packaging from the capsid interior to outer surface for initiating nuclear egress. Differences in folding and orientation of subunit domains between herpesvirus capsids suggest that common elements have been modified for specific functions. PMID:27111889

  3. Nucleotide-Protectable Labeling of Sulfhydryl Groups in Subunit I of the ATPhase from Halobacterium Saccharovorum

    NASA Technical Reports Server (NTRS)

    Sulzner, Michael; Stan-Lotter, Helga; Hochstein, Lawrence I.

    1992-01-01

    A membrane-bound ATPase from the archaebacterium Halobacterium saccharovorum is inhibited by N-ethyl-maleimide in a nucleotide-protectable manner. When the enzyme was incubated with N-[C-14]jethylmaleimide, the bulk of radioactivity was as- sociated with the 87,000-Da subunit (subunit 1). ATP, ADP, or AMP reduced incorporation of the inhibitor. No charge shift of subunit I was detected following labeling with N-ethylmaleimide, indicating an electroneutral reaction. The results are consistent with the selective modification of sulfhydryl groups in subunit I at or near the catalytic site and are further evidence of a resemblance between this archaebacterial ATPase and the vacuolar-type ATPases.

  4. [Nucleotide sequence of genes for alpha- and beta-subunits of luciferase from Photobacterium leiognathi].

    PubMed

    Illarionov, B A; Protopopova, M V; Karginov, V A; Mertvetsov, N P; Gitel'zon, I I

    1988-03-01

    Nucleotide sequence of the Photobacterium leiognathi DNA containing genes of alpha and beta subunits of luciferase has been determined. We also deduced amino acid sequence and molecular mass of luciferase and localized luciferase genes in the sequenced DNA fragment.

  5. High degree of coordination and division of labor among subunits in a homomeric ring ATPase.

    PubMed

    Chistol, Gheorghe; Liu, Shixin; Hetherington, Craig L; Moffitt, Jeffrey R; Grimes, Shelley; Jardine, Paul J; Bustamante, Carlos

    2012-11-21

    Ring NTPases of the ASCE superfamily perform a variety of cellular functions. An important question about the operation of these molecular machines is how the ring subunits coordinate their chemical and mechanical transitions. Here, we present a comprehensive mechanochemical characterization of a homomeric ring ATPase-the bacteriophage φ29 packaging motor-a homopentamer that translocates double-stranded DNA in cycles composed of alternating dwells and bursts. We use high-resolution optical tweezers to determine the effect of nucleotide analogs on the cycle. We find that ATP hydrolysis occurs sequentially during the burst and that ADP release is interlaced with ATP binding during the dwell, revealing a high degree of coordination among ring subunits. Moreover, we show that the motor displays an unexpected division of labor: although all subunits of the homopentamer bind and hydrolyze ATP during each cycle, only four participate in translocation, whereas the remaining subunit plays an ATP-dependent regulatory role.

  6. Subunit connectivity, assembly determinants, and architecture of the yeast exocyst complex

    PubMed Central

    Heider, Margaret R.; Gu, Mingyu; Duffy, Caroline M.; Mirza, Anne M.; Marcotte, Laura L.; Walls, Alexandra C.; Farrall, Nicholas; Hakhverdyan, Zhanna; Field, Mark C.; Rout, Michael P.; Frost, Adam; Munson, Mary

    2016-01-01

    The exocyst is a hetero-octameric complex proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst from Saccharomyces cerevisiae, and show that the purified complexes are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin-induced degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer, and demonstrated that several known exocyst binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex using negative stain electron microscopy; our results indicate that exocyst exists predominantly as a stable, octameric complex with an elongated architecture that suggests the subunits are contiguous helical bundles packed together into a bundle of long rods. PMID:26656853

  7. Complex regulation of γ-secretase: from obligatory to modulatory subunits

    PubMed Central

    Gertsik, Natalya; Chiu, Danica; Li, Yue-Ming

    2014-01-01

    γ-Secretase is a four subunit, 19-pass transmembrane enzyme that cleaves amyloid precursor protein (APP), catalyzing the formation of amyloid beta (Aβ) peptides that form amyloid plaques, which contribute to Alzheimer’s disease (AD) pathogenesis. γ-Secretase also cleaves Notch, among many other type I transmembrane substrates. Despite its seemingly promiscuous enzymatic capacity, γ-secretase activity is tightly regulated. This regulation is a function of many cellular entities, including but not limited to the essential γ-secretase subunits, nonessential (modulatory) subunits, and γ-secretase substrates. Regulation is also accomplished by an array of cellular events, such as presenilin (active subunit of γ-secretase) endoproteolysis and hypoxia. In this review we discuss how γ-secretase is regulated with the hope that an advanced understanding of these mechanisms will aid in the development of effective therapeutics for γ-secretase-associated diseases like AD and Notch-addicted cancer. PMID:25610395

  8. Differential Targeting of Gβγ-Subunit Signaling with Small Molecules

    NASA Astrophysics Data System (ADS)

    Bonacci, Tabetha M.; Mathews, Jennifer L.; Yuan, Chujun; Lehmann, David M.; Malik, Sundeep; Wu, Dianqing; Font, Jose L.; Bidlack, Jean M.; Smrcka, Alan V.

    2006-04-01

    G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor-dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein-coupled receptor signaling that may represent an important therapeutic strategy.

  9. Separation of glutathione transferase subunits from Proteus vulgaris by two-dimensional gel electrophoresis.

    PubMed

    Hong, Giaming; Chien, Yi-Chih; Chien, Cheng-I

    2003-10-01

    Cytosolic glutathione transferases of Proteus vulgaris were purified by affinity chromatography and characterized by two-dimensional gel electrophoresis. Four different subunits were identified, and each subunit contained a different molecular mass, ranging from 26.2 kDa to 28.5 kDa; a different pI value, ranging from 8.2 to 9.4; and a different amount of protein fraction, ranging from 10% to 56%. All four subunits existed as basic proteins (pI > 7.0). From these results, we concluded that multiple forms of glutathione transferase enzymes existed in Proteus vulgaris, and four different glutathione transferase subunits were separated by 2-D gel electrophoresis.

  10. Cooperative Subunit Refolding of a Light-Harvesting Protein through a Self-Chaperone Mechanism.

    PubMed

    Laos, Alistair J; Dean, Jacob C; Toa, Zi S D; Wilk, Krystyna E; Scholes, Gregory D; Curmi, Paul M G; Thordarson, Pall

    2017-01-27

    The fold of a protein is encoded by its amino acid sequence, but how complex multimeric proteins fold and assemble into functional quaternary structures remains unclear. Here we show that two structurally different phycobiliproteins refold and reassemble in a cooperative manner from their unfolded polypeptide subunits, without biological chaperones. Refolding was confirmed by ultrafast broadband transient absorption and two-dimensional electronic spectroscopy to probe internal chromophores as a marker of quaternary structure. Our results demonstrate a cooperative, self-chaperone refolding mechanism, whereby the β-subunits independently refold, thereby templating the folding of the α-subunits, which then chaperone the assembly of the native complex, quantitatively returning all coherences. Our results indicate that subunit self-chaperoning is a robust mechanism for heteromeric protein folding and assembly that could also be applied in self-assembled synthetic hierarchical systems.

  11. Experimental ulcerative herpetic keratitis. IV. Preliminary observations on the efficacy of a herpes simplex subunit vaccine.

    PubMed

    Carter, C A; Hartley, C E; Skinner, G R; Turner, S P; Easty, D L

    1981-10-01

    Systemic vaccination of rabbits with an inactivated type 1 virus subunit vaccine induced humoral and cell-mediated immune responses. Following ocular infection with type 1 herpes virus corneal ulceration and virus excretion were reduced in the vaccinated rabbits.

  12. Functional protein expression of multiple sodium channel alpha- and beta-subunit isoforms in neonatal cardiomyocytes.

    PubMed

    Kaufmann, Susann G; Westenbroek, Ruth E; Zechner, Christoph; Maass, Alexander H; Bischoff, Sebastian; Muck, Jenny; Wischmeyer, Erhard; Scheuer, Todd; Maier, Sebastian K G

    2010-01-01

    Voltage-gated sodium channels are composed of pore-forming alpha- and auxiliary beta-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel alpha-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na(v)1.5 sodium channel alpha-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel alpha- and beta-subunits. alpha-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel alpha-subunit isoforms Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4 and Na(v)1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac alpha-subunit isoform, Na(v)1.5. Each of the beta-subunit isoforms (beta1-beta4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the alpha-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel alpha-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na(v)1.5 alpha-subunit and they contribute to the total sodium current.

