Science.gov

Sample records for protein mutant r234w

  1. Structure of mutant human oncogene protein determined

    SciTech Connect

    Baum, R.

    1989-01-16

    The protein encoded by a mutant human oncogene differs only slightly in structure from the native protein that initiates normal cell division, a finding that may complicate efforts to develop inhibitors of the mutant protein. Previously, the x-ray structure of the protein encoded by the normal c-Ha-ras gene, a protein believed to signal cells to start or stop dividing through its interaction with guanosine triphosphate (GTP), was reported. The structure of the protein encoded by a transforming c-Ha-ras oncogene, in which a valine codon replaces the normal glycine codon at position 12 in the gene, has now been determined. The differences in the structures of the mutant and normal proteins are located primarily in a loop that interacts with the /beta/-phosphate of a bound guanosine diphosphate (GDP) molecule.

  2. Regulation of Mutant p53 Protein Expression

    PubMed Central

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation. PMID:26734569

  3. Proteasome inhibitors suppress the protein expression of mutant p53.

    PubMed

    Halasi, Marianna; Pandit, Bulbul; Gartel, Andrei L

    2014-01-01

    Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53.

  4. Proteasome inhibitors suppress the protein expression of mutant p53

    PubMed Central

    Halasi, Marianna; Pandit, Bulbul; Gartel, Andrei L

    2014-01-01

    Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53. PMID:25485499

  5. Construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins.

    PubMed Central

    Peng, D; Koetzner, C A; McMahon, T; Zhu, Y; Masters, P S

    1995-01-01

    Targeted RNA recombination was used to construct mouse hepatitis virus (MHV) mutants containing chimeric nucleocapsid (N) protein genes in which segments of the bovine coronavirus N gene were substituted in place of their corresponding MHV sequences. This defined portions of the two N proteins that, despite evolutionary divergence, have remained functionally equivalent. These regions included most of the centrally located RNA-binding domain and two putative spacers that link the three domains of the N protein. By contrast, the amino terminus of N, the acidic carboxy-terminal domain, and a serine- and arginine-rich segment of the central domain could not be transferred from bovine coronavirus to MHV, presumably because these parts of the molecule participate in protein-protein interactions that are specific for each virus (or, possibly, each host). Our results demonstrate that targeted recombination can be used to make extensive substitutions in the coronavirus genome and can generate recombinants that could not otherwise be made between two viruses separated by a species barrier. The implications of these findings for N protein structure and function as well as for coronavirus RNA recombination are discussed. PMID:7636993

  6. Tn5 insertion mutants of Pseudomonas aeruginosa deficient in surface expression of ferripyochelin-binding protein

    SciTech Connect

    Sokol, P.A.

    1987-07-01

    Transposon (Tn5) insertion mutants were isolated in Pseudomonas aeruginosa PAO. These mutants were screened for expression of the ferripyochelin-binding protein with monoclonal antibody in a whole-cell immunoblot assay. Fourteen mutants were identified which did not express ferripyochelin-binding protein on the cell surface. These mutants did not take up /sup 59/Fe-labeled pyochelin and grew slowly in the presence of iron chelators.

  7. scyllo-Inositol promotes robust mutant Huntingtin protein degradation.

    PubMed

    Lai, Aaron Y; Lan, Cynthia P; Hasan, Salwa; Brown, Mary E; McLaurin, Joanne

    2014-02-01

    Huntington disease is characterized by neuronal aggregates and inclusions containing polyglutamine-expanded huntingtin protein and peptide fragments (polyQ-Htt). We have used an established cell-based assay employing a PC12 cell line overexpressing truncated exon 1 of Htt with a 103-residue polyQ expansion that yields polyQ-Htt aggregates to investigate the fate of polyQ-Htt-drug complexes. scyllo-Inositol is an endogenous inositol stereoisomer known to inhibit accumulation and toxicity of the amyloid-β peptide and α-synuclein. In light of these properties, we investigated the effect of scyllo-inositol on polyQ-Htt accumulation. We show that scyllo-inositol lowered the number of visible polyQ-Htt aggregates and robustly decreased polyQ-Htt protein abundance without concomitant cellular toxicity. We found that scyllo-inositol-induced polyQ-Htt reduction was by rescue of degradation pathways mediated by the lysosome and by the proteasome but not autophagosomes. The rescue of degradation pathways was not a direct result of scyllo-inositol on the lysosome or proteasome but due to scyllo-inositol-induced reduction in mutant polyQ-Htt protein levels.

  8. Subcellular distribution of mutant movement proteins of Cucumber mosaic virus fused to green fluorescent proteins.

    PubMed

    Canto, Tomas; Palukaitis, Peter

    2005-04-01

    The subcellular distribution of the movement proteins (MPs) of nine alanine-scanning mutants of Cucumber mosaic virus (CMV), fused to the green fluorescent protein (GFP) and expressed from CMV, was determined by confocal microscopy of infected epidermal cells of Nicotiana tabacum and Nicotiana benthamiana, as well as infected N. benthamiana protoplasts. Only those mutant MPs that were functional for movement in all host species tested localized to plasmodesmata of infected epidermal cells and to tubules extending from the surface of infected protoplasts, as for wild-type CMV 3a MP. Various mutant MPs that were either conditionally functional for movement or dysfunctional for movement did not localize to plasmodesmata and did not form tubules on the surface of infected protoplasts. Rather, they showed distribution to different extents throughout the infected cells, including the cytoplasm, nucleus or the plasma membrane. The CMV 3a MP also did not associate with microtubules.

  9. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    SciTech Connect

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; Rathore, Rajendra; Ramchandran, Ramani

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

  10. Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins

    DOE PAGES

    Nayak, Jaladhi; Gastonguay, Adam J.; Talipov, Marat R.; Vakeel, Padmanabhan; Span, Elise A.; Kalous, Kelsey S.; Kutty, Raman G.; Jensen, Davin R.; Pokkuluri, Phani Raj; Sem, Daniel S.; et al

    2014-12-18

    Background: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. Results: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function.more » We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. Conclusion: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.« less

  11. Oxysterol-binding protein ORP3 rescues the Amyotrophic Lateral Sclerosis-linked mutant VAPB phenotype.

    PubMed

    Darbyson, Angie; Ngsee, Johnny K

    2016-02-01

    A mutation in VAPB causes a familial form of Amyotrophic Lateral Sclerosis. The mutant protein (VAPB-P56S) is aggregate prone and blocks retrograde traffic from the endoplasmic reticulum (ER) Golgi intermediate compartment (ERGIC) including trafficking to the nuclear envelope (NE). Here we report a morphological screen where overexpression of oxysterol binding protein-related protein-3 (ORP3) rescued the mutant VAPB phenotype. It resolved the mutant VAPB-induced membrane expansions, restored solubility of the mutant protein in non-ionic detergent, and restored trafficking of Emerin to the NE. Knockdown of ORP3 or VAPB increased the intracellular level of phosphatidylinositol 4-phosphate (PtdIns4P). Decreasing PtdIns4P levels by inhibiting its synthesis reduced the severity of the mutant VAPB-induced membrane expansions and restored Emerin trafficking to the NE. Thus, VAPB and its interacting partners cooperatively regulate protein trafficking through the ERGIC by modulating PtdIns4P levels.

  12. Familial prion protein mutants inhibit Hrd1-mediated retrotranslocation of misfolded proteins by depleting misfolded protein sensor BiP.

    PubMed

    Peters, Sarah L; Déry, Marc-André; LeBlanc, Andrea C

    2016-03-01

    Similar to many proteins trafficking through the secretory pathway, cellular prion protein (PrP) partly retrotranslocates from the endoplasmic reticulum to the cytosol through the endoplasmic reticulum-associated degradation (ERAD) pathway in an attempt to alleviate accumulation of cellular misfolded PrP. Surprisingly, familial PrP mutants fail to retrotranslocate and simultaneously block normal cellular PrP retrotranslocation. That impairments in retrotranslocation of misfolded proteins could lead to global disruptions in cellular homeostasis prompted further investigations into PrP mutant retrotranslocation defects. A gain- and loss-of-function approach identified human E3 ubiquitin ligase, Hrd1, as a critical regulator of PrP retrotranslocation in mammalian cells. Expression of familial human PrP mutants, V210I(129V) and M232R(129V), not only abolished PrP retrotranslocation, but also that of Hrd1-dependent ERAD substrates, transthyretin TTR(D18G) and α1-anti-trypsin A1AT(NHK). Mutant PrP expression decreased binding immunoglobulin protein (BiP) levels by 50% and attenuated ER stress-induced BiP by increasing BiP turnover 6-fold. Overexpression of BiP with PrP mutants rescued retrotranslocation of PrP, TTR(D18G) and A1AT(NHK). PrP mutants-induced cell death was also rescued by co-expression of BiP. These results show that PrP mutants highjack the Hrd1-dependent ERAD pathway, an action that would result in misfolded protein accumulation especially in terminally differentiated neurons. This could explain the age-dependent neuronal degeneration in familial prion diseases. PMID:26740554

  13. Crystal structure of a mutant of archaeal ribosomal protein L1 from Methanococcus jannaschii

    NASA Astrophysics Data System (ADS)

    Sarskikh, A. V.; Gabdulkhakov, A. G.; Kostareva, O. S.; Shklyaeva, A. A.; Tishchenko, S. V.

    2014-05-01

    The crystal structure of a mutant of archaeal ribosomal protein L1 from Methanococcus jannaschii with the deletion of a nonconserved positively charged cluster consisting of eight C-terminal amino acid residues is determined by the molecular replacement method at 1.75 Å resolution. This mutant is shown to form more stable and ordered crystals belonging to a space group other than that of the wild-type protein crystals. The positively charged C-terminal region has only a slight effect on the interaction between protein L1 and RNA molecules. Hence, this mutant can be used to prepare protein-RNA complexes and obtain their crystals.