  13. Genetic disruption of the autism spectrum disorder risk gene PLAUR induces GABAA receptor subunit changes

    PubMed Central

    Eagleson, Kathie L.; Gravielle, Maria C.; SchlueterMcFadyen-Ketchum, Lisa J.; Russek, Shelley J.; Farb, David H.; Levitt, Pat

    2010-01-01

    Disruption of the GABAergic system has been implicated in multiple developmental disorders, including epilepsy, autism spectrum disorder and schizophrenia. The human gene encoding uPAR (PLAUR) has been shown recently to be associated with the risk of autism. The uPAR-/- mouse exhibits a regionally selective reduction in GABAergic interneurons in frontal and parietal regions of the cerebral cortex as well as in the CA1 and dentate gyrus subfields of the hippocampus. Behaviorally, these mice exhibit increased sensitivity to pharmacologically-induced seizures, heightened anxiety, and atypical social behavior. Here, we explore potential alterations in GABAergic circuitry that may occur in the context of altered interneuron development. Analysis of gene expression for 13 GABAA receptor subunits using quantitative real-time PCR indicates seven subunit mRNAs (α1, α2, α3, β2, β3, γ2S and γ2L) of interest. Semi-quantitative in situ hybridization analysis focusing on these subunit mRNAs reveals a complex pattern of potential gene regulatory adaptations. The levels of α2 subunit mRNAs increase in frontal cortex, CA1 and CA3, while those of α3 decrease in frontal cortex and CA1. In contrast, α1 subunit mRNAs are unaltered in any region examined. β2 subunit mRNAs are increased in frontal cortex whereas β3 subunit mRNAs are decreased in parietal cortex. Finally, γ2S subunit mRNAs are increased in parietal cortex while γ2L subunit mRNAs are increased in the dentate gyrus, potentially altering the γ2S:γ2L ratio in these two regions. For all subunits, no changes were observed in forebrain regions where GABAergic interneuron numbers are normal. We propose that disrupted differentiation of GABAergic neurons specifically in frontal and parietal cortices leads to regionally-selective alterations in local circuitry and subsequent adaptive changes in receptor subunit composition. Future electrophysiological studies will be useful in determining how alterations in network

  14. Distribution of NMDA and AMPA receptor subunits at thalamo-amygdaloid dendritic spines.

    PubMed

    Radley, Jason J; Farb, Claudia R; He, Yong; Janssen, William G M; Rodrigues, Sarina M; Johnson, Luke R; Hof, Patrick R; LeDoux, Joseph E; Morrison, John H

    2007-02-23

    Synapses onto dendritic spines in the lateral amygdala formed by afferents from the auditory thalamus represent a site of plasticity in Pavlovian fear conditioning. Previous work has demonstrated that thalamic afferents synapse onto LA spines expressing glutamate receptor (GluR) subunits, but the GluR subunit distribution at the synapse and within the cytoplasm has not been characterized. Therefore, we performed a quantitative analysis for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits GluR2 and GluR3 and N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B by combining anterograde labeling of thalamo-amygdaloid afferents with postembedding immunoelectron microscopy for the GluRs in adult rats. A high percentage of thalamo-amygdaloid spines was immunoreactive for GluR2 (80%), GluR3 (83%), and NR1 (83%), while a smaller proportion of spines expressed NR2B (59%). To compare across the various subunits, the cytoplasmic to synaptic ratios of GluRs were measured within thalamo-amygdaloid spines. Analyses revealed that the cytoplasmic pool of GluR2 receptors was twice as large compared to the GluR3, NR1, and NR2B subunits. Our data also show that in the adult brain, the NR2B subunit is expressed in the majority of in thalamo-amygdaloid spines and that within these spines, the various GluRs are differentially distributed between synaptic and non-synaptic sites. The prevalence of the NR2B subunit in thalamo-amygdaloid spines provides morphological evidence supporting its role in the fear conditioning circuit while the differential distribution of the GluR subtypes may reflect distinct roles for their involvement in this circuitry and synaptic plasticity.

  15. 3D reconstruction of the hemocyanin subunit dimer from the chiton Acanthochiton fascicularis.

    PubMed

    Harris, J Robin; Meissner, Ulrich; Gebauer, Wolfgang; Markl, Jürgen

    2004-01-01

    Procedures are presented for the purification of the subunit dimer from Acanthochiton fasicularis hemocyanin. Electron microscopy of negatively stained specimens revealed a uniform population of macromolecules possessing the characteristic "boat shape". A 3D reconstruction from this EM data generated a approximately 3 nm resolution model that correlates well with earlier data of the purported subunit dimer, extracted from the 3D reconstruction of the didecamer of Haliotis tuberculata hemocyanin type 1.

  16. Type B Heterotrimeric G Protein γ-Subunit Regulates Auxin and ABA Signaling in Tomato.

    PubMed

    Subramaniam, Gayathery; Trusov, Yuri; Lopez-Encina, Carlos; Hayashi, Satomi; Batley, Jacqueline; Botella, José Ramón

    2016-02-01

    Heterotrimeric G proteins composed of α, β, and γ subunits are central signal transducers mediating the cellular response to multiple stimuli in most eukaryotes. Gγ subunits provide proper cellular localization and functional specificity to the heterotrimer complex. Plant Gγ subunits, divided into three structurally distinct types, are more diverse than their animal counterparts. Type B Gγ subunits, lacking a carboxyl-terminal isoprenylation motif, are found only in flowering plants. We present the functional characterization of type B Gγ subunit (SlGGB1) in tomato (Solanum lycopersicum). We show that SlGGB1 is the most abundant Gγ subunit in tomato and strongly interacts with the Gβ subunit. Importantly, the green fluorescent protein-SlGGB1 fusion protein as well as the carboxyl-terminal yellow fluorescent protein-SlGGB1/amino-terminal yellow fluorescent protein-Gβ heterodimer were localized in the plasma membrane, nucleus, and cytoplasm. RNA interference-mediated silencing of SlGGB1 resulted in smaller seeds, higher number of lateral roots, and pointy fruits. The silenced lines were hypersensitive to exogenous auxin, while levels of endogenous auxins were lower or similar to those of the wild type. SlGGB1-silenced plants also showed strong hyposensitivity to abscisic acid (ABA) during seed germination but not in other related assays. Transcriptome analysis of the transgenic seeds revealed abnormal expression of genes involved in ABA sensing, signaling, and response. We conclude that the type B Gγ subunit SlGGB1 mediates auxin and ABA signaling in tomato.

  17. Studies on the murine Ss protein. I. Purification, molecular weight, and subunit structure

    PubMed Central

    1975-01-01

    The murine Ss protein has been isolated and purified. Using specific antisera, the radiolabeled protein has a mol wt of 120,000 in sodium dodecyl sulfate polyacrylamide gels. It is composed of two basic subunits of 23,000 and 14,000 daltons. The smaller molecular weight subunit contains a single disulfide bridge, is devoid of carbohydrate, and may represent the murine equivalent of beta2-microglobulin. PMID:809530

  18. Differential proteolysis of the subunits of pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase.

    PubMed

    Cheng, H F; Tao, M

    1990-02-05

    Antibodies against the alpha (Mr 67,000) and beta (Mr 60,000) subunits of wheat seedling Fru-2,6-P2-stimulated pyrophosphate-dependent 6-phosphofructo-1-phosphotransferase (PFP) were used to probe the subunit structures of several partially purified plant PFPs after tryptic digestion. Antisera to the alpha and beta subunits of wheat seedling PFP cross-reacted with the corresponding alpha and beta subunits of PFP preparations from wheat germ, potato tubers, and lettuce leaves. With the mung bean PFP, both antisera reacted with a protein band of Mr 60,000. A protein band corresponding to the Mr 67,000 alpha subunit was not detected in the mung bean PFP preparation. Tryptic digestion of wheat seedling and potato tuber PFPs resulted in the preferential cleavage of the alpha subunit. The trypsinized PFP retained most of its Fru-2,6-P2-stimulated activity but not its basal activity. The proteolyzed enzyme also exhibited a 2-fold increase in Ka for Fru-2,6-P2. Studies with the mung bean enzyme revealed that the anti-alpha immunoreactive component was more sensitive to trypsinization than the anti-beta immunoreactive component of the Mr 60,000 protein band. Thus, the Mr 60,000 protein band of the mung bean PFP appears to be heterogeneous and contains both alpha and beta-like proteins. The above observations indicate that the alpha and beta subunits of PFP are two distinct polypeptides and that alpha acts as a regulatory protein in regulating both the catalytic activity and the Fru-2,6-P2-binding affinity of the beta subunit.

  19. Neutron Scattering and the 30 S Ribosomal Subunit of E. Coli

    DOE R&D Accomplishments Database

    Moore, P. B.; Engelman, D. M.; Langer, J. A.; Ramakrishnan, V. R.; Schindler, D. G.; Schoenborn, B. P.; Sillers, I. Y.; Yabuki, S.

    1982-06-01

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today.

  20. Pharmacological consequences of the coexpression of BK channel α and auxiliary β subunits

    PubMed Central

    Torres, Yolima P.; Granados, Sara T.; Latorre, Ramón

    2014-01-01

    Coded by a single gene (Slo1, KCM) and activated by depolarizing potentials and by a rise in intracellular Ca2+ concentration, the large conductance voltage- and Ca2+-activated K+ channel (BK) is unique among the superfamily of K+ channels. BK channels are tetramers characterized by a pore-forming α subunit containing seven transmembrane segments (instead of the six found in voltage-dependent K+ channels) and a large C terminus composed of two regulators of K+ conductance domains (RCK domains), where the Ca2+-binding sites reside. BK channels can be associated with accessory β subunits and, although different BK modulatory mechanisms have been described, greater interest has recently been placed on the role that the β subunits may play in the modulation of BK channel gating due to its physiological importance. Four β subunits have currently been identified (i.e., β1, β2, β3, and β4) and despite the fact that they all share the same topology, it has been shown that every β subunit has a specific tissue distribution and that they modify channel kinetics as well as their pharmacological properties and the apparent Ca2+ sensitivity of the α subunit in different ways. Additionally, different studies have shown that natural, endogenous, and synthetic compounds can modulate BK channels through β subunits. Considering the importance of these channels in different pathological conditions, such as hypertension and neurological disorders, this review focuses on the mechanisms by which these compounds modulate the biophysical properties of BK channels through the regulation of β subunits, as well as their potential therapeutic uses for diseases such as those mentioned above. PMID:25346693

  1. Neutron scattering and the 30 S ribosomal subunit of E. coli

    SciTech Connect

    Moore, P.B.; Engelman, D.M.; Langer, J.A.; Ramakrishnan, V.R.; Schindler, D.G.; Schoenborn, B.P.; Sillers, I.Y.; Yabuki, S.

    1982-01-01

    This paper reviews the progress made in the study of the internal organization of the 30 S ribosomal subunit of E. coli by neutron scattering since 1975. A map of that particle showing the position of 14 of the subunit's 21 proteins is presented, and the methods currently used for collecting and analyzing such data are discussed. Also discussed is the possibility of extending the interpretation of neutron mapping data beyond the limits practical today. 30 references, 5 figures.

  2. Ricin, ricin agglutinin, and the ricin binding subunit structural comparison by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Brandt, N. N.; Chikishev, A. Yu.; Sotnikov, A. I.; Savochkina, Yu. A.; Agapov, I. I.; Tonevitsky, A. G.