  14. The global translation profile in a ribosomal protein mutant resembles that of an eIF3 mutant

    PubMed Central

    2013-01-01

    Background Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mRNAs to examine the defects in translation in a poly(A) binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1. Results The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins. Detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mRNAs. First, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. Second, a group of mRNAs with incomplete coding sequences appeared to be uncoupled from the regulon, since their dependence on RPL24B differed from regular mRNAs. Third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild-type. Some sister paralogs also differed in their response to the rpl24b mutation. In contrast to rpl24b, the pab2 pab8 mutant revealed few gene specific translational defects, but a group of seed storage protein mRNAs were stimulated in their ribosome occupancy. In the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected 12 biological replicates of translation states from wild-type seedlings. We defined 20% of mRNAs as having a high variance in their translation state. Many of these mRNAs were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mRNAs. Conclusions These data

  15. Apolipoprotein A-I mutant proteins having cysteine substitutions and polynucleotides encoding same

    DOEpatents

    Oda, Michael N.; Forte, Trudy M.

    2007-05-29

    Functional Apolipoprotein A-I mutant proteins, having one or more cysteine substitutions and polynucleotides encoding same, can be used to modulate paraoxonase's arylesterase activity. These ApoA-I mutant proteins can be used as therapeutic agents to combat cardiovascular disease, atherosclerosis, acute phase response and other inflammatory related diseases. The invention also includes modifications and optimizations of the ApoA-I nucleotide sequence for purposes of increasing protein expression and optimization.

  16. Accelerated Disease Onset with Stabilized Familial Amyotrophic Lateral Sclerosis (ALS)-linked Mutant TDP-43 Proteins*

    PubMed Central

    Watanabe, Shoji; Kaneko, Kumi; Yamanaka, Koji

    2013-01-01

    Abnormal protein accumulation is a pathological hallmark of neurodegenerative diseases, including accumulation of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS). Dominant mutations in the TDP-43 gene are causative for familial ALS; however, the relationship between mutant protein biochemical phenotypes and disease course and their significance to disease pathomechanism are not known. Here, we found that longer half-lives of mutant proteins correlated with accelerated disease onset. Based on our findings, we established a cell model in which chronic stabilization of wild-type TDP-43 protein provoked cytotoxicity and recapitulated pathogenic protein cleavage and insolubility to the detergent Sarkosyl, TDP-43 properties that have been observed in sporadic ALS lesions. Furthermore, these cells showed proteasomal impairment and dysregulation of their own mRNA levels. These results suggest that chronically increased stability of mutant or wild-type TDP-43 proteins results in a gain of toxicity through abnormal proteostasis. PMID:23235148

  17. Proteomic profiling of maize opaque endosperm mutants reveals selective accumulation of lysine-enriched proteins

    PubMed Central

    Morton, Kyla J.; Jia, Shangang; Zhang, Chi; Holding, David R.

    2016-01-01

    Reduced prolamin (zein) accumulation and defective endoplasmic reticulum (ER) body formation occurs in maize opaque endosperm mutants opaque2 (o2), floury2 (fl2), defective endosperm*B30 (DeB30), and Mucronate (Mc), whereas other opaque mutants such as opaque1 (o1) and floury1 (fl1) are normal in these regards. This suggests that other factors contribute to kernel texture. A liquid chromatography approach coupled with tandem mass spectrometry (LC-MS/MS) proteomics was used to compare non-zein proteins of nearly isogenic opaque endosperm mutants. In total, 2762 proteins were identified that were enriched for biological processes such as protein transport and folding, amino acid biosynthesis, and proteolysis. Principal component analysis and pathway enrichment suggested that the mutants partitioned into three groups: (i) Mc, DeB30, fl2 and o2; (ii) o1; and (iii) fl1. Indicator species analysis revealed mutant-specific proteins, and highlighted ER secretory pathway components that were enriched in selected groups of mutants. The most significantly changed proteins were related to stress or defense and zein partitioning into the soluble fraction for Mc, DeB30, o1, and fl1 specifically. In silico dissection of the most significantly changed proteins revealed novel qualitative changes in lysine abundance contributing to the overall lysine increase and the nutritional rebalancing of the o2 and fl2 endosperm. PMID:26712829

  18. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    SciTech Connect

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M.; Wehnert, Manfred; Huebner, Stefan

    2009-08-15

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

  19. Mutant p53 proteins counteract autophagic mechanism sensitizing cancer cells to mTOR inhibition.

    PubMed

    Cordani, Marco; Oppici, Elisa; Dando, Ilaria; Butturini, Elena; Dalla Pozza, Elisa; Nadal-Serrano, Mercedes; Oliver, Jordi; Roca, Pilar; Mariotto, Sofia; Cellini, Barbara; Blandino, Giovanni; Palmieri, Marta; Di Agostino, Silvia; Donadelli, Massimo

    2016-08-01

    Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy.

  20. microRNAs: short non-coding bullets of gain of function mutant p53 proteins

    PubMed Central

    Donzelli, Sara; Strano, Sabrina; Blandino, Giovanni

    2014-01-01

    TP53 gene mutations are present in more than half of all human cancers. The resulting proteins are mostly full-length with a single aminoacid change and are abundantly present in cancer cells. Some of mutant p53 proteins gain oncogenic activities through which actively contribute to the aberrant cell proliferation, increased resistance to apoptotic stimuli and ability to metastatize of cancer cells. Gain of function mutant p53 proteins can transcriptionally regulate the expression of a large plethora of target genes. This mainly occurs through the formation of oncogenic transcriptional competent complexes that include mutant p53 protein, known transcription factors, posttranslational modifiers and scaffold proteins. Mutant p53 protein can also transcriptionally regulate the expression of microRNAs, small non-coding RNAs that regulate gene expression at the posttranscriptional level. Each microRNA can putatively target the expression of hundred mRNAs and consequently impact on many cellular functions. Thus, gain of function mutant p53 proteins can exert their oncogenic activities through the modulation of both non-coding and coding regions of human genome. PMID:25594041

  1. Stabilization of mutant BRCA1 protein confers PARP inhibitor and platinum resistance

    PubMed Central

    Johnson, Neil; Johnson, Shawn F.; Yao, Wei; Li, Yu-Chen; Choi, Young-Eun; Bernhardy, Andrea J.; Wang, Yifan; Capelletti, Marzia; Sarosiek, Kristopher A.; Moreau, Lisa A.; Chowdhury, Dipanjan; Wickramanayake, Anneka; Harrell, Maria I.; Liu, Joyce F.; D’Andrea, Alan D.; Miron, Alexander; Swisher, Elizabeth M.; Shapiro, Geoffrey I.

    2013-01-01

    Breast Cancer Type 1 Susceptibility Protein (BRCA1)-deficient cells have compromised DNA repair and are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Despite initial responses, the development of resistance limits clinical efficacy. Mutations in the BRCA C-terminal (BRCT) domain of BRCA1 frequently create protein products unable to fold that are subject to protease-mediated degradation. Here, we show HSP90-mediated stabilization of a BRCT domain mutant BRCA1 protein under PARP inhibitor selection pressure. The stabilized mutant BRCA1 protein interacted with PALB2-BRCA2-RAD51, was essential for RAD51 focus formation, and conferred PARP inhibitor as well as cisplatin resistance. Treatment of resistant cells with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibition. Resistant cells also acquired a TP53BP1 mutation that facilitated DNA end resection in the absence of a BRCA1 protein capable of binding CtIP. Finally, concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occur in clinical samples of BRCA1-mutated recurrent ovarian carcinomas that have developed resistance to platinum. These results provide evidence for a two-event mechanism by which BRCA1-mutant tumors acquire anticancer therapy resistance. PMID:24085845

  2. Reexamination of alcohol dehydrogenase structural mutants in Drosophila using protein blotting

    SciTech Connect

    Hollocher, H.; Place, A.R.

    1987-06-01

    Using protein blotting and an immuno-overlay procedure, the authors have reexamined the cross-reacting material produced by ADH null-activity mutants generated with ethyl methanesulfonate (EMS). Of the 13 mutants, 11 have an immunodetectable polypeptide of wild-type size. The native and urea denatured isoelectric points (pI) establish that 7 of 13 of the mutations have no effect on protein charge. The electrophoretic mobilities of each variant on increasing percent acrylamide gels (Ferguson analysis), reveal that 9 of the 11 immunodetectable mutations have retained the ability form dimers under native conditions. None of the inactive mutant proteins has the ability to form the adduct-bound isozyme. The authors have found no correlation between protein pI and i vivo stability. The observed frequencies of specific charge class alterations do not dispute the propensity of G:A transitions previously found for EMS mutagenesis.

  3. Expression of mutant bone morphogenetic protein receptor II worsens pulmonary hypertension secondary to pulmonary fibrosis.

    PubMed

    Bryant, Andrew J; Robinson, Linda J; Moore, Christy S; Blackwell, Thomas R; Gladson, Santhi; Penner, Niki L; Burman, Ankita; McClellan, Lucas J; Polosukhin, Vasiliy V; Tanjore, Harikrishna; McConaha, Melinda E; Gleaves, Linda A; Talati, Megha A; Hemnes, Anna R; Fessel, Joshua P; Lawson, William E; Blackwell, Timothy S; West, James D

    2015-12-01

    Pulmonary fibrosis is often complicated by pulmonary hypertension (PH), and previous studies have shown a potential link between bone morphogenetic protein receptor II (BMPR2) and PH secondary to pulmonary fibrosis. We exposed transgenic mice expressing mutant BMPR2 and control mice to repetitive intraperitoneal injections of bleomycin for 4 weeks. The duration of transgene activation was too short for mutant BMPR2 mice to develop spontaneous PH. Mutant BMPR2 mice had increased right ventricular systolic pressure compared to control mice, without differences in pulmonary fibrosis. We found increased hypoxia-inducible factor (HIF)1-α stabilization in lungs of mutant-BMPR2-expressing mice compared to controls following bleomycin treatment. In addition, expression of the hypoxia response element protein connective tissue growth factor was increased in transgenic mice as well as in a human pulmonary microvascular endothelial cell line expressing mutant BMPR2. In mouse pulmonary vascular endothelial cells, mutant BMPR2 expression resulted in increased HIF1-α and reactive oxygen species production following exposure to hypoxia, both of which were attenuated with the antioxidant TEMPOL. These data suggest that expression of mutant BMPR2 worsens secondary PH through increased HIF activity in vascular endothelium. This pathway could be therapeutically targeted in patients with PH secondary to pulmonary fibrosis.