    2005-02-01

    Raman spectroscopy is used to study conformation-sensitive vibrational bands of the plant toxins ricin and ricin agglutinin and the ricin binding subunit in aqueous solution. The analysis of the Raman data yields the conformational state of the protein molecules differing from that predicted by the X-ray data. The differences and similarities in the conformational state of ricin, ricin agglutinin, and ricin binding subunit are discussed.

  3. Molecular Analyses of a Three-Subunit Euryarchaeal Clamp Loader Complex from Methanosarcina acetivorans▿ †

    PubMed Central

    Chen, Yi-Hsing; Lin, Yuyen; Yoshinaga, Aya; Chhotani, Benazir; Lorenzini, Jenna L.; Crofts, Alexander A.; Mei, Shou; Mackie, Roderick I.; Ishino, Yoshizumi; Cann, Isaac K. O.

    2009-01-01

    Chromosomal DNA replication is dependent on processive DNA synthesis. Across the three domains of life and in certain viruses, a toroidal sliding clamp confers processivity to replicative DNA polymerases by encircling the DNA and engaging the polymerase in protein/protein interactions. Sliding clamps are ring-shaped; therefore, they have cognate clamp loaders that open and load them onto DNA. Here we use biochemical and mutational analyses to study the structure/function of the Methanosarcina acetivorans clamp loader or replication factor C (RFC) homolog. M. acetivorans RFC (RFCMa), which represents an intermediate between the common archaeal RFC and the eukaryotic RFC, comprises two different small subunits (RFCS1 and RFCS2) and a large subunit (RFCL). Size exclusion chromatography suggested that RFCS1 exists in oligomeric states depending on protein concentration, while RFCS2 exists as a monomer. Protein complexes of RFCS1/RFCS2 formed in solution; however, they failed to stimulate DNA synthesis by a cognate DNA polymerase in the presence of its clamp. Determination of the subunit composition and previous mutational analysis allowed the prediction of the spatial distribution of subunits in this new member of the clamp loader family. Three RFCS1 subunits are flanked by an RFCS2 and an RFCL. The spatial distribution is, therefore, reminiscent of the minimal Escherichia coli clamp loader that exists in space as three γ-subunits (motor) flanked by the δ′ (stator) and the δ (wrench) subunits. Mutational analysis, however, suggested that the similarity between the two clamp loaders does not translate into the complete conservation of the functions of individual subunits within the RFCMa complex. PMID:19717601

  4. Crystal Structure of the Oxazolidinone Antibiotic Linezolid Bound to the 50S Ribosomal Subunit

    SciTech Connect

    Ippolito,J.; Kanyo, Z.; Wang, D.; Franceschi, F.; Moore, P.; Steitz, T.; Duffy, E.

    2008-01-01

    The oxazolidinone antibacterials target the 50S subunit of prokaryotic ribosomes. To gain insight into their mechanism of action, the crystal structure of the canonical oxazolidinone, linezolid, has been determined bound to the Haloarcula marismortui 50S subunit. Linezolid binds the 50S A-site, near the catalytic center, which suggests that inhibition involves competition with incoming A-site substrates. These results provide a structural basis for the discovery of improved oxazolidinones active against emerging drug-resistant clinical strains.

  5. Two temporally synthesized charge subunits interact to form the five isoforms of cottonseed (Gossypium hirsutum) catalase.

    PubMed Central

    Ni, W; Trelease, R N; Eising, R

    1990-01-01

    Five charge isoforms of tetrameric catalase were isolated from cotyledons of germinated cotton (Gossypium hirsutum L.) seedlings. Denaturing isoelectric focusing of the individual isoforms in polyacrylamide gels indicated that isoforms A (most anodic) and E (most cathodic) consisted of one subunit of different charge, whereas isoforms B, C and D each consisted of a mixture of these two subunits. Thus the five isoforms apparently were formed through combinations of two subunits in different ratios. Labelling cotyledons in vivo with [35S]methionine at three daily intervals in the dark, and translation in vivo of polyadenylated RNA isolated from cotyledons at the same ages, revealed synthesis of two different subunits. One of the subunits was synthesized in cotyledons at all ages studied (days 1-3), whereas the other subunit was detected only at days 2 and 3. This differential expression of two catalase subunits helped explain previous results from this laboratory showing that the two anodic forms (A and B) found in maturing seeds were supplemented with three cathodic forms (C-E) after the seeds germinated. These subunit data also helped clarify our new findings that proteins of isoforms A, B and C (most active isoforms) accumulated in cotyledons of plants kept in the dark for 3 days, then gradually disappeared during the next several days, whereas isoforms D and E (least active isoforms) remained in the cells. This shift in isoform pattern occurred whether seedlings were kept in the dark or exposed to continuous light after day 3, although exposure to light enhanced this process. These sequential molecular events were responsible for the characteristic developmental changes (rise and fall) in total catalase activity. We believe that the isoform changeover is physiologically related to the changeover in glyoxysome to leaf-type-peroxisome metabolism. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1695843

  6. The Evolution of the Four Subunits of Voltage-Gated Calcium Channels: Ancient Roots, Increasing Complexity, and Multiple Losses

    PubMed Central

    Moran, Yehu; Zakon, Harold H.

    2014-01-01

    The alpha subunits of voltage-gated calcium channels (Cavs) are large transmembrane proteins responsible for crucial physiological processes in excitable cells. They are assisted by three auxiliary subunits that can modulate their electrical behavior. Little is known about the evolution and roles of the various subunits of Cavs in nonbilaterian animals and in nonanimal lineages. For this reason, we mapped the phyletic distribution of the four channel subunits and reconstructed their phylogeny. Although alpha subunits have deep evolutionary roots as ancient as the split between plants and opistokonths, beta subunits appeared in the last common ancestor of animals and their close-relatives choanoflagellates, gamma subunits are a bilaterian novelty and alpha2/delta subunits appeared in the lineage of Placozoa, Cnidaria, and Bilateria. We note that gene losses were extremely common in the evolution of Cavs, with noticeable losses in multiple clades of subfamilies and also of whole Cav families. As in vertebrates, but not protostomes, Cav channel genes duplicated in Cnidaria. We characterized by in situ hybridization the tissue distribution of alpha subunits in the sea anemone Nematostella vectensis, a nonbilaterian animal possessing all three Cav subfamilies common to Bilateria. We find that some of the alpha subunit subtypes exhibit distinct spatiotemporal expression patterns. Further, all six sea anemone alpha subunit subtypes are conserved in stony corals, which separated from anemones 500 MA. This unexpected conservation together with the expression patterns strongly supports the notion that these subtypes carry unique functional roles. PMID:25146647

  7. Lifelong ethanol consumption and brain regional GABAA receptor subunit mRNA expression in alcohol-preferring rats.

    PubMed

    Sarviharju, Maija; Hyytiä, Petri; Hervonen, Antti; Jaatinen, Pia; Kiianmaa, Kalervo; Korpi, Esa R

    2006-11-01

    Brain regional gamma-aminobutyric acid type A (GABAA) receptor subunit mRNA expression was studied in ethanol-preferring AA (Alko, Alcohol) rats after moderate ethanol drinking for up to 2 years of age. In situ hybridization with oligonucleotide probes specific for 13 different subunits was used with coronal cryostat sections of the brains. Selective alterations were observed by ethanol exposure and/or aging in signals for several subunits. Most interestingly, the putative highly ethanol-sensitive alpha4 and beta3 subunit mRNAs were significantly decreased in several brain regions. The age-related alterations in alpha4 subunit expression were parallel to those caused by lifelong ethanol drinking, whereas aging had no significant effect on beta3 subunit expression. The results suggest that prolonged ethanol consumption leading to blood concentrations of about 10 mM may downregulate the mRNA expression of selected GABAA receptor subunits and that aging might have partly similar effects.

  8. Regulation of the Telomerase Reverse Transcriptase Subunit through Epigenetic Mechanisms

    PubMed Central

    Lewis, Kayla A.; Tollefsbol, Trygve O.

    2016-01-01

    Chromosome-shortening is characteristic of normal cells, and is known as the end replication problem. Telomerase is the enzyme responsible for extending the ends of the chromosomes in de novo synthesis, and occurs in germ cells as well as most malignant cancers. There are three subunits of telomerase: human telomerase RNA (hTERC), human telomerase associated protein (hTEP1), or dyskerin, and human telomerase reverse transcriptase (hTERT). hTERC and hTEP1 are constitutively expressed, so the enzymatic activity of telomerase is dependent on the transcription of hTERT. DNA methylation, histone methylation, and histone acetylation are basic epigenetic regulations involved in the expression of hTERT. Non-coding RNA can also serve as a form of epigenetic control of hTERT. This epigenetic-based regulation of hTERT is important in providing a mechanism for reversibility of hTERT control in various biological states. These include embryonic down-regulation of hTERT contributing to aging and the upregulation of hTERT playing a critical role in over 90% of cancers. Normal human somatic cells have a non-methylated/hypomethylated CpG island within the hTERT promoter region, while telomerase-positive cells paradoxically have at least a partially methylated promoter region that is opposite to the normal roles of DNA methylation. Histone acetylation of H3K9 within the promoter region is associated with an open chromatin state such that transcription machinery has the space to form. Histone methylation of hTERT has varied control of the gene, however. Mono- and dimethylation of H3K9 within the promoter region indicate silent euchromatin, while a trimethylated H3K9 enhances gene transcription. Non-coding RNAs can target epigenetic-modifying enzymes, as well as transcription factors involved in the control of hTERT. An epigenetics diet that can affect the epigenome of cancer cells is a recent fascination that has received much attention. By combining portions of this diet with

  9. Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes.

    PubMed Central

    Muntz, K H; Sternweis, P C; Gilman, A G; Mumby, S M

    1992-01-01

    Two approaches were taken to address the possible role of gamma-subunit prenylation in dictating the cellular distribution of guanine nucleotide-binding regulatory proteins. Prenylation of gamma subunits was prevented by site-directed mutagenesis or by inhibiting the synthesis of mevalonate, the precursor of cellular isoprenoids. When beta or gamma subunits were transiently expressed in COS-M6 simian kidney cells (COS) cells, the proteins were found in the membrane fraction by immunoblotting. Immunofluorescence experiments indicated that the proteins were distributed to intracellular structures in addition to plasma membranes. Replacement of Cys68 of gamma with Ser prevented prenylation of the mutant protein and association of the protein with the membrane fraction of COS cells. Immunoblotting results demonstrated that some of the beta subunits were found in the cytoplasm when coexpressed with the nonprenylated mutant gamma subunit. When Neuro 2A cells were treated with compactin to inhibit protein prenylation, a fraction of endogenous beta and gamma was distributed in the cytoplasm. It is concluded that prenylation facilitates association of gamma subunits with membranes, that the cellular location of gamma influences the distribution of beta, and that prenylation is not an absolute requirement for interaction of beta and gamma. Images PMID:1550955

  10. Expression, purification, and characterization of a biologically active bovine enterokinase catalytic subunit in Escherichia coli.

    PubMed

    Yuan, Liu-Di; Hua, Zi-Chun

    2002-07-01

    Enterokinase (EC 3.4.21.9) is a serine proteinase in the duodenum that exhibits specificity for the sequence (Asp)(4)-Lys. It converts trypsinogen to trypsin. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for in vitro cleavage of fusion proteins. cDNA encoding the catalytic chain of Chinese bovine enterokinase was cloned and its encoding amino acid sequence is identical to the previously reported sequence although there are two one-base mutations which do not change the encoded amino acid. The EK catalytic subunit cDNA was cloned into plasmid pET32a, and fused downstream to the fusion partner thioredoxin (Trx) and the following DDDDK enterokinase recognition sequence. The recombinant bovine enterokinase catalytic subunit was expressed in Escherichia coli BL21(DE3), and most products existed in soluble form. After an in vivo autocatalytic cleavage of the recombinant Trx-EK catalytic domain fusion protein, intact, biologically active EK catalytic subunit was released from the fusion protein. The recombinant intact EK catalytic subunit was purified to homogeneity with a specific activity of 720 AUs/mg protein through ammonium sulfate precipitation, DEAE chromatography, and gel filtration. The purified intact EK catalytic subunit has a K(m) of 0.17 mM, and K(cat) is 20.8s(-1). From 100 ml flask culture, 4.3 mg pure active EK catalytic subunits were obtained.

  11. Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 acetyltransferase complex.

    PubMed

    Setiaputra, Dheva; Ross, James D; Lu, Shan; Cheng, Derrick T; Dong, Meng-Qiu; Yip, Calvin K

    2015-04-17

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility.

  12. Evidence for an unusual transmembrane configuration of AGG3, a Class C Gγ Subunit, of Arabidopsis

    PubMed Central

    Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan; Urano, Daisuke; Trusov, Yuri; McCurdy, David W.; Assmann, Sarah M.; Jones, Alan M.; Botella, Jose R.

    2015-01-01

    SUMMARY Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex is comprised of one Gα, one Gβ and one Gγ subunit. However, in addition to the canonical Gγ subunits (Class A), plants also possess two unusual, plant-specific classes of Gγ subunits (Classes B and C) not yet found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (Class B) which is important for membrane anchoring of the protein, and thus give rise to a flexible subpopulation of Gβ/γ heterodimers that is not necessarily restricted to the plasma membrane. Even more interesting, plants also contain Class C Gγ subunits which are twice the size of canonical Gγs, with a predicted transmembrane domain, and a large cysteine-rich, extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology has been unequivocally demonstrated. Here, we provide compelling evidence that AGG3, a Class C Ggamma subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular. PMID:25430066

  13. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

    PubMed Central

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

    2014-01-01

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

  14. Two classes of regulatory subunits coassemble in the same BK channel and independently regulate gating

    NASA Astrophysics Data System (ADS)

    Gonzalez-Perez, Vivian; Xia, Xiao-Ming; Lingle, Christopher J.

    2015-09-01

    High resolution proteomics increasingly reveals that most native ion channels are assembled in macromolecular complexes. However, whether different partners have additive or cooperative functional effects, or whether some combinations of proteins may preclude assembly of others are largely unexplored topics. The large conductance Ca2+-and-voltage activated potassium channel (BK) is well-suited to discern nuanced differences in regulation arising from combinations of subunits. Here we examine whether assembly of two different classes of regulatory proteins, β and γ, in BK channels is exclusive or independent. Our results show that both γ1 and up to four β2-subunits can coexist in the same functional BK complex, with the gating shift caused by β2-subunits largely additive with that produced by the γ1-subunit(s). The multiplicity of β:γ combinations that can participate in a BK complex therefore allow a range of BK channels with distinct functional properties tuned by the specific stoichiometry of the contributing subunits.

  15. Ricin and Ricinus communis agglutinin subunits are all derived from a single-size polypeptide precursor.

    PubMed

    Butterworth, A G; Lord, J M

    1983-12-01

    Antibodies have been raised in rabbits against the individually purified A and B subunits of the toxic castor bean lectin, ricin, and against the A' and B' subunits of Ricinus communis agglutinin type I. Each of the antisera recognised a single polypeptide species of Mr 60 500 when maturing castor bean endosperm mRNA was translated in vitro in a rabbit-reticulocyte-derived system. When dog pancreatic microsomal vesicles were included in the translational system, each subunit antiserum precipitated a group of 66 000-68 000-Mr core-glycosylated polypeptides which had been translocated into the lumen of the vesicles. The 60 500-Mr polypeptide appeared to be a common precursor to all four individual lectin subunits since (a) its glycosylated (66 000-68 000-Mr) forms were readily detected in the endoplasmic reticulum fraction isolated from maturing castor bean endosperm and (b) pulse-chase studies showed that the glycosylated precursors disappeared from the endoplasmic reticulum fraction with the concomittant appearance of authentic lectin subunits in a soluble protein fraction which included protein body matrix components. Antiserum prepared against whole R. communis agglutinin, type I, also precipitated the 65 000-Mr precursor in vitro and in vivo, but in addition precipitated a non-glycosylated 34 000-Mr polypeptide. This smaller protein is not a lectin subunit precursor, contradicting an earlier suggestion. It is most probably a precursor to the 2-S albumin storage proteins found in castor bean endosperm protein bodies.

  16. The TCP1γ subunit of Leishmania donovani forms a biologically active homo-oligomeric complex.

    PubMed

    Bhaskar; Mitra, Kalyan; Kuldeep, Jitendra; Siddiqi, Mohammad Imran; Goyal, Neena

    2015-12-01

    Chaperonins are a class of molecular chaperons that encapsulate nascent or stress-denatured proteins and assist their intracellular assembly and folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP1 ring complex is a hetero-oligomeric complex comprising two rings, each formed of eight subunits that may have distinct substrate recognition and ATP hydrolysis properties. In Leishmania, only the TCP1γ subunit has been cloned and characterized. It exhibited differential expression at various growth stages of promastigotes. In the present study, we expressed the TCP1γ subunit in Escherichia coli to investigate whether it forms chaperonin-like complexes and plays a role in protein folding. LdTCP1γ formed high-molecular-weight complexes within E. coli cells as well as in Leishmania cell lysates. The recombinant protein is arranged into two back-to-back rings of seven subunits each, as predicted by homology modelling and observed by negative staining electron microscopy. This morphology is consistent with that of the oligomeric double-ring group I chaperonins found in mitochondria. The LdTCP1γ homo-oligomeric complex hydrolysed ATP, and was active as assayed by luciferase refolding. Thus, the homo-oligomer performs chaperonin reactions without partner subunit(s). Further, co-immunoprecipitation studies revealed that LdTCP1γ interacts with actin and tubulin proteins, suggesting that the complex may have a role in maintaining the structural dynamics of the cytoskeleton of parasites.

  17. Evaluation of human antibody responses to diphtheria toxin subunits A and B in various age groups.

    PubMed

    Karakus, R; Caglar, K; Aybay, C

    2007-11-01

    This study aimed to evaluate human antibody responses to diphtheria toxin subunits in various age groups. Antibodies against the intact diphtheria toxin and the diphtheria toxin subunits A and B were evaluated in 1319 individuals using a double-antigen ELISA. Although high levels of protection (83.6%, 95% CI 79.2-87.4) were found in children and adolescents, the middle-aged adult population was less protected (28.8%, 95% CI 24.3-33.6). An increase in age was associated with a decrease in the frequency of protected individuals in the 0-39-year age group (p <0.001). Anti-subunit B levels correlated well (p <0.01) with levels of antibodies against the intact toxin. In children aged < or =16 years, the intervals at which the peaks in geometric mean titres of anti-subunit B antibodies were observed were found to correlate with the ages at which booster doses are administered. Overall, males appeared to be more protected than females (OR 1.67, 95% CI 1.34-2.08, p <0.001). A small group of individuals had antibody levels of > or =0.1 IU/mL against the intact toxin, but did not have protective antibody against subunit B. Determination of anti-subunit B antibody levels should help in evaluating the effectiveness of diphtheria boosters and other aspects of diphtheria immunity.

  18. Energetic Contributions to Channel Gating of Residues in the Muscle Nicotinic Receptor β1 Subunit

    PubMed Central

    Akk, Gustav; Eaton, Megan; Li, Ping; Zheng, Steven; Lo, Joshua; Steinbach, Joe Henry

    2013-01-01

    In the pentameric ligand-gated ion channel family, transmitter binds in the extracellular domain and conformational changes result in channel opening in the transmembrane domain. In the muscle nicotinic receptor and other heteromeric members of the family one subunit does not contribute to the canonical agonist binding site for transmitter. A fundamental question is whether conformational changes occur in this subunit. We used records of single channel activity and rate-equilibrium free energy relationships to examine the β1 (non-ACh-binding) subunit of the muscle nicotinic receptor. Mutations to residues in the extracellular domain have minimal effects on the gating equilibrium constant. Positions in the channel lining (M2 transmembrane) domain contribute strongly and relatively late during gating. Positions thought to be important in other subunits in coupling the transmitter-binding to the channel domains have minimal effects on gating. We conclude that the conformational changes involved in channel gating propagate from the binding-site to the channel in the ACh-binding subunits and subsequently spread to the non-binding subunit. PMID:24194945

  19. A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits.

    PubMed

    Strunk, Bethany S; Novak, Megan N; Young, Crystal L; Karbstein, Katrin

    2012-07-06

    Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.