  4. Expression of mutant bone morphogenetic protein receptor II worsens pulmonary hypertension secondary to pulmonary fibrosis

    PubMed Central

    Robinson, Linda J.; Moore, Christy S.; Blackwell, Thomas R.; Gladson, Santhi; Penner, Niki L.; Burman, Ankita; McClellan, Lucas J.; Polosukhin, Vasiliy V.; Tanjore, Harikrishna; McConaha, Melinda E.; Gleaves, Linda A.; Talati, Megha A.; Hemnes, Anna R.; Fessel, Joshua P.; Lawson, William E.; Blackwell, Timothy S.; West, James D.

    2015-01-01

    Abstract Pulmonary fibrosis is often complicated by pulmonary hypertension (PH), and previous studies have shown a potential link between bone morphogenetic protein receptor II (BMPR2) and PH secondary to pulmonary fibrosis. We exposed transgenic mice expressing mutant BMPR2 and control mice to repetitive intraperitoneal injections of bleomycin for 4 weeks. The duration of transgene activation was too short for mutant BMPR2 mice to develop spontaneous PH. Mutant BMPR2 mice had increased right ventricular systolic pressure compared to control mice, without differences in pulmonary fibrosis. We found increased hypoxia-inducible factor (HIF)1-α stabilization in lungs of mutant-BMPR2-expressing mice compared to controls following bleomycin treatment. In addition, expression of the hypoxia response element protein connective tissue growth factor was increased in transgenic mice as well as in a human pulmonary microvascular endothelial cell line expressing mutant BMPR2. In mouse pulmonary vascular endothelial cells, mutant BMPR2 expression resulted in increased HIF1-α and reactive oxygen species production following exposure to hypoxia, both of which were attenuated with the antioxidant TEMPOL. These data suggest that expression of mutant BMPR2 worsens secondary PH through increased HIF activity in vascular endothelium. This pathway could be therapeutically targeted in patients with PH secondary to pulmonary fibrosis. PMID:26697175

  5. Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.

    PubMed

    Loo, T W; Clarke, D M

    1997-01-10

    There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.

  6. Oxysterol-binding protein ORP3 rescues the Amyotrophic Lateral Sclerosis-linked mutant VAPB phenotype.

    PubMed

    Darbyson, Angie; Ngsee, Johnny K

    2016-02-01

    A mutation in VAPB causes a familial form of Amyotrophic Lateral Sclerosis. The mutant protein (VAPB-P56S) is aggregate prone and blocks retrograde traffic from the endoplasmic reticulum (ER) Golgi intermediate compartment (ERGIC) including trafficking to the nuclear envelope (NE). Here we report a morphological screen where overexpression of oxysterol binding protein-related protein-3 (ORP3) rescued the mutant VAPB phenotype. It resolved the mutant VAPB-induced membrane expansions, restored solubility of the mutant protein in non-ionic detergent, and restored trafficking of Emerin to the NE. Knockdown of ORP3 or VAPB increased the intracellular level of phosphatidylinositol 4-phosphate (PtdIns4P). Decreasing PtdIns4P levels by inhibiting its synthesis reduced the severity of the mutant VAPB-induced membrane expansions and restored Emerin trafficking to the NE. Thus, VAPB and its interacting partners cooperatively regulate protein trafficking through the ERGIC by modulating PtdIns4P levels. PMID:26812496

  7. Interaction of interleukin-2 (IL-2) mutant proteins with interleukin-2 receptors

    SciTech Connect

    Liang, S.M.; Lee, N.; Chollet, A.

    1987-05-01

    The authors have previously produced several human IL-2 mutant proteins by site specific mutagenesis. Deletion or substitution of alanine for cysteine at positions 58 and 105 results in the decrease of biological activities. Substitution of serine for cysteine at position 125 does not affect the activity, however, deletion of this cysteine or amino acids in its vicinity causes a dramatic loss of activity. In this study, the interaction of these mutant proteins with IL-2 receptors has been analyzed by evaluating the competition between these mutant proteins and recombinant DNA derived IL-2 (rIL-2) for the binding to murine CTLL-2, an IL-2 dependent cell line. Addition of unlabeled rIL-2 (1 x 10/sup -11/ to 10/sup -7/M) inhibited the binding of I/sup 125/-labeled rIL-2 (1 x 10/sup -10/M, specific activity 39.6 uCi/mg) to CTLL-2 cells in a concentration dependent manner. Mutant proteins with substitution of alanine for cysteine at position 58 (Ala 58) or deletion of cysteine at position 125 (Des-Cys 125) required a 100-fold higher concentration than rIL-2 to reach 50% inhibition. These results indicate that the decrease of biological activity in mutant proteins is partly, if not primarily, due to the attenuation in their abilities to bind IL-2 receptors.

  8. A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens

    PubMed Central

    Konar, Monica; Rossi, Raffaella; Walter, Helen; Pajon, Rolando; Beernink, Peter T.

    2015-01-01

    Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins. PMID:26057742

  9. Identification of trans-dominant HIV-1 rev protein mutants by direct transfer of bacterially produced proteins into human cells.

    PubMed Central

    Mermer, B; Felber, B K; Campbell, M; Pavlakis, G N

    1990-01-01

    A synthetic rev gene containing substitutions which introduced unique restriction sites but did not alter the deduced amino acid sequence was used as a vehicle to construct mutations in rev. Insertion or substitution mutations within a domain of Rev resulted in proteins able to inhibit the function of Rev protein in trans. Rev function was monitored in a cell line, HLfB, which contained a rev- mutant provirus. HLfB cells require the presence of rev for virus production, which was conveniently monitored by immunoblot detection of p24gag. Trans-dominant mutants were identified after expression in bacteria and delivery into HLfB cells by protoplast fusion. In addition, the trans-dominant phenotype was verified by expression of the mutant proteins in HLfB cells after cotransfection. These studies define a region between amino acid residues 81 and 88 of rev, in which different mutations result in proteins capable of inhibiting Rev function. Images PMID:2186373

  10. Systematic phenotypic screen of Arabidopsis peroxisomal mutants identifies proteins involved in β-oxidation.

    PubMed

    Cassin-Ross, Gaëlle; Hu, Jianping

    2014-11-01

    Peroxisomes are highly dynamic and multifunctional organelles essential to development. Plant peroxisomes accommodate a multitude of metabolic reactions, many of which are related to the β-oxidation of fatty acids or fatty acid-related metabolites. Recently, several dozens of novel peroxisomal proteins have been identified from Arabidopsis (Arabidopsis thaliana) through in silico and experimental proteomic analyses followed by in vivo protein targeting validations. To determine the functions of these proteins, we interrogated their transfer DNA insertion mutants with a series of physiological, cytological, and biochemical assays to reveal peroxisomal deficiencies. Sugar dependence and 2,4-dichlorophenoxybutyric acid and 12-oxo-phytodienoic acid response assays uncovered statistically significant phenotypes in β-oxidation-related processes in mutants for 20 of 27 genes tested. Additional investigations uncovered a subset of these mutants with abnormal seed germination, accumulation of oil bodies, and delayed degradation of long-chain fatty acids during early seedling development. Mutants for seven genes exhibited deficiencies in multiple assays, strongly suggesting the involvement of their gene products in peroxisomal β-oxidation and initial seedling growth. Proteins identified included isoforms of enzymes related to β-oxidation, such as acyl-CoA thioesterase2, acyl-activating enzyme isoform1, and acyl-activating enzyme isoform5, and proteins with functions previously unknown to be associated with β-oxidation, such as Indigoidine synthase A, Senescence-associated protein/B12D-related protein1, Betaine aldehyde dehydrogenase, and Unknown protein5. This multipronged phenotypic screen allowed us to reveal β-oxidation proteins that have not been discovered by single assay-based mutant screens and enabled the functional dissection of different isoforms of multigene families involved in β-oxidation. PMID:25253886

  11. Systematic phenotypic screen of Arabidopsis peroxisomal mutants identifies proteins involved in β-oxidation.

    PubMed

    Cassin-Ross, Gaëlle; Hu, Jianping

    2014-11-01

    Peroxisomes are highly dynamic and multifunctional organelles essential to development. Plant peroxisomes accommodate a multitude of metabolic reactions, many of which are related to the β-oxidation of fatty acids or fatty acid-related metabolites. Recently, several dozens of novel peroxisomal proteins have been identified from Arabidopsis (Arabidopsis thaliana) through in silico and experimental proteomic analyses followed by in vivo protein targeting validations. To determine the functions of these proteins, we interrogated their transfer DNA insertion mutants with a series of physiological, cytological, and biochemical assays to reveal peroxisomal deficiencies. Sugar dependence and 2,4-dichlorophenoxybutyric acid and 12-oxo-phytodienoic acid response assays uncovered statistically significant phenotypes in β-oxidation-related processes in mutants for 20 of 27 genes tested. Additional investigations uncovered a subset of these mutants with abnormal seed germination, accumulation of oil bodies, and delayed degradation of long-chain fatty acids during early seedling development. Mutants for seven genes exhibited deficiencies in multiple assays, strongly suggesting the involvement of their gene products in peroxisomal β-oxidation and initial seedling growth. Proteins identified included isoforms of enzymes related to β-oxidation, such as acyl-CoA thioesterase2, acyl-activating enzyme isoform1, and acyl-activating enzyme isoform5, and proteins with functions previously unknown to be associated with β-oxidation, such as Indigoidine synthase A, Senescence-associated protein/B12D-related protein1, Betaine aldehyde dehydrogenase, and Unknown protein5. This multipronged phenotypic screen allowed us to reveal β-oxidation proteins that have not been discovered by single assay-based mutant screens and enabled the functional dissection of different isoforms of multigene families involved in β-oxidation.