  20. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex.

    PubMed

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B; Webb, Kristofor; Bennett, Eric J; Vinterbo, Staal; Potter, Clinton S; Carragher, Bridget; Joazeiro, Claudio A P

    2014-11-11

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.

  1. Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids.

    PubMed

    Werner, David F; Porcu, Patrizia; Boyd, Kevin N; O'Buckley, Todd K; Carter, Jenna M; Kumar, Sandeep; Morrow, A Leslie

    2016-04-01

    GABAA receptors containing α4 subunits are widely implicated in acute ethanol sensitivity, and their spatial and temporal regulation prominently contributes to ethanol-induced neuroplasticity in hippocampus and cortex. However, it is unknown if α4-containing GABAA receptors in the thalamus, an area of high α4 expression, display similar regulatory patterns following ethanol administration, and if so, by which molecular mechanisms. In the current study, thalamic GABAA receptor α4 subunit levels were increased following a 6-week-, but not a 2-week chronic ethanol diet. Following acute high-dose ethanol administration, thalamic GABAA receptor α4 subunit levels were regulated in a temporal fashion, as a decrease was observed at 2h followed by a delayed transient increase. PKCγ and PKCδ levels paralleled α4 temporal expression patterns following ethanol exposure. Initial decreases in α4 subunit expression were associated with reduced serine phosphorylation. Delayed increases in expression were not associated with a change in phosphorylation state, but were prevented by inhibiting neuroactive steroid production with the 5α-reductase inhibitor finasteride. Overall, these studies indicate that thalamic GABAA receptor α4 subunit expression following acute and chronic ethanol administration exhibits similar regulatory patterns as other regions and that transient expression patterns following acute exposure in vivo are likely dependent on both subunit phosphorylation state and neuroactive steroids.

  2. Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis

    SciTech Connect

    Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan; Urano, Daisuke; Trusov, Yuri; Sheahan, Michael B.; McCurdy, David W.; Assmann, Sarah M.; Jones, Alan M.; Botella, Jose R.

    2014-12-22

    Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex is comprised of one Gα, one Gβ and one Gγ subunit. However, in addition to the canonical Gγ subunits (Class A), plants also possess two unusual, plant-specific classes of Gγ subunits (Classes B and C) not yet found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (Class B) which is important for membrane anchoring of the protein, and thus give rise to a flexible subpopulation of Gβ/γ heterodimers that is not necessarily restricted to the plasma membrane. Even more interesting, plants also contain Class C Gγ subunits which are twice the size of canonical Gγs, with a predicted transmembrane domain, and a large cysteine-rich, extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology has been unequivocally demonstrated. Finally, we provide compelling evidence that AGG3, a Class C Ggamma subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.

  3. Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis

    DOE PAGES

    Wolfenstetter, Susanne; Chakravorty, David; Kula, Ryan; ...

    2014-12-22

    Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex is comprised of one Gα, one Gβ and one Gγ subunit. However, in addition to the canonical Gγ subunits (Class A), plants also possess two unusual, plant-specific classes of Gγ subunits (Classes B and C) not yet found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (Class B) which is important for membrane anchoring of the protein, and thus give rise to a flexible subpopulation of Gβ/γ heterodimers that is notmore » necessarily restricted to the plasma membrane. Even more interesting, plants also contain Class C Gγ subunits which are twice the size of canonical Gγs, with a predicted transmembrane domain, and a large cysteine-rich, extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology has been unequivocally demonstrated. Finally, we provide compelling evidence that AGG3, a Class C Ggamma subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.« less

  4. Pleiotropic Effects of Loss of the Dα1 Subunit in Drosophila melanogaster: Implications for Insecticide Resistance.

    PubMed

    Somers, Jason; Luong, Hang Ngoc Bao; Mitchell, Judith; Batterham, Philip; Perry, Trent

    2017-01-01

    Nicotinic acetylcholine receptors (nAChRs) are a highly conserved gene family that form pentameric receptors involved in fast excitatory synaptic neurotransmission. The specific roles individual nAChR subunits perform in Drosophila melanogaster and other insects are relatively uncharacterized. Of the 10 D. melanogaster nAChR subunits, only three have described roles in behavioral pathways; Dα3 and Dα4 in sleep, and Dα7 in the escape response. Other subunits have been associated with resistance to several classes of insecticides. In particular, our previous work has demonstrated that an allele of the Dα1 subunit is associated with resistance to neonicotinoid insecticides. We used ends-out gene targeting to create a knockout of the Dα1 gene to facilitate phenotypic analysis in a controlled genetic background. To our knowledge, this is the first report of a native function for any nAChR subunits known to be targeted by insecticides. Loss of Dα1 function was associated with changes in courtship, sleep, longevity, and insecticide resistance. While acetylcholine signaling had previously been linked with mating behavior and reproduction in D. melanogaster, no specific nAChR subunit had been directly implicated. The role of Dα1 in a number of behavioral phenotypes highlights the importance of understanding the biological roles of nAChRs and points to the fitness cost that may be associated with neonicotinoid resistance.

  5. Archaeal Mo-Containing Glyceraldehyde Oxidoreductase Isozymes Exhibit Diverse Substrate Specificities through Unique Subunit Assemblies

    PubMed Central

    Miyake, Masayuki; Fushinobu, Shinya

    2016-01-01

    Archaea use glycolytic pathways distinct from those found in bacteria and eukaryotes, where unique enzymes catalyze each reaction step. In this study, we isolated three isozymes of glyceraldehyde oxidoreductase (GAOR1, GAOR2 and GAOR3) from the thermoacidophilic archaeon Sulfolobus tokodaii. GAOR1–3 belong to the xanthine oxidoreductase superfamily, and are composed of a molybdo-pyranopterin subunit (L), a flavin subunit (M), and an iron-sulfur subunit (S), forming an LMS hetero-trimer unit. We found that GAOR1 is a tetramer of the STK17810/STK17830/STK17820 hetero-trimer, GAOR2 is a dimer of the STK23390/STK05620/STK05610 hetero-trimer, and GAOR3 is the STK24840/STK05620/STK05610 hetero-trimer. GAOR1–3 exhibited diverse substrate specificities for their electron donors and acceptors, due to their different L-subunits, and probably participate in the non-phosphorylative Entner-Doudoroff glycolytic pathway. We determined the crystal structure of GAOR2, as the first three-dimensional structure of an archaeal molybdenum-containing hydroxylase, to obtain structural insights into their substrate specificities and subunit assemblies. The gene arrangement and the crystal structure suggested that the M/S-complex serves as a structural scaffold for the binding of the L-subunit, to construct the three enzymes with different specificities. Collectively, our findings illustrate a novel principle of a prokaryotic multicomponent isozyme system. PMID:26808202

  6. Immunohistochemical localization of inhibin/activin subunits in the wild ground squirrel (Citellus dauricus Brandt) ovary.

    PubMed

    Sheng, Xia; Weng, Jiaju; Zhang, Haolin; Li, Xiaonan; Zhang, Mengyuan; Xu, Meiyu; Weng, Qiang; Watanabe, Gen; Taya, Kazuyoshi

    2012-01-01

    The intraovarian function of gonadally produced inhibin and activin has been extensively studied in experimental models for decades, yet their presence and function have been rarely reported in wild rodents. With our seasonal breeding model, the wild ground squirrel, we aimed to investigate the possible roles of these peptides in the seasonal folliculogenesis. Immunohistochemical staining and Western blotting have been used to detect the cellular localization and expression patterns of inhibin/activin subunits (α, β(A) and β(B)). In the breeding season ovary, all three subunits were present in granulosa cells, theca cells of antral follicles and interstitial cells, with the strongest immunostaining in granulosa cells. Following ovulation, the corpora lutea become a major site of inhibin/activin synthesis. In the nonbreeding season ovary, inhibin/activin α and β(A) subunits were weakly immunopositive in granulosa cells of early stage follicles, while β(B) subunit was undetectable. The expression level of inhibin/activin subunit proteins were generally higher in the ovaries of the breeding season, and then decreased to a relatively low level during the nonbreeding season. The dynamic expression of inhibin/activin subunits indicated that they might play important paracrine and/or autocrine roles during the seasonal folliculogenesis of the wild ground squirrel.

  7. The 2.3 {angstrom} crystal structure of cholera toxin B subunit pentamer: Choleragenoid

    SciTech Connect

    Zhang, Rong-Guang; Westbrook, M.L.; Maulik, P.R.; Reed, R.A.; Shipley, G.; Westbrook, E.M. |; Scott, D.L.; Otwinowski, Z.

    1996-02-01

    Cholera toxin, a heterohexameric AB{sub 5} enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding to GM{sub 1} gangliosides exposed on the luminal surface of intestinal epithelial cells. We have solved the crystal structure of choleragenoid at 2.3 {Angstrom} resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin (choleragen), the heat-labile enterotoxin from E. coli, and for a choleragenoid-GM{sub 1} pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of the A subunit or the receptor pentasaccharide to choleragenoid has only a modest effect on the local stereochemistry and does not perceptibly alter the subunit interface.

  8. The subunit structure of potato tuber ADPglucose pyrophosphorylase. [Solanum tuberosum L

    SciTech Connect

    Okita, T.W.; Nakata, P.A.; Anderson, J.M. ); Sowokinos, J. ); Morell, M.; Preiss, J. )

    1990-06-01

    ADPglucose pyrophosphorylase has been extensively purified from potato (Solanum tuberosum L.) tuber tissue to study its structure. By employing a modified published procedure together with Mono Q chromatography, a near homogeneous enzyme preparation was obtained with substantial improvement in enzyme yield and specific activity. In single dimensional sodium dodecyl sulfate polyacrylamide gels, the enzyme migrated as a single polypeptide band with a mobility of about 50,000 daltons. Analysis by two-dimensional polyacrylamide gel electrophoresis, however, revealed the presence of two types of subunits which could be distinguished by their slight differences in net charge and molecular weight. The smaller potato tuber subunit was recognized by antiserum prepared against the smaller spinach leaf 51 kilodalton ADPglucose pyrophosphorylase subunit. In contrast, the anti-54 kilodalton raised against the spinach leaf subunit did not significantly react to the tuber enzyme subunits. The results are consistent with the hypothesis that the potato tuber ADPglucose pyrophosphorylase is not composed of a simple homotetramer as previously suggested, but is a product of two separate and distinct subunits as observed for the spinach leaf and maize enzymes.

  9. Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement.

    PubMed

    Setiaputra, Dheva T; Cheng, Derrick Th; Lu, Shan; Hansen, Jesse M; Dalwadi, Udit; Lam, Cindy Hy; To, Jeffrey L; Dong, Meng-Qiu; Yip, Calvin K

    2017-02-01

    Elongator is a ~850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique subunits (Elp1 to Elp6). Despite the wealth of structural data on the individual subunits, the overall architecture and subunit organization of the full Elongator and the molecular mechanisms of how it exerts its multiple activities remain unclear. Using single-particle electron microscopy (EM), we revealed that yeast Elongator adopts a bilobal architecture and an unexpected asymmetric subunit arrangement resulting from the hexameric Elp456 subassembly anchored to one of the two Elp123 lobes that form the structural scaffold. By integrating the EM data with available subunit crystal structures and restraints generated from cross-linking coupled to mass spectrometry, we constructed a multiscale molecular model that showed the two Elp3, the main catalytic subunit, are located in two distinct environments. This work provides the first structural insights into Elongator and a framework to understand the molecular basis of its multifunctionality.

  10. Wheat gluten: high molecular weight glutenin subunits--structure, genetics, and relation to dough elasticity.

    PubMed

    Anjum, Faqir Muhammad; Khan, Moazzam Rafiq; Din, Ahmad; Saeed, Muhammad; Pasha, Imran; Arshad, Muhammad Umair

    2007-04-01

    Gluten proteins, representing the major protein fraction of the starchy endosperm, are predominantly responsible for the unique position of wheat amongst cereals. These form a continuous proteinaceous matrix in the cells of the mature dry grain and form a continuous viscoelastic network during the mixing process of dough development. These viscoelastic properties underline the utilization of wheat to prepare bread and other wheat flour based foodstuffs. One group of gluten proteins is glutenin, which consists of high molecular weight (HMW) and low molecular weight (LMW) subunits. The HMW glutenin subunits (HMW-GS) are particularly important for determining dough elasticity. The common wheat possesses 3 to 5 HMW subunits encoded at the Glu-1 loci on the long arms of group 1 chromosomes (1A, 1B, and 1D). The presence of certain HMW subunits is positively correlated with good bread-making quality. Glutamine-rich repetitive sequences that comprise the central part of the HMW subunits are actually responsible for the elastic properties due to extensive arrays of interchain hydrogen bonds. Genetic engineering can be used to manipulate the amount and composition of the HMW subunits, leading to either increased dough strength or more drastic changes in gluten structure and properties.

  11. Functional characterisation of the regulatory subunit of cyclic AMP-dependent protein kinase A homologue of Giardia lamblia: Differential expression of the regulatory and catalytic subunits during encystation.

    PubMed

    Gibson, Candace; Schanen, Brian; Chakrabarti, Debopam; Chakrabarti, Ratna

    2006-06-01

    To understand the functional roles of protein kinase A (PKA) during vegetative and differentiating states of Giardia parasites, we studied the structural and functional characteristics of the regulatory subunit of PKA (gPKAr) and its involvement in the giardial encystment process. Molecular cloning and characterisation showed that gPKAr contains two tandem 3'5'-cyclic adenosine monphosphate (cyclic AMP) binding domains at the C-terminal end and the interaction domain for the catalytic subunit. A number of consensus residues including in vivo phosphorylation site for PKAc and dimerisation/docking domain are present in gPKAr. The regulatory subunit physically interacts with the catalytic subunit and inhibits its kinase activity in the absence of cyclic AMP, which could be partially restored upon addition of cyclic AMP. Western blot analysis showed a marked reduction in the endogenous gPKAr concentration during differentiation of Giardia into cysts. An increased activity of gPKAc was also detected during encystation without any significant change in the protein concentration. Distinct localisations of gPKAc to the anterior flagella, basal bodies and caudal flagella as noted in trophozoites were absent in encysting cells at later stages. Instead, PKAc staining was punctate and located mostly to the cell periphery. Our study indicates possible enrichment of the active gPKAc during late stages of encystation, which may have implications in completion of the encystment process or priming of cysts for efficient excystation.

  12. A 102 kDa subunit of a Golgi-associated particle has homology to beta subunits of trimeric G proteins.

    PubMed Central

    Harrison-Lavoie, K J; Lewis, V A; Hynes, G M; Collison, K S; Nutland, E; Willison, K R

    1993-01-01

    We have identified a 102 kDa protein, p102, which is found on the cytoplasmic face of Golgi membranes, exocytic transport vesicles and in the cytosol. A monoclonal antibody that cross-reacts with p102 is able to immunoprecipitate a 500-600 kDa protein complex containing p102 and additional subunits. The composition of this p102-containing protein complex resembles that of the Golgi coatomer complex, which constitutes the coat of non-clathrin coated vesicles. One of the subunits of the p102 complex reacts with a monoclonal antibody that detects beta-COP, a subunit of the Golgi coatomer complex. Like beta-COP, p102 exists in a brefeldin A-sensitive association with Golgi membranes. The sequence of p102 contains an N-terminal domain composed of six repeats which are similar to those found in the beta subunit of trimeric G proteins and other regulatory proteins. We suggest that p102 may be involved in regulating membrane traffic in the constitutive exocytic pathway. Images PMID:8335000

  13. Allele-specific interactions between the yeast RFC1 and RFC5 genes suggest a basis for RFC subunit-subunit interactions.

    PubMed

    Beckwith, W; McAlear, M A

    2000-11-01

    Replication factor C (RFC) is an essential, multi-subunit ATPase that functions in DNA replication, DNA repair, and DNA metabolism-related checkpoints. In order to investigate how the individual RFC subunits contribute to these functions in vivo, we undertook a genetic analysis of RFC genes from budding yeast. We isolated and characterized mutations in the RFC5 gene that could suppress the cold-sensitive phenotype of rfc1-1 mutants. Analysis of the RFC5 suppressors revealed that they could not suppress the elongated telomere phenotype, the sensitivity to DNA damaging agents, or the mutator phenotype of rfc1-1 mutants. Unlike the checkpoint-defective rfc5-1 mutation, the RFC5 suppressor mutations did not interfere with the methylmethane sulfonate- or hydroxyurea-induced phosphorylation of Rad53p. The Rfc5p suppressor substitutions mapped to amino acid positions in the conserved RFC box motifs IV-VII. Comparisons of the structures of related RFC box-containing proteins suggest that these RFC motifs may function to coordinate interactions between neighboring subunits of multi-subunit ATPases.

  14. Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis.

    PubMed

    Brunk, I; Pahner, I; Maier, U; Jenner, B; Veh, R W; Nürnberg, B; Ahnert-Hilger, G

    1999-05-01

    Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of

  15. Relative activities and stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.

    PubMed Central

    Lim, W K; Shin, H J; Milton, D L; Hardman, J K

    1991-01-01

    In vitro mutagenesis of the Escherichia coli trpA gene has yielded 66 mutant tryptophan synthase alpha subunits containing single amino acid substitutions at 49 different residue sites and 29 double and triple amino acid substitutions at 16 additional sites, all within the first 121 residues of the protein. The 66 singly altered mutant alpha subunits encoded from overexpression vectors have been examined for their ability to support growth in trpA mutant host strains and for their enzymatic and stability properties in crude extracts. With the exception of mutant alpha subunits altered at catalytic residue sites Glu-49 and Asp-60, all support growth; this includes those (48 of 66) that have no enzymatic defects and those (18 of 66) that do. The majority of the enzymatically defective mutant alpha subunits have decreased capacities for substrate (indole-3-glycerol phosphate) utilization, typical of the early trpA missense mutants isolated by in vivo selection methods. These defects vary in severity from complete loss of activity for mutant alpha subunits altered at residue positions 49 and 60 to those, altered elsewhere, that are partially (up to 40 to 50%) defective. The complete inactivation of the proteins altered at the two catalytic residue sites suggest that, as found via in vitro site-specific mutagenesis of the Salmonella typhimurium tryptophan synthetase alpha subunit, both residues probably also participate in a push-pull general acid-base catalysis of indole-3-glycerol phosphate breakdown for the E. coli enzyme as well. Other classes of mutant alpha subunits include some novel types that are defective in their functional interaction with the other tryptophan synthetase component, the beta 2 subunit. Also among the mutant alpha subunits, 19 were found altered at one or another of the 34 conserved residue sites in this portion of the alpha polypeptide sequence; surprisingly, 10 of these have wild-type enzymatic activity, and 16 of these can satisfy growth

  16. The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1

    PubMed Central

    Basu, Debaleena; Li, Xiao-Ping; Kahn, Jennifer N.; May, Kerrie L.; Kahn, Peter C.