  12. Blood cellular mutant LXR-α protein stability governs initiation of coronary heart disease

    PubMed Central

    Arora, Mansi; Kaul, Deepak; Sharma, Yash Paul

    2013-01-01

    AIM: To investigate the role of [breast and ovarian cancer susceptibility 1 (BRCA1)-associated RING domain 1 (BARD1)]/BRCA1 E3-ubiquitin ligase complex in governing the stability of mutant liver X receptor-α (LXR-α) protein in coronary heart disease (CHD) subjects. METHODS: The expression analysis of various genes was carried out by quantitative real time polymerase chain reaction and western blotting within blood mononuclear cells of human CHD subjects at various stages of coronary occlusion and their corresponding normal healthy counterparts. Immunoprecipitation experiments were performed to establish protein interactions between LXR-α and BARD1. Peripheral blood mononuclear cells were cultured and exposed to Vitamin D3 and Cisplatin to validate the degradation of mutant LXR-α protein in CHD subjects by BARD1/BRCA1 complex. RESULTS: The expression of mutant LXR-α protein in CHD subjects was found to decrease gradually with the severity of coronary occlusion exhibiting a strong negative correlation, r = -0.975 at P < 0.001. Further, the expression of BARD1 and BRCA1 also increased with the disease severity, r = 0.895 and 0.873 respectively (P < 0.001). Immunoprecipitation studies established that BARD1/BRCA1 complex degrades mutant LXR-α via ubiquitination. The absence of functional LXR-α protein resulted in increased expression of inflammatory cytokines such as interleukin (IL)-6, IL-8 and interferon-γ and decreased expression of ABCA1 (ATP-binding cassette A1) (r = 0.932, 0.949, 0.918 and -0.902 with respect to Gensini score; P < 0.001). Additionally, cell culture experiments proved that Vitamin D3 could prevent the degradation of mutant LXR-α and restore its functional activity to some extent. CONCLUSION: Mutant LXR-α protein in CHD subjects is degraded by BARD1/BRCA1 complex and Vitamin D3 can rescue and restore its function. PMID:24009820

  13. Mutant p53 proteins counteract autophagic mechanism sensitizing cancer cells to mTOR inhibition.

    PubMed

    Cordani, Marco; Oppici, Elisa; Dando, Ilaria; Butturini, Elena; Dalla Pozza, Elisa; Nadal-Serrano, Mercedes; Oliver, Jordi; Roca, Pilar; Mariotto, Sofia; Cellini, Barbara; Blandino, Giovanni; Palmieri, Marta; Di Agostino, Silvia; Donadelli, Massimo

    2016-08-01

    Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy. PMID:27118659

  14. Plant resistance to fungal infection induced by nontoxic pokeweed antiviral protein mutants.

    PubMed

    Zoubenko, O; Uckun, F; Hur, Y; Chet, I; Tumer, N

    1997-10-01

    Pokeweed antiviral protein (PAP), a 29-kD protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. Transgenic plants expressing PAP are resistant to a broad spectrum of plant viruses. Nontoxic PAP mutants have been isolated by random mutagenesis and selection in yeast. One of these mutants, PAP-X, had a point mutation at the active-site (E176V) that abolished enzymatic activity, and another mutant, delta C25PAP, had a nonsense mutation near the C-terminus (W237stop) that deleted 25 C-terminal amino acids. Unlike the wild-type PAP, expression of neither mutant was toxic to transgenic plants. We show that both class I (basic) and class II (acidic) isoforms of pathogenesis-related (PR) proteins are overexpressed in transgenic plants expressing PAP and the nontoxic PAP mutants. Although PR-proteins are constitutively expressed, no increase in salicylic acid levels was detected. Homozygous progeny of transgenic plants expressing either PAP or the nontoxic PAP mutants displayed resistance to the fungal pathogen Rhizoctonia solani. These results show that expression of PAP or the nontoxic PAP mutants activates multiple plant defense pathways independently of salicylic acid and confers resistance to fungal infection. The C-terminal 25 amino acids of PAP, which are required for toxicity in vivo, are not critical for resistance to viral or fungal infection, indicating that toxicity of PAP can be separated from pathogen resistance.

  15. The Activating Transcription Factor 3 Protein Suppresses the Oncogenic Function of Mutant p53 Proteins*

    PubMed Central

    Wei, Saisai; Wang, Hongbo; Lu, Chunwan; Malmut, Sarah; Zhang, Jianqiao; Ren, Shumei; Yu, Guohua; Wang, Wei; Tang, Dale D.; Yan, Chunhong

    2014-01-01

    Mutant p53 proteins (mutp53) often acquire oncogenic activities, conferring drug resistance and/or promoting cancer cell migration and invasion. Although it has been well established that such a gain of function is mainly achieved through interaction with transcriptional regulators, thereby modulating cancer-associated gene expression, how the mutp53 function is regulated remains elusive. Here we report that activating transcription factor 3 (ATF3) bound common mutp53 (e.g. R175H and R273H) and, subsequently, suppressed their oncogenic activities. ATF3 repressed mutp53-induced NFKB2 expression and sensitized R175H-expressing cancer cells to cisplatin and etoposide treatments. Moreover, ATF3 appeared to suppress R175H- and R273H-mediated cancer cell migration and invasion as a consequence of preventing the transcription factor p63 from inactivation by mutp53. Accordingly, ATF3 promoted the expression of the metastasis suppressor SHARP1 in mutp53-expressing cells. An ATF3 mutant devoid of the mutp53-binding domain failed to disrupt the mutp53-p63 binding and, thus, lost the activity to suppress mutp53-mediated migration, suggesting that ATF3 binds to mutp53 to suppress its oncogenic function. In line with these results, we found that down-regulation of ATF3 expression correlated with lymph node metastasis in TP53-mutated human lung cancer. We conclude that ATF3 can suppress mutp53 oncogenic function, thereby contributing to tumor suppression in TP53-mutated cancer. PMID:24554706

  16. Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins.

    PubMed

    Larsen, Sasha; Weaver, Jun; de Sa Campos, Katherine; Bulahan, Rhobe; Nguyen, Jackson; Grove, Heather; Huang, Amy; Low, Lauren; Tran, Namphuong; Gomez, Seth; Yau, Jennifer; Ilustrisimo, Thomas; Kawilarang, Jessica; Lau, Jonathan; Tranphung, Maivi; Chen, Irene; Tran, Christina; Fox, Marcia; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2013-11-01

    Although Pichia pastoris is a popular protein expression system, it exhibits limitations in its ability to secrete heterologous proteins. Therefore, a REMI (restriction enzyme mediated insertion) strategy was utilized to select mutant beta-g alactosidase s upersecretion (bgs) strains that secreted increased levels of a β-galactosidase reporter. Many of the twelve BGS genes may have functions in intracellular signaling or vesicle transport. Several of these strains also appeared to contain a more permeable cell wall. Preliminary characterization of four bgs mutants showed that they differed in the ability to enhance the export of other reporter proteins. bgs13, which has a disruption in a gene homologous to Saccharomyces cerevisiae protein kinase C (PKC1), gave enhanced secretion of most recombinant proteins that were tested, raising the possibility that it has the universal super-secreter phenotype needed in an industrial production strain of P. pastoris. PMID:23881328

  17. Understanding protein lids: kinetic analysis of active hinge mutants in triosephosphate isomerase.

    PubMed

    Sun, J; Sampson, N S

    1999-08-31

    In previous work we tested what three amino acid sequences could serve as a protein hinge in triosephosphate isomerase [Sun, J., and Sampson, N. S. (1998) Protein Sci. 7, 1495-1505]. We generated a genetic library encoding all 8000 possible 3 amino acid combinations at the C-terminal hinge and selected for those combinations of amino acids that formed active mutants. These mutants were classified into six phylogenetic families. Two families resembled wild-type hinges, and four families represented new types of hinges. In this work, the kinetic characteristics and thermal stabilities of mutants representing each of these families were determined in order to understand what properties make an efficient protein hinge, and why all of the families are not observed in nature. From a steady-state kinetic analysis of our mutants, it is clear that the partitioning between protonation of intermediate to form product and intermediate release from the enzyme surface to form methylglyoxal (a decomposition product) is not affected. The two most impaired mutants undergo a change in rate-limiting step from enediol formation to dihydroxyacetone phosphate binding. Thus, it appears that k(cat)/K(m)'s are reduced relative to wild type as a result of slower Michaelis complex formation and dissociation, rather than increased loop opening speed.

  18. Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

    SciTech Connect

    Takanashi, Keisuke; Yamaguchi, Atsushi

    2014-09-26

    Highlights: • Aggregation of ALS-linked FUS mutant sequesters ALS-associated RNA-binding proteins (FUS wt, hnRNP A1, and hnRNP A2). • Aggregation of ALS-linked FUS mutant sequesters SMN1 in the detergent-insoluble fraction. • Aggregation of ALS-linked FUS mutant reduced the number of speckles in the nucleus. • Overproduced ALS-linked FUS mutant reduced the number of processing-bodies (PBs). - Abstract: Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.

  19. Protein overexport in a Saccharomyces cerevisiae mutant is not due to facilitated release of cell-surface proteins.

    PubMed

    Alexieva, K I; Venkov, P V

    2000-01-01

    Saccharomyces cerevisiae strain MW11 is a temperature-sensitive mutant which exports twenty times more proteins at 37 degrees C than parental or wild-type strains do. To understand the mechanism underlying the protein overexport in the mutant the possibility of an altered cell-wall structure leading to facilitated release of cell-surface proteins was studied. Data on calcofluor white and zymolyase sensitivities, resistance to killer 1 toxin and determination of exported acid phosphatase and invertase did not provide evidence for alterations in the cell-wall structure that could explain the protein overexport phenotype. The results were obtained in experiments when transcription of mutated gene was discontinued which permits the full expression of the protein overexport phenotype.