    2015-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1. PMID:26483409

  17. The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1.

    PubMed

    Basu, Debaleena; Li, Xiao-Ping; Kahn, Jennifer N; May, Kerrie L; Kahn, Peter C; Tumer, Nilgun E

    2015-10-19

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infections can lead to life-threatening complications, including hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS), which is the most common cause of acute renal failure in children in the United States. Stx1 and Stx2 are AB5 toxins consisting of an enzymatically active A subunit associated with a pentamer of receptor binding B subunits. Epidemiological evidence suggests that Stx2-producing E. coli strains are more frequently associated with HUS than Stx1-producing strains. Several studies suggest that the B subunit plays a role in mediating toxicity. However, the role of the A subunits in the increased potency of Stx2 has not been fully investigated. Here, using purified A1 subunits, we show that Stx2A1 has a higher affinity for yeast and mammalian ribosomes than Stx1A1. Biacore analysis indicated that Stx2A1 has faster association and dissociation with ribosomes than Stx1A1. Analysis of ribosome depurination kinetics demonstrated that Stx2A1 depurinates yeast and mammalian ribosomes and an RNA stem-loop mimic of the sarcin/ricin loop (SRL) at a higher catalytic rate and is a more efficient enzyme than Stx1A1. Stx2A1 depurinated ribosomes at a higher level in vivo and was more cytotoxic than Stx1A1 in Saccharomyces cerevisiae. Stx2A1 depurinated ribosomes and inhibited translation at a significantly higher level than Stx1A1 in human cells. These results provide the first direct evidence that the higher affinity for ribosomes in combination with higher catalytic activity toward the SRL allows Stx2A1 to depurinate ribosomes, inhibit translation, and exhibit cytotoxicity at a significantly higher level than Stx1A1.

  18. [Preparation of phycocyanin subunits liposomes and the photodynamic experiment on cancer cells].

    PubMed

    Guo, Rui-yong; Huang, Bei; Zuo, Man-man; Wang, Yong-zhong; Hu, Ling

    2008-10-01

    Phycocyanin subunits liposomes (PCS-lip) were prepared and its cellular uptake and photodynamic therapy (PDT) effect on cancer cells were studied. In the experiment, film dispersion method was used to prepare phycocyanin subunits liposomes; particle size and distribution were detected by zetasizer and transmission electric microscope; the effects of liposome as carrier on cell uptake in vitro were evaluated in S180 by using fluorescence microscope; and photodynamic therapy effect was assessed with MTT method. As shown in the results, the particle size mainly ranged from 80 nm to 160 nm, and average encapsulation rate was 42.3%. In the concentration of 100 microg x mL(-1), transfection rate reached (18.5 +/- 0.8)% at 2 h, (23.1 +/- 0.9)% at 4 h, keeping a balance in 5-6 h, and its photodynamic therapy effect in vitro improved with the increasing of concentration of phycocyanin subunits liposomes. In the concentration of 200 micro x mL(-1) cell survival rate of BGC-823 and S180 reached (45 +/- 5.2)% and (36 +/- 5.5)%, respectively, and the cell survival rate differentiation between PCS-PDT group and PCS-lip-PDT group reached 7%-11% (P < 0.05). In this study film dispersion method could keep the biological activity of phycocyanin subunits very well. Phycocyanin subunits liposomes will transfect cells more quickly than phycocyanin subunits in the same concentration, and in the same conditions, phycocyanin subunits liposomes have the better PDT effect on cancer cells as they were incubated with cells for 4 h.

  19. Functional characterization of Kv channel beta-subunits from rat brain.

    PubMed Central

    Heinemann, S H; Rettig, J; Graack, H R; Pongs, O

    1996-01-01

    1. The potassium channel beta-subunit from rat brain, Kv beta 1.1, is known to induce inactivation of the delayed rectifier channel Kv1.1 and Kv1.4 delta 1-110. 2. Kv beta 1.1 was co-expressed in Xenopus oocytes with various other potassium channel alpha-subunits. Kv beta 1.1 induced inactivation in members of the Kv1 subfamily with the exception of Kv 1.6; no inactivation of Kv 2.1, Kv 3.4 delta 2-28 and Kv4.1 channels could be observed. 3. The second member of the beta-subunit subfamily, Kv beta 2, had a shorter N-terminal end, accelerated inactivation of the A-type channel Kv 1.4, but did not induce inactivation when co-expressed with delayed rectifiers of the Kv1 channel family. 4. To test whether this subunit co-assembles with Kv alpha-subunits, the N-terminal inactivating domains of Kv beta 1.1 and Kv beta 3 were spliced to the N-terminus of Kv beta 2. The chimaeric beta-subunits (beta 1/ beta 2 and beta 3/ beta 2) induced fast inactivation of several Kv1 channels, indicating that Kv beta 2 associates with these alpha-subunits. No inactivation was induced in Kv 1.3, Kv 1.6, Kv2.1 and Kv3.4 delta 2-28 channels. 5. Kv beta 2 caused a voltage shift in the activation threshold of Kv1.5 of about -10 mV, indicating a putative physiological role. Kv beta 2 had a smaller effect on Kv 1.1 channels. 6. Kv beta 2 accelerated the activation time course of Kv1.5 but had no marked effect on channel deactivation. PMID:8799886

  20. Functional characterization of Kv channel beta-subunits from rat brain.

    PubMed

    Heinemann, S H; Rettig, J; Graack, H R; Pongs, O

    1996-06-15

    1. The potassium channel beta-subunit from rat brain, Kv beta 1.1, is known to induce inactivation of the delayed rectifier channel Kv1.1 and Kv1.4 delta 1-110. 2. Kv beta 1.1 was co-expressed in Xenopus oocytes with various other potassium channel alpha-subunits. Kv beta 1.1 induced inactivation in members of the Kv1 subfamily with the exception of Kv 1.6; no inactivation of Kv 2.1, Kv 3.4 delta 2-28 and Kv4.1 channels could be observed. 3. The second member of the beta-subunit subfamily, Kv beta 2, had a shorter N-terminal end, accelerated inactivation of the A-type channel Kv 1.4, but did not induce inactivation when co-expressed with delayed rectifiers of the Kv1 channel family. 4. To test whether this subunit co-assembles with Kv alpha-subunits, the N-terminal inactivating domains of Kv beta 1.1 and Kv beta 3 were spliced to the N-terminus of Kv beta 2. The chimaeric beta-subunits (beta 1/ beta 2 and beta 3/ beta 2) induced fast inactivation of several Kv1 channels, indicating that Kv beta 2 associates with these alpha-subunits. No inactivation was induced in Kv 1.3, Kv 1.6, Kv2.1 and Kv3.4 delta 2-28 channels. 5. Kv beta 2 caused a voltage shift in the activation threshold of Kv1.5 of about -10 mV, indicating a putative physiological role. Kv beta 2 had a smaller effect on Kv 1.1 channels. 6. Kv beta 2 accelerated the activation time course of Kv1.5 but had no marked effect on channel deactivation.

  1. Coexpression of striatal dopamine receptor subtypes and excitatory amino acid subunits.

    PubMed

    Ariano, M A; Larson, E R; Noblett, K L; Sibley, D R; Levine, M S

    1997-08-01

    The striatal cellular coexpression patterns for the D(1A) and D2 dopamine (DA) receptor subtypes and the ionotropic excitatory amino acid (EAA) subunits of the N-methyl-D-aspartate (NMDA-R1) and the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) (GluR1 and GluR2/3) receptor subunits were examined morphologically. Their coincidence was assessed by visualization of mRNA transcripts, localization of encoded receptor proteins, and binding analysis using concurrently paired methods of fluorescence detection. The findings indicated that 1) mRNA transcripts for both receptor systems were detected in the medium-sized neuron population, and the distribution of receptor message closely reflected protein and binding patterns, with the exception of the GluR1 subunit; 2) both DA receptor mRNA transcripts were coexpressed with each ionotropic EAA receptor subunit examined and with each other, and NMDA and AMPA receptor subunits also showed coincident expression; 3) D(1A) DA receptor protein was detected in neurons which coexpressed EAA subunit proteins; and 4) GluR2/3 and NMDA-R1 subunit proteins were coexpressed in medium-sized neurons which also demonstrated D2 DA receptor binding sites. These findings suggest morphological receptor "promiscuity" since the coexpression patterns between DA and EAA receptors were found in all permutations. The results provide a spatial framework for physiological findings describing functional interactions between the two DA receptor types and between specific DA and EAA receptors in the striatum.

  2. Distribution of Kv3.3 potassium channel subunits in distinct neuronal populations of mouse brain.

    PubMed

    Chang, Su Ying; Zagha, Edward; Kwon, Elaine S; Ozaita, Andres; Bobik, Marketta; Martone, Maryann E; Ellisman, Mark H; Heintz, Nathaniel; Rudy, Bernardo

    2007-06-20

    Kv3.3 proteins are pore-forming subunits of voltage-dependent potassium channels, and mutations in the gene encoding for Kv3.3 have recently been linked to human disease, spinocerebellar ataxia 13, with cerebellar and extracerebellar symptoms. To understand better the functions of Kv3.3 subunits in brain, we developed highly specific antibodies to Kv3.3 and analyzed immunoreactivity throughout mouse brain. We found that Kv3.3 subunits are widely expressed, present in important forebrain structures but particularly prominent in brainstem and cerebellum. In forebrain and midbrain, Kv3.3 expression was often found colocalized with parvalbumin and other Kv3 subunits in inhibitory neurons. In brainstem, Kv3.3 was strongly expressed in auditory and other sensory nuclei. In cerebellar cortex, Kv3.3 expression was found in Purkinje and granule cells. Kv3.3 proteins were observed in axons, terminals, somas, and, unlike other Kv3 proteins, also in distal dendrites, although precise subcellular localization depended on cell type. For example, hippocampal dentate granule cells expressed Kv3.3 subunits specifically in their mossy fiber axons, whereas Purkinje cells of the cerebellar cortex strongly expressed Kv3.3 subunits in axons, somas, and proximal and distal, but not second- and third-order, dendrites. Expression in Purkinje cell dendrites was confirmed by immunoelectron microscopy. Kv3 channels have been demonstrated to rapidly repolarize action potentials and support high-frequency firing in various neuronal populations. In this study, we identified additional populations and subcellular compartments that are likely to sustain high-frequency firing because of the expression of Kv3.3 and other Kv3 subunits.