  20. Destabilizing protein polymorphisms in the genetic background direct phenotypic expression of mutant SOD1 toxicity.

    PubMed

    Gidalevitz, Tali; Krupinski, Thomas; Garcia, Susana; Morimoto, Richard I

    2009-03-01

    Genetic background exerts a strong modulatory effect on the toxicity of aggregation-prone proteins in conformational diseases. In addition to influencing the misfolding and aggregation behavior of the mutant proteins, polymorphisms in putative modifier genes may affect the molecular processes leading to the disease phenotype. Mutations in SOD1 in a subset of familial amyotrophic lateral sclerosis (ALS) cases confer dominant but clinically variable toxicity, thought to be mediated by misfolding and aggregation of mutant SOD1 protein. While the mechanism of toxicity remains unknown, both the nature of the SOD1 mutation and the genetic background in which it is expressed appear important. To address this, we established a Caenorhabditis elegans model to systematically examine the aggregation behavior and genetic interactions of mutant forms of SOD1. Expression of three structurally distinct SOD1 mutants in C. elegans muscle cells resulted in the appearance of heterogeneous populations of aggregates and was associated with only mild cellular dysfunction. However, introduction of destabilizing temperature-sensitive mutations into the genetic background strongly enhanced the toxicity of SOD1 mutants, resulting in exposure of several deleterious phenotypes at permissive conditions in a manner dependent on the specific SOD1 mutation. The nature of the observed phenotype was dependent on the temperature-sensitive mutation present, while its penetrance reflected the specific combination of temperature-sensitive and SOD1 mutations. Thus, the specific toxic phenotypes of conformational disease may not be simply due to misfolding/aggregation toxicity of the causative mutant proteins, but may be defined by their genetic interactions with cellular pathways harboring mildly destabilizing missense alleles. PMID:19266020

  1. CHEMOSENSITIZATION BY A NON-APOPTOGENIC HEAT SHOCK PROTEIN 70-BINDING APOPTOSIS INDUCING FACTOR MUTANT

    EPA Science Inventory

    Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis inducing factor mutant

    Abstract
    HSP70 inhibits apoptosis by neutralizing the caspase activator Apaf-1 and by interacting with apoptosis inducing factor (AIF), a mitochondrial flavoprotein wh...

  2. Isolation and characterization of pediocin AcH chimeric protein mutants with altered bactericidal activity.

    PubMed

    Miller, K W; Schamber, R; Osmanagaoglu, O; Ray, B

    1998-06-01

    A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to approximately 60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed approximately 2. 8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.

  3. Distinct tumor protein p53 mutants in breast cancer subgroups.

    PubMed

    Dumay, Anne; Feugeas, Jean-Paul; Wittmer, Evelyne; Lehmann-Che, Jacqueline; Bertheau, Philippe; Espié, Marc; Plassa, Louis-François; Cottu, Paul; Marty, Michel; André, Fabrice; Sotiriou, Christos; Pusztai, Lajos; de Thé, Hugues

    2013-03-01

    Tumor protein p53 (TP53) is mutated in approximately 30% of breast cancers, but this frequency fluctuates widely between subclasses. We investigated the p53 mutation status in 572 breast tumors, classified into luminal, basal and molecular apocrine subgroups. As expected, the lowest mutation frequency was observed in luminal (26%), and the highest in basal (88%) tumors. Luminal tumors showed significantly higher frequency of substitutions (82 vs. 65%), notably A/T to G/C transitions (31 vs. 15%), whereas molecular apocrine and basal tumors presented much higher frequencies of complex mutations (deletions/insertions) (36 and 33%, respectively, vs. 18%). Accordingly, missense mutations were significantly more frequent in luminal tumors (75 vs. 54%), whereas basal tumors displayed significantly increased rates of TP53 truncations (43 vs. 25%), resulting in loss of function and/or expression. Interestingly, as basal tumors, molecular apocrine tumors presented with a high rate of complex mutations, but paradoxically, these were not associated with increased frequency of p53 truncation. As in luminal tumors, this could reflect a selective pressure for p53 gain of function, possibly through P63/P73 inactivation. Collectively, these observations point not only to different mechanisms of TP53 alterations, but also to different functional consequences in the different breast cancer subtypes.

  4. Fluorescence studies of the binding of bacteriophage M13 gene V mutant proteins to polynucleotides.

    PubMed

    Stassen, A P; Harmsen, B J; Schoenmakers, J G; Hilbers, C W; Konings, R N

    1992-06-15

    This investigation describes how the binding characteristics of the single-stranded DNA-binding protein encoded by gene V of bacteriophage M13, are affected by single-site amino acid substitutions. The series of mutant proteins tested includes mutations in the purported monomer-monomer interaction region as well as mutations in the DNA-binding domain at positions which are thought to be functionally involved in monomer-monomer interaction or single-stranded DNA binding. The characteristics of the binding of the mutant proteins to the homopolynucleotides poly(dA), poly(dU) and poly(dT), were studied by means of fluorescence-titration experiments. The binding stoichiometry and fluorescence quenching of the mutant proteins are equal to, or lower than, the wild-type gene V protein values. In addition, all proteins measured bind a more-or-less co-operative manner to single-stranded DNA. The binding affinities for poly(dA) decrease in the following order: Y61H greater than wild-type greater than F68L and R16H greater than Y41F and Y41H greater than F73L greater than R21C greater than Y34H greater than G18D/Y56H. Possible explanations for the observed differences are discussed. The conservation of binding affinity, also for mutations in the single-stranded DNA-binding domain, suggests that the binding to homopolynucleotides is largely non-specific.

  5. Glucose-induced production of recombinant proteins in Hansenula polymorpha mutants deficient in catabolite repression.

    PubMed

    Krasovska, Olena S; Stasyk, Olena G; Nahorny, Viktor O; Stasyk, Oleh V; Granovski, Nikolai; Kordium, Vitaliy A; Vozianov, Oleksandr F; Sibirny, Andriy A

    2007-07-01

    The most commonly used expression platform for production of recombinant proteins in the methylotrophic yeast Hansenula polymorpha relies on the strong and strictly regulated promoter from the gene encoding peroxisomal enzyme alcohol (or methanol) oxidase (P(MOX)). Expression from P(MOX) is induced by methanol and is partially derepressed in glycerol or xylose medium, whereas in the presence of hexoses, disaccharides or ethanol, it is repressed. The need for methanol for maximal induction of gene expression in large-scale fermentation is a significant drawback, as this compound is toxic, flammable, supports a slow growth rate and requires extensive aeration. We isolated H. polymorpha mutants deficient in glucose repression of P(MOX) due to an impaired HpGCR1 gene, and other yet unidentified secondary mutations. The mutants exhibited pronounced defects in P(MOX) regulation only by hexoses and xylose, but not by disaccharides or ethanol. With one of these mutant strains as hosts, we developed a modified two-carbon source mode expression platform that utilizes convenient sugar substrates for growth (sucrose) and induction of recombinant protein expression (glucose or xylose). We demonstrate efficient regulatable by sugar carbon sources expression of three recombinant proteins: a secreted glucose oxidase from the fungus Aspergillus niger, a secreted mini pro-insulin, and an intracellular hepatitis B virus surface antigen in these mutant hosts. The modified expression platform preserves the favorable regulatable nature of P(MOX) without methanol, making a convenient alternative to the traditional system. PMID:17163508

  6. Hepatitis B virus X protein mutants exhibit distinct biological activities in hepatoma Huh7 cells

    SciTech Connect

    Liu Xiaohong; Zhang Shuhui; Lin Jing; Zhang Shunmin; Feitelson, Mark A.; Gao Hengjun; Zhu Minghua

    2008-09-05

    The role of the hepatitis B virus X protein (HBx) in hepatocarcinogenesis remains controversial. To investigate the biological impact of hepatitis B virus x gene (HBx) mutation on hepatoma cells, plasmids expressing the full-length HBx or HBx deletion mutants were constructed. The biological activities in these transfectants were analyzed by a series of assays. Results showed that HBx3'-20 and HBx3'-40 amino acid deletion mutants exhibited an increase in cellular proliferation, focus formation, tumorigenicity, and invasive growth and metastasis through promotion of the cell cycle from G0/G1 to the S phase, when compared with the full-length HBx. In contrast, HBx3'-30 amino acid deletion mutant repressed cell proliferation by blocking in G1 phase. The expression of P53, p21{sup WAF1}, p14{sup ARF}, and MDM2 proteins was regulated by expression of HBx mutants. In conclusions, HBx variants showed different effects and functions on cell proliferation and invasion by regulation of the cell cycle progression and its associated proteins expression.

  7. Streptomyces relC mutants with an altered ribosomal protein ST-L11 and genetic analysis of a Streptomyces griseus relC mutant.

    PubMed Central

    Ochi, K

    1990-01-01

    Several relaxed (rel) mutants have been obtained from Streptomyces species by selecting colonies resistant to thiopeptin, an analogue of thiostrepton. Using two-dimensional gel electrophoresis, I compared the ribosomal proteins from rel and rel+ pairs of S. antibioticus, S. lavendulae, S. griseoflavus, and S. griseus. It was found that all of the Streptomyces rel mutants thus examined had an altered or missing ribosomal protein, designated tentatively ST-L11. These rel mutants therefore could be classified as relC mutants and were highly sensitive to erythromycin or high temperature. A relC mutant of S. griseus was defective in streptomycin production, but phenotypic reversion of this defect to normal productivity was found at high incidence among progeny of the relC mutant. This phenotypic reversion did not accompany a reappearance of ribosomal protein ST-L11, and furthermore the ability of accumulating ppGpp still remained at a low level, thus suggesting existence of a mutation (named sup) which suppresses the streptomycin deficiency phenotype exhibited by the relC mutant. Genetic analysis revealed that there is a correlation between the rel mutation and the inability to produce streptomycin or aerial mycelia. The sup mutation was found to lie at a chromosomal locus distinct from that of the relC mutation. It was therefore concluded that the dependence of streptomycin production on the normal function of the relC gene could be entirely bypassed by a mutation at the suppressor locus (sup). The suppressing effect of the sup mutation on the relC mutation was blocked when the afs mutation (defective in A-factor synthesis) was introduced into a relC sup double mutant. It is proposed that the sup gene or its product can be direct or indirect target for ppGpp. Images PMID:2113916

  8. An improved reversibly dimerizing mutant of the FK506-binding protein FKBP

    PubMed Central

    Barrero, Juan J.; Papanikou, Effrosyni; Casler, Jason C.; Day, Kasey J.; Glick, Benjamin S.

    2016-01-01

    ABSTRACT FK506-binding protein (FKBP) is a monomer that binds to FK506, rapamycin, and related ligands. The F36M substitution, in which Phe36 in the ligand-binding pocket is changed to Met, leads to formation of antiparallel FKBP dimers, which can be dissociated into monomers by ligand binding. This FKBP(M) mutant has been employed in the mammalian secretory pathway to generate aggregates that can be dissolved by ligand addition to create cargo waves. However, when testing this approach in yeast, we found that dissolution of FKBP(M) aggregates was inefficient. An improved reversibly dimerizing FKBP formed aggregates that dissolved more readily. This FKBP(L,V) mutant carries the F36L mutation, which increases the affinity of ligand binding, and the I90V mutation, which accelerates ligand-induced dissociation of the dimers. The FKBP(L,V) mutant expands the utility of reversibly dimerizing FKBP.

  9. Genetic Properties of Temperature-Sensitive Folding Mutants of the Coat Protein of Phage P22

    PubMed Central

    Gordon, C. L.; King, J.

    1994-01-01

    Temperature-sensitive mutations fall into two general classes: those generating thermolabile proteins; and those generating defects in protein synthesis, folding or assembly. Temperature-sensitive mutations at 17 sites in the gene for the coat protein of Phage P22 are of the latter class, preventing the productive folding of the polypeptide chain at restrictive temperature. We show here that, though the coat subunits interact intimately to form the viral shell, these temperature-sensitive folding (TSF) mutations were all recessive to wild type. The mutant polypeptide chains were not rescued by the presence of wild-type polypeptide chains. Missense substitutions in multimeric proteins frequently exhibit intragenic complementation; however, all pairs of coat protein TSF mutants tested failed to complement. The recessive phenotypes, absence of rescue and absence of intragenic complementation are all accounted for by the TSF defect, in which destabilization of a folding intermediate at restrictive temperature prevents the mutant chain from reaching the conformation required for subunit/subunit recognition. We suggest that absence of intragenic complementation should be a general property of TSF mutations in genes encoding multimeric proteins. The spectra of new loci identified by isolating second-site suppressors and synthetic lethals of temperature sensitive mutants will also differ depending on the nature of the defect. In the case of TSF mutations, where folding intermediates are defective rather than the native molecule, the spectra of other genes identified should shift from those whose products interact with the native molecule to those whose products influence the folding process. PMID:8150274

  10. Death-associated protein kinase 1 promotes growth of p53-mutant cancers

    PubMed Central

    Zhao, Jing; Zhao, Dekuang; Poage, Graham M.; Mazumdar, Abhijit; Zhang, Yun; Hill, Jamal L.; Hartman, Zachary C.; Savage, Michelle I.; Mills, Gordon B.; Brown, Powel H.

    2015-01-01

    Estrogen receptor–negative (ER-negative) breast cancers are extremely aggressive and associated with poor prognosis. In particular, effective treatment strategies are limited for patients diagnosed with triple receptor–negative breast cancer (TNBC), which also carries the worst prognosis of all forms of breast cancer; therefore, extensive studies have focused on the identification of molecularly targeted therapies for this tumor subtype. Here, we sought to identify molecular targets that are capable of suppressing tumorigenesis in TNBCs. Specifically, we found that death-associated protein kinase 1 (DAPK1) is essential for growth of p53-mutant cancers, which account for over 80% of TNBCs. Depletion or inhibition of DAPK1 suppressed growth of p53-mutant but not p53-WT breast cancer cells. Moreover, DAPK1 inhibition limited growth of other p53-mutant cancers, including pancreatic and ovarian cancers. DAPK1 mediated the disruption of the TSC1/TSC2 complex, resulting in activation of the mTOR pathway. Our studies demonstrated that high DAPK1 expression causes increased cancer cell growth and enhanced signaling through the mTOR/S6K pathway; evaluation of multiple breast cancer patient data sets revealed that high DAPK1 expression associates with worse outcomes in individuals with p53-mutant cancers. Together, our data support targeting DAPK1 as a potential therapeutic strategy for p53-mutant cancers. PMID:26075823

  11. IBMPFD Disease-Causing Mutant VCP/p97 Proteins Are Targets of Autophagic-Lysosomal Degradation

    PubMed Central

    Bayraktar, Oznur; Akkoc, Yunus; Eberhart, Karin; Kosar, Ali

    2016-01-01

    The ubiquitin-proteasome system (UPS) degrades soluble proteins and small aggregates, whereas macroautophagy (autophagy herein) eliminates larger protein aggregates, tangles and even whole organelles in a lysosome-dependent manner. VCP/p97 was implicated in both pathways. VCP/p97 mutations cause a rare multisystem disease called IBMPFD (Inclusion Body Myopathy with Paget’s Disease and Frontotemporal Dementia). Here, we studied the role IBMPFD-related mutants of VCP/p97 in autophagy. In contrast with the wild-type VCP/p97 protein or R155C or R191Q mutants, the P137L mutant was aggregate-prone. We showed that, unlike commonly studied R155C or R191Q mutants, the P137L mutant protein stimulated both autophagosome and autolysosome formation. Moreover, P137L mutant protein itself was a substrate of autophagy. Starvation- and mTOR inhibition-induced autophagy led to the degradation of the P137L mutant protein, while preserving the wild-type and functional VCP/p97. Strikingly, similar to the P137L mutant, other IBMPFD-related VCP/p97 mutants, namely R93C and G157R mutants induced autophagosome and autolysosome formation; and G157R mutant formed aggregates that could be cleared by autophagy. Therefore, cellular phenotypes caused by P137L mutant expression were not isolated observations, and some other IBMPFD disease-related VCP/p97 mutations could lead to similar outcomes. Our results indicate that cellular mechanisms leading to IBMPFD disease may be various, and underline the importance of studying different disease-associated mutations in order to better understand human pathologies and tailor mutation-specific treatment strategies. PMID:27768726

  12. Abelson murine leukemia virus transformation-defective mutants with impaired P120-associated protein kinase activity.

    PubMed Central

    Reynolds, F H; Van de Ven, W J; Stephenson, J R

    1980-01-01

    Several transformation-defective (td) mutants of Abelson murine leukemia virus (AbLV) are described. Cells nonproductively infected with such mutants exhibited a high degree of growth contact inhibition, failed to form colonies in soft agar, lacked rescuable transforming virus, and were as susceptible as uninfected control cells to transformation by wild-type (wt) AbLV pseudotype virus. In addition, each of several td AbLV nonproductively infected cell clones analyzed was found to be nontumorigenic in vivo. Biochemical analysis of td mutant AbLV-infected clones revealed levels of expression of the major AbLV translational product, P120, and a highly related 80,000-Mr AbLV-encoded protein, P80, at concentrations analogous to those in wt AbLV-transformed cells. Although the AbLV-specific 120,000-Mr polyproteins expressed in td mutant AbLV-infected clones were indistinguishable from those in wt AbLV-transformed lines with respect to molecular weight and [35S]methionine tryptic peptide composition, they each differed from wt AbLV P120 in their patterns of post-translational phosphorylation. A previously described AbLV-associated protein kinase activity is shown to recognize as substrate a major tyrosine-specific acceptor site(s) contained within a single well-resolved tryptic peptide common to both AbLV P120 and P80. In vitro [gamma-32P]ATP-mediated labeling of this phosphorylation site was reduced to below detectable levels in td mutant nonproductively infected cell clones. These findings establish that the AbLV-encoded polyprotein P120 and its associated protein kinase activity are involved in AbLV tumorigenesis. Images PMID:6253663

  13. Co-occurence of filamentation defects and impaired biofilms in Candida albicans protein kinase mutants.

    PubMed

    Konstantinidou, Nina; Morrissey, John Patrick

    2015-12-01

    Pathogenicity of Candida albicans is linked with its developmental stages, notably the capacity switch from yeast-like to hyphal growth, and to form biofilms on surfaces. To better understand the cellular processes involved in C. albicans development, a collection of 63 C. albicans protein kinase mutants was screened for biofilm formation in a microtitre plate assay. Thirty-eight mutants displayed some degree of biofilm impairment, with 20 categorised as poor biofilm formers. All the poor biofilm formers were also defective in the switch from yeast to hyphae, establishing it as a primary defect. Five genes, VPS15, IME2, PKH3, PGA43 and CEX1, encode proteins not previously reported to influence hyphal development or biofilm formation. Network analysis established that individual components of some processes, most interestingly MAP kinase pathways, are not required for biofilm formation, most likely indicating functional redundancy. Mutants were also screened for their response to bacterial supernatants and it was found that Pseudomonas aeruginosa supernatants inhibited biofilm formation in all mutants, regardless of the presence of homoserine lactones (HSLs). In contrast, Candida morphology was only affected by supernatant containing HSLs. This confirms the distinct HSL-dependent inhibition of filamentation and the HSL-independent impairment of biofilm development by P. aeruginosa.

  14. Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20.

    PubMed Central

    Priestley, A; Beamish, H J; Gell, D; Amatucci, A G; Muhlmann-Diaz, M C; Singleton, B K; Smith, G C; Blunt, T; Schalkwyk, L C; Bedford, J S; Jackson, S P; Jeggo, P A; Taccioli, G E

    1998-01-01

    The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase. To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes. Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus. This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity. PMID:9518490

  15. Genomic polymorphism and protein changes of soybean mutant induced by space environment

    NASA Astrophysics Data System (ADS)

    He, J.; Gao, Y.; Sun, Y.

    Soybean 194 4126 of excellent agricultural qualities such as high yield and rounder and wider leaf was selected in six generation after abroad recoverable satellite 15 days in 1996 from Soybean 72163 featured with long-leaf white-blossom grey-hair and infinitude-poding To explore the mechanisms of plant mutation induced by space environment we have experimented at genome and proteome level on Soybean 194 4126 and its control Soybean 72163 Amplified Fragment Length Polymorphism AFLP was used to identify mutated sits and the result shows that 36 polymorphic bands varying between 100 and 900 bp in 2022 DNA bands varying between 100 and 1500 bp have been amplified out of 64 pairs of primer combinations between mutant Soybean 194 4126 and the control plant So the mutation degree of DNA is 3 56 The protein two-dimensional electrophoresis 2-DE and peptide mass fingerprint PMF assays were used to investigate the difference of proteins in fruits and leaves between Soybean 194 4126 and its control Results indicate that 62 protein dots specially appear in Soybean 72163 and 39 dots specially in the mutant Soybean 194 4126 by image analysis software PDQuest in the 2-DE maps of soybean seeds Using PMF assay and protein data-base searching to investigate two distinct protein dots we found that the protein specially expressed in the seed of mutant Soybean 194 4126 may be Dehydrin and the other protein specially expressed in the seed of the control Soybean 72163 may be maturation-associated protein MAT1 Because Dehydrin and MAT1 are

  16. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.