  3. Cloning and characterization of the ionotropic GABA receptor subunit ρ1 from pig (Sus scrofa).

    PubMed

    Reyes-Ruiz, Jorge Mauricio; Limon, Agenor; Miledi, Ricardo

    2014-01-13

    Since human and pig eyes have remarkably anatomical and physiological similitudes swine models have been broadly used for functional studies and therapeutic research. Recently, a GABAρ-mediated relaxation of retinal vascularity suggested that GABAρ signaling may be used to improve retinal blood flow in vascular-driven impaired vision, and a further molecular characterization of GABAρ receptors would be beneficial. However, none of the GABAρ type subunits from pigs has been yet cloned; Among the 19 subunits that compose the family of GABAA receptors, ρ1-3 subunits are capable of forming homomeric channels. These homomeric receptors are particularly interesting because their pharmacological and kinetic properties are notably different from receptors composed by other GABAA subunits. Here we report the cloning of the GABAρ1subunit from the pig and the functional expression of homomeric channels in Xenopus oocytes. The most notable difference found in the pig GABAρ1 receptor was the absence of a stretch of 17 amino acids near the amino terminus (R41-V58) conserved in the rat and the human. This sequence has a higher nucleotidic match with the transcript variant 2 of the human GABAρ1 subunit. Xenopus oocytes injected with cRNA from the receptor generated currents when exposed to GABA that shared all the characteristics of other GABAρ1 subunits in mammals, including its modulation by dopamine. This study will help to increase the knowledge of the genetics of the pig, further the understanding of this important neurotransmitter receptor family and will shed some light in the evolution of these genes among mammals.

  4. Influence of subunit composition on desensitization of neuronal acetylcholine receptors at low concentrations of nicotine.

    PubMed

    Fenster, C P; Rains, M F; Noerager, B; Quick, M W; Lester, R A

    1997-08-01

    The influence of alpha and beta subunits on the properties of nicotine-induced activation and desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes was examined. Receptors containing alpha4 subunits were more sensitive to activation by nicotine than alpha3-containing receptors. At low concentrations of nicotine, nAChRs containing beta2 subunits reached near-maximal desensitization more rapidly than beta4-containing receptors. The concentration of nicotine producing half-maximal desensitization was influenced by the particular alpha subunit expressed; similar to results for activation, alpha4-containing receptors were more sensitive to desensitizing levels of nicotine than alpha3-containing receptors. The alpha subunit also influenced the rate of recovery from desensitization; this rate was approximately inversely proportional to the apparent nicotine affinity for the desensitized state. The homomeric alpha7 receptor showed the lowest sensitivity to nicotine for both activation and desensitization; alpha7 nAChRs also demonstrated the fastest desensitization kinetics. These subunit-dependent properties remained in the presence of external calcium, although subtle, receptor subtype-specific effects on both the apparent affinities for activation and desensitization and the desensitization kinetics were noted. These data imply that the subunit composition of various nAChRs determines the degree to which receptors are desensitized and/or activated by tobacco-related levels of nicotine. The subtype-specific balance between receptor activation and desensitization should be considered important when the cellular and behavioral actions of nicotine are interpreted.

  5. FtsZ Filament Dynamics at Steady State: Subunit Exchange with and without Nucleotide Hydrolysis†

    PubMed Central

    Chen, Yaodong; Erickson, Harold P.

    2009-01-01

    We have measured three aspects of FtsZ filament dynamics at steady state: rates of GTP hydrolysis, subunit exchange between protofilaments, and disassembly induced by dilution or excess GDP. All three reactions were slowed with an increase in the potassium concentration from 100 to 500 mM, via replacement of potassium with rubidium, or with an increase in the magnesium concentration from 5 to 20 mM. Electron microscopy showed that the polymers assembled under the conditions of fastest assembly were predominantly short, one-stranded protofilaments, whereas under conditions of slower dynamics, the protofilaments tended to associate into long, thin bundles. We suggest that exchange of subunits between protofilaments at steady state involves two separate mechanisms: (1) fragmentation or dissociation of subunits from protofilament ends following GTP hydrolysis and (2) reversible association and dissociation of subunits from protofilament ends independent of hydrolysis. Exchange of nucleotides on these recycling subunits could give the appearance of exchange directly into the polymer. Several of our observations suggest that exchange of nucleotide can take place on these recycling subunits, but not directly into the FtsZ polymer. Annealing of protofilaments was demonstrated for the L68W mutant in EDTA buffer but not in Mg buffer, where rapid cycling of subunits may obscure the effect of annealing. We also reinvestigated the nucleotide composition of FtsZ polymers at steady state. We found that the GDP:GTP ratio was 50:50 for concentrations of GTP > 100 μM, significantly higher than the 20:80 ratio previously reported at 20 μM GTP. PMID:19527070

  6. Identification and characterization of high-molecular-weight glutenin subunits from Agropyron intermedium.

    PubMed

    Cao, Shuanghe; Li, Zhixin; Gong, Caiyan; Xu, Hong; Yang, Ran; Hao, Shanting; Wang, Xianping; Wang, Daowen; Zhang, Xiangqi

    2014-01-01

    High-molecular-weight glutenin subunit (HMW-GS) is a primary determinant of processing quality of wheat. Considerable progress has been made in understanding the structure, function and genetic regulation of HMW-GS in wheat and some of its related species, but less is known about their orthologs in Agropyron intermedium, a useful related species for wheat improvement. Here seven HMW-GSs in Ag. intermedium were identified using SDS-PAGE and Western blotting experiments. Subsequently, the seven genes (Glu-1Aix1 ∼ 4 and Glu-1Aiy1 ∼ 3) encoding the seven HMW-GSs were isolated using PCR technique with degenerate primers, and confirmed by bacterial expression and Western blotting. Sequence analysis indicated that the seven Ag. intermedium HMW-GSs shared high similarity in primary structure to those of wheat, but four of the seven subunits were unusually small compared to the representatives of HMW-GS from wheat and two of them possessed extra cysteine residues. The alignment and clustering analysis of deduced amino acid sequences revealed that 1Aix1 and 1Aiy1 subunits had special molecular structure, belonging to the hybrid type compounding between typical x- and y-type subunit. The xy-type subunit 1Aix1 is composed of the N-terminal of x-type and C-terminal of y-type, whereas yx-type subunit 1Aiy1 comprises the N-terminal of y-type and C-terminal of x-type. This result strongly supported the hypothesis of unequal crossover mechanism that might generate the novel coding sequence for the hybrid type of HMW-GSs. In addition to the aforementioned, the other novel characteristics of the seven subunits were also discussed. Finally, phylogenetic analysis based on HMW-GS genes was carried out and provided new insights into the evolutionary biology of Ag. intermedium.

  7. Assembly of immunoglobulin M. Blocked thiol groups of intracellular 7S subunits

    PubMed Central

    Askonas, Brigitte A.; Parkhouse, R. M. E.

    1971-01-01

    We have shown previously that immunoglobulin M (IgM) is present within IgM-forming cells mainly in its 7S subunit form (IgMs), whereas only fully assembled IgM pentamers are secreted. There is no spontaneous polymerization of intracellular IgMs in cell lysates, suggesting that the 7S subunits had blocked cysteine residues. This suggestion was explored and confirmed in the present paper. Radioactive IgM (secreted) and IgMs (intracellular) were prepared by sucrose-density-gradient centrifugation after incubation of cells of the IgM-producing mouse myeloma MOPC 104E with [3H]leucine. We investigated the susceptibility to reduction of fully assembled mouse IgM and its reconstitution from subunits by analysis by polyacrylamide-gel electrophoresis under dissociating conditions. With increasing concentrations of dithioerythritol, interchain disulphide bonds were cleaved in the following order: inter-IgMs subunit, intra-IgMs subunit H-H, intra-IgMs subunit H-L. Removal of the reducing agent from IgM-reduction mixtures by filtration through Sephadex G-25 caused partial reconstitution of IgM at low protein concentrations (5–100μg/ml) and total reconstitution at higher protein concentrations (300μg/ml or more). Isolated radioactive intracellular IgMs showed no tendency to polymerize unless first treated with a reducing agent; under optimum conditions removal of the reducing agent caused 70% of the subunits to be assembled into IgM. Similar assembly occurred when IgMs was isolated from cells that had been lysed in the presence of an irreversible alkylating reagent (iodoacetamide). The intracellular IgMs cysteine residues responsible for inter-IgMs linkage therefore appear to be reversibly blocked within the cells. Assembly into IgM is thus controlled by removal of this block during secretion. ImagesFig. 1. PMID:5126912

  8. Relationship between auxiliary gamma subunits and mallotoxin on BK channel modulation

    PubMed Central

    Guan, Xin; Li, Qin; Yan, Jiusheng

    2017-01-01

    The large-conductance, calcium- and voltage-activated K+(BK) channel consists of the pore-forming α subunits (BKα) and auxiliary subunits. The auxiliary γ1-3 subunits potently modulate the BK channel by shifting its voltage-dependence of channel activation toward the hyperpolarizing direction by approximately 145 mV (γ1), 100 mV (γ2), and 50 mV (γ3). Mallotoxin is a potent small-molecule BK channel activator. We analyzed the relationship between mallotoxin and the γ subunits in their BK channel-activating effects in membrane patches excised from HEK-293 cells. We found that mallotoxin, when applied extracellularly, shifted the half-activation voltage (V1/2) of BKα channels by −72 mV. The channel-activating effect of mallotoxin was greatly attenuated in the presence of the γ1, γ2, or γ3 subunit, with resultant ΔV1/2 (+/− mallotoxin) values of −9, −28, or −15 mV, respectively. Most examined γ1 mutant subunits antagonized mallotoxin’s channel-activating effect in a manner that was largely dependent on its own modulatory function. However, mallotoxin caused an irreversible functional and structural disengagement of the γ1-F273S mutant from BK channels. We infer that the auxiliary γ subunit effectively interferes with mallotoxin on BK channel modulation via either a direct steric competition or an indirect allosteric influence on mallotoxin’s binding and action on BKα. PMID:28165042

  9. Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

    PubMed Central

    Matsuda, K; Buckingham, S D; Freeman, J C; Squire, M D; Baylis, H A; Sattelle, D B

    1998-01-01

    Imidacloprid is a new insecticide with selective toxicity for insects over