    PubMed Central

    Robinson, J S; Klionsky, D J; Banta, L M; Emr, S D

    1988-01-01

    Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature

  17. Cyclin-dependent kinase 5 phosphorylation of familial prion protein mutants exacerbates conversion into amyloid structure.

    PubMed

    Rouget, Raphaël; Sharma, Gyanesh; LeBlanc, Andréa C

    2015-02-27

    Familial prion protein (PrP) mutants undergo conversion from soluble and protease-sensitive to insoluble and partially protease-resistant proteins. Cyclin-dependent kinase 5 (Cdk5) phosphorylation of wild type PrP (pPrP) at serine 43 induces a conversion of PrP into aggregates and fibrils. Here, we investigated whether familial PrP mutants are predisposed to Cdk5 phosphorylation and whether phosphorylation of familial PrP mutants increases conversion. PrP mutants representing three major familial PrP diseases and different PrP structural domains were studied. We developed a novel in vitro kinase reaction coupled with Thioflavin T binding to amyloid structure assay to monitor phosphorylation-dependent amyloid conversion. Although non-phosphorylated full-length wild type or PrP mutants did not convert into amyloid, Cdk5 phosphorylation rapidly converted these into Thioflavin T-positive structures following first order kinetics. Dephosphorylation partially reversed conversion. Phosphorylation-dependent conversion of PrP from α-helical structures into β-sheet structures was confirmed by circular dichroism. Relative to wild type pPrP, most PrP mutants showed increased rate constants of conversion. In contrast, non-phosphorylated truncated PrP Y145X (where X represents a stop codon) and Q160X mutants converted spontaneously into Thioflavin T-positive fibrils after a lag phase of over 20 h, indicating nucleation-dependent polymerization. Phosphorylation reduced the lag phase by over 50% and thus accelerated the formation of the nucleating event. Consistently, phosphorylated Y145X and phosphorylated Q160X exacerbated conversion in a homologous seeding reaction, whereas WT pPrP could not seed WT PrP. These results demonstrate an influence of both the N terminus and the C terminus of PrP on conversion. We conclude that post-translational modifications of the flexible N terminus of PrP can cause or exacerbate PrP mutant conversion. PMID:25572400

  18. Molecular pathogenesis of Spondylocheirodysplastic Ehlers-Danlos syndrome caused by mutant ZIP13 proteins

    PubMed Central

    Bin, Bum-Ho; Hojyo, Shintaro; Hosaka, Toshiaki; Bhin, Jinhyuk; Kano, Hiroki; Miyai, Tomohiro; Ikeda, Mariko; Kimura-Someya, Tomomi; Shirouzu, Mikako; Cho, Eun-Gyung; Fukue, Kazuhisa; Kambe, Taiho; Ohashi, Wakana; Kim, Kyu-Han; Seo, Juyeon; Choi, Dong-Hwa; Nam, Yeon-Ju; Hwang, Daehee; Fukunaka, Ayako; Fujitani, Yoshio; Yokoyama, Shigeyuki; Superti-Furga, Andrea; Ikegawa, Shiro; Lee, Tae Ryong; Fukada, Toshiyuki

    2014-01-01

    The zinc transporter protein ZIP13 plays critical roles in bone, tooth, and connective tissue development, and its dysfunction is responsible for the spondylocheirodysplastic form of Ehlers-Danlos syndrome (SCD-EDS, OMIM 612350). Here, we report the molecular pathogenic mechanism of SCD-EDS caused by two different mutant ZIP13 proteins found in human patients: ZIP13G64D, in which Gly at amino acid position 64 is replaced by Asp, and ZIP13ΔFLA, which contains a deletion of Phe-Leu-Ala. We demonstrated that both the ZIP13G64D and ZIP13ΔFLA protein levels are decreased by degradation via the valosin-containing protein (VCP)-linked ubiquitin proteasome pathway. The inhibition of degradation pathways rescued the protein expression levels, resulting in improved intracellular Zn homeostasis. Our findings uncover the pathogenic mechanisms elicited by mutant ZIP13 proteins. Further elucidation of these degradation processes may lead to novel therapeutic targets for SCD-EDS. PMID:25007800

  19. Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities.

    PubMed Central

    Weis, K; Dingwall, C; Lamond, A I

    1996-01-01

    The small nuclear GTP binding protein Ran is required for transport of nuclear proteins through the nuclear pore complex (NPC). Although it is known that GTP hydrolysis by Ran is essential for this reaction, it has been unclear whether additional energy-consuming steps are also required. To uncouple the energy requirements for Ran from other nucleoside triphosphatases, we constructed a mutant derivative of Ran that has an altered nucleotide specificity from GTP to xanthosine 5' triphosphate. Using this Ran mutant, we demonstrate that nucleotide hydrolysis by Ran is sufficient to promote efficient nuclear protein import in vitro. Under these conditions, protein import could no longer be inhibited with non-hydrolysable nucleotide analogues, indicating that no Ran-independent energy-requiring steps are essential for the protein translocation reaction through the NPC. We further provide evidence that nuclear protein import requires Ran in the GDP form in the cytoplasm. This suggests that a coordinated exchange reaction from Ran-GDP to Ran-GTP at the pore is necessary for translocation into the nucleus. Images PMID:9003787

  20. Mutant p53 protein expression and antioxidant status deficiency in breast cancer

    PubMed Central

    Milicevic, Zorka; Kasapovic, Jelena; Gavrilovic, Ljubica; Milovanovic, Zorka; Bajic, Vladan; Spremo-Potparevic, Biljana

    2014-01-01

    It is well recognized that cancers develop and grow as a result of disordered function of tumor suppressor genes and oncogenes, which may be exploited for screening purposes. Extensive evidence indicated tumor suppressor protein p53 as candidate marker for mutation identification. We have investigated mutant p53 protein expression in human breast tumors in relation to antioxidant status deficiency. The study included 100 breast cancer patients. p53 protein expression was evaluated by Western blot assay and immunostaining using a CM-1, DO-7 and Pab240 antibodies. Antioxidant parameters and lipid peroxidation were estimated by biochemical analyses. Western blotting with epitopespecific monoclonal antibody Pab240 strongly suggests that nuclear extracts from breast cancer cells express mutant forms of p53. It is of interest that the mutant forms of p53 overexpression in conjunction with the appearance of nuclear bodies are observed in highly aggressive carcinomas. Expression of isoform Δp53 (45 kDa) and isoform of ~ 29 kDa were more common in cases with LN metastasis. These studies point out the molecular consequences of oxidative stress (lipid peroxides, LP, p<0.001) and antioxidant status deficiency (copper, zinc superoxid dismutase, SOD, p<0.001; catalase, CAT, p<0.01; glutathione reductase, GR, p<0.001; glutathione, GSH, p<0.05) and indicate the importance of p53 mutation as the commonest genetic alteration detected in breast cancer cells. The expression of mutant p53 is correlated to increased lipid peroxides (0.346, p<0.05 ) and lowered antioxidant activity of CAT (- 0.437, p<0.01) in the breast cancer patients. PMID:26417293

  1. In vivo functional protein-protein interaction: nuclear targeted hsp90 shifts cytoplasmic steroid receptor mutants into the nucleus.

    PubMed Central

    Kang, K I; Devin, J; Cadepond, F; Jibard, N; Guiochon-Mantel, A; Baulieu, E E; Catelli, M G

    1994-01-01

    In target tissue extracts, heat shock protein hsp90 has been found associated to all unliganded steroid receptors. Modulation of important functions of these receptors, including prevention of DNA binding and optimization of transcriptional activity, has been attributed to hsp90. However no unequivocal in vivo demonstration of interaction between receptors and hsp90 has been presented. We targeted chicken hsp90, a mainly cytoplasmic protein, with the nucleoplasmin nuclear localization signal (90NLS). After transfection into COS-7 cells, 90NLS was found in the nucleus with specific immunofluorescence and confocal microscopy techniques. A human glucocorticosteroid receptor mutant devoid of NLS sequence was also expressed in COS-7 cells and found exclusively cytoplasmic. Coexpression of 90NLS and of the cytoplasmic human glucocorticosteroid receptor mutant led to complete nuclear localization of the receptor, indicating its piggyback transport by 90NLS and thus physical and functional interaction between the two proteins in the absence of hormone. The same nuclear localization was obtained after cotransfection of 90NLS and a cytoplasmic rabbit progesterone receptor mutant. Finally, coexpression of wild-type rabbit progesterone receptor (nuclear) and wildtype hsp90 (cytoplasmic) into COS-7 cells provoked partial relocalization of hsp90 into the nucleus. These experiments lay the groundwork on which to study hsp90 as a chaperone, regulating activities of steroid receptors and possibly participating in their nuclear-cytoplasmic shuttling. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8278390

  2. Strep-tag II Mutant Maltose-binding Protein for Reagentless Fluorescence Sensing

    PubMed Central

    Hasmoni, Siti Halimah; Mau, Goh Kian; Karsani, Saiful Anuar; Cass, Anthony; Shahir, Shafinaz

    2016-01-01

    Maltose-binding protein (MBP) is a periplasmic binding protein found in Gram negative bacteria. MBP is involved in maltose transport and bacterial chemotaxis; it binds to maltose and maltodextrins comprising α(1–4)-glucosidically linked linear glucose polymers and α(1–4)-glucosidically linked cyclodextrins. Upon ligand binding, MBP changes its conformation from an open to a closed form. This molecular recognition—transducing a ligand-binding event into a physical one—renders MBP an ideal candidate for biosensor development. Here, we describe the construction of a Strep-tag II mutant MBP for reagentless fluorescence sensing. malE, which encodes MBP, was amplified. A cysteine residue was introduced by site-directed mutagenesis to ensure a single label attachment at a specific site with a thiol-specific fluorescent probe. An environmentally sensitive fluorophore (IANBD amide) was covalently attached to the introduced thiol group and analysed by fluorescence sensing. The tagged mutant MBP (D95C) was purified (molecular size, ∼42 kDa). The fluorescence measurements of the IANBD-labelled Strep-tag II–D95C in the solution phase showed an appreciable change in fluorescence intensity (dissociation constant, 7.6±1.75 μM). Our mutant MBP retains maltose-binding activity and is suitable for reagentless fluorescence sensing. PMID:27019682

  3. Strep-tag II Mutant Maltose-binding Protein for Reagentless Fluorescence Sensing.

    PubMed

    Hasmoni, Siti Halimah; Mau, Goh Kian; Karsani, Saiful Anuar; Cass, Anthony; Shahir, Shafinaz

    2016-02-01

    Maltose-binding protein (MBP) is a periplasmic binding protein found in Gram negative bacteria. MBP is involved in maltose transport and bacterial chemotaxis; it binds to maltose and maltodextrins comprising α(1-4)-glucosidically linked linear glucose polymers and α(1-4)-glucosidically linked cyclodextrins. Upon ligand binding, MBP changes its conformation from an open to a closed form. This molecular recognition-transducing a ligand-binding event into a physical one-renders MBP an ideal candidate for biosensor development. Here, we describe the construction of a Strep-tag II mutant MBP for reagentless fluorescence sensing. malE, which encodes MBP, was amplified. A cysteine residue was introduced by site-directed mutagenesis to ensure a single label attachment at a specific site with a thiol-specific fluorescent probe. An environmentally sensitive fluorophore (IANBD amide) was covalently attached to the introduced thiol group and analysed by fluorescence sensing. The tagged mutant MBP (D95C) was purified (molecular size, ∼42 kDa). The fluorescence measurements of the IANBD-labelled Strep-tag II-D95C in the solution phase showed an appreciable change in fluorescence intensity (dissociation constant, 7.6±1.75 μM). Our mutant MBP retains maltose-binding activity and is suitable for reagentless fluorescence sensing.

  4. Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish

    PubMed Central

    Grone, Brian P.; Marchese, Maria; Hamling, Kyla R.; Kumar, Maneesh G.; Krasniak, Christopher S.; Sicca, Federico; Santorelli, Filippo M.; Patel, Manisha; Baraban, Scott C.

    2016-01-01

    Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing “dark-flash” visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations. PMID:26963117

  5. Food safety assessment of Cry8Ka5 mutant protein using Cry1Ac as a control Bt protein.

    PubMed

    Farias, Davi Felipe; Viana, Martônio Ponte; Oliveira, Gustavo Ramos; Santos, Vanessa Olinto; Pinto, Clidia Eduarda Moreira; Viana, Daniel Araújo; Vasconcelos, Ilka Maria; Grossi-de-Sa, Maria Fátima; Carvalho, Ana Fontenele Urano

    2015-07-01

    Cry8Ka5 is a mutant protein from Bacillus thuringiensis (Bt) that has been proposed for developing transgenic plants due to promising activity against coleopterans, like Anthonomus grandis (the major pest of Brazilian cotton culture). Thus, an early food safety assessment of Cry8Ka5 protein could provide valuable information to support its use as a harmless biotechnological tool. This study aimed to evaluate the food safety of Cry8Ka5 protein following the two-tiered approach, based on weights of evidence, proposed by ILSI. Cry1Ac protein was used as a control Bt protein. The history of safe use revealed no convincing hazard reports for Bt pesticides and three-domain Cry proteins. The bioinformatics analysis with the primary amino acids sequence of Cry8Ka5 showed no similarity to any known toxic, antinutritional or allergenic proteins. The mode of action of Cry proteins is well understood and their fine specificity is restricted to insects. Cry8Ka5 and Cry1Ac proteins were rapidly degraded in simulated gastric fluid, but were resistant to simulated intestinal fluid and heat treatment. The LD50 for Cry8Ka5 and Cry1Ac was >5000 mg/kg body weight when administered by gavage in mice. Thus, no expected relevant risks are associated with the consumption of Cry8Ka5 protein.

  6. Food safety assessment of Cry8Ka5 mutant protein using Cry1Ac as a control Bt protein.

    PubMed

    Farias, Davi Felipe; Viana, Martônio Ponte; Oliveira, Gustavo Ramos; Santos, Vanessa Olinto; Pinto, Clidia Eduarda Moreira; Viana, Daniel Araújo; Vasconcelos, Ilka Maria; Grossi-de-Sa, Maria Fátima; Carvalho, Ana Fontenele Urano

    2015-07-01

    Cry8Ka5 is a mutant protein from Bacillus thuringiensis (Bt) that has been proposed for developing transgenic plants due to promising activity against coleopterans, like Anthonomus grandis (the major pest of Brazilian cotton culture). Thus, an early food safety assessment of Cry8Ka5 protein could provide valuable information to support its use as a harmless biotechnological tool. This study aimed to evaluate the food safety of Cry8Ka5 protein following the two-tiered approach, based on weights of evidence, proposed by ILSI. Cry1Ac protein was used as a control Bt protein. The history of safe use revealed no convincing hazard reports for Bt pesticides and three-domain Cry proteins. The bioinformatics analysis with the primary amino acids sequence of Cry8Ka5 showed no similarity to any known toxic, antinutritional or allergenic proteins. The mode of action of Cry proteins is well understood and their fine specificity is restricted to insects. Cry8Ka5 and Cry1Ac proteins were rapidly degraded in simulated gastric fluid, but were resistant to simulated intestinal fluid and heat treatment. The LD50 for Cry8Ka5 and Cry1Ac was >5000 mg/kg body weight when administered by gavage in mice. Thus, no expected relevant risks are associated with the consumption of Cry8Ka5 protein. PMID:25890087

  7. Resistance to β-Lactamase Inhibitor Protein Does Not Parallel Resistance to Clavulanic Acid in TEM β-Lactamase Mutants

    PubMed Central

    Schroeder, William A.; Locke, Troy R.; Jensen, Susan E.

    2002-01-01

    In order to compare patterns of resistance to inhibition by clavulanic acid with patterns of resistance to inhibition by a β-lactamase inhibitor protein (BLIP), R164S, R244S, and R164S/R244S mutant forms of TEM β-lactamase were prepared by site-directed mutagenesis. When kinetic parameters were determined for these mutant and wild-type forms of TEM, the single mutants showed properties that were similar to those in the literature but the double mutant showed properties that were very different. The R164S/R244S double mutant form of TEM retained its resistance to inhibition by clavulanic acid (characteristic of the R244S mutation) but lost all its ability to hydrolyze ceftazidime (characteristic of the R164S mutation). While these characteristics are contrary to those previously reported for an R164S/R244S double mutant, this discrepancy resulted from the use of a defective mutant in the earlier study. Both the single and double mutant forms of TEM remained highly sensitive when tested for inhibition by BLIP, showing only slightly increased resistance compared to that of the wild type; this pattern of resistance is quite different from the pattern of clavulanic acid resistance. The slight increases in resistance to inhibition by BLIP seen in the mutants may have been related to the fact that all of the mutations effected changes in the net charge on the TEM protein that could impede interactions with BLIP. PMID:12384366

  8. Conductance and block of hair-cell mechanotransducer channels in transmembrane channel-like protein mutants.

    PubMed

    Beurg, Maryline; Kim, Kyunghee X; Fettiplace, Robert

    2014-07-01

    Transmembrane channel-like (TMC) proteins TMC1 and TMC2 are crucial to the function of the mechanotransducer (MT) channel of inner ear hair cells, but their precise function has been controversial. To provide more insight, we characterized single MT channels in cochlear hair cells from wild-type mice and mice with mutations in Tmc1, Tmc2, or both. Channels were recorded in whole-cell mode after tip link destruction with BAPTA or after attenuating the MT current with GsMTx-4, a peptide toxin we found to block the channels with high affinity. In both cases, the MT channels in outer hair cells (OHCs) of wild-type mice displayed a tonotopic gradient in conductance, with channels from the cochlear base having a conductance (110 pS) nearly twice that of those at the apex (62 pS). This gradient was absent, with channels at both cochlear locations having similar small conductances, with two different Tmc1 mutations. The conductance of MT channels in inner hair cells was invariant with cochlear location but, as in OHCs, was reduced in either Tmc1 mutant. The gradient of OHC conductance also disappeared in Tmc1/Tmc2 double mutants, in which a mechanically sensitive current could be activated by anomalous negative displacements of the hair bundle. This "reversed stimulus-polarity" current was seen with two different Tmc1/Tmc2 double mutants, and with Tmc1/Tmc2/Tmc3 triple mutants, and had a pharmacological sensitivity comparable to that of native MT currents for most antagonists, except dihydrostreptomycin, for which the affinity was less, and for curare, which exhibited incomplete block. The existence in the Tmc1/Tmc2 double mutants of MT channels with most properties resembling those of wild-type channels indicates that proteins other than TMCs must be part of the channel pore. We suggest that an external vestibule of the MT channel may partly account for the channel's large unitary conductance, high Ca(2+) permeability, and pharmacological profile, and that this vestibule

  9. Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans

    NASA Astrophysics Data System (ADS)

    Adachi, R.; Takaya, T.; Kuriyama, K.; Higashibata, A.; Ishioka, N.; Kagawa, H.

    We have investigated the effect of microgravity during spaceflight on body-wall muscle fiber size and muscle proteins in the paramyosin mutant of Caenorhabditis elegans. Both mutant and wild-type strains were subjected to 10 days of microgravity during spaceflight and compared to ground control groups. No significant change in muscle fiber size or quantity of the protein was observed in wild-type worms; where as atrophy of body-wall muscle and an increase in thick filament proteins were observed in the paramyosin mutant unc-15(e73) animals after spaceflight. We conclude that the mutant with abnormal muscle responded to microgravity by increasing the total amount of muscle protein in order to compensate for the loss of muscle function.

  10. Selective modulation of wild type receptor functions by mutants of G-protein-coupled receptors.

    PubMed

    Le Gouill, C; Parent, J L; Caron, C A; Gaudreau, R; Volkov, L; Rola-Pleszczynski, M; Stanková, J

    1999-04-30

    Members of the G-protein-coupled receptor (GPCR) family are involved in most aspects of higher eukaryote biology, and mutations in their coding sequence have been linked to several diseases. In the present study, we report that mutant GPCR can affect the functional properties of the co-expressed wild type (WT) receptor. Mutants of the human platelet-activating factor receptor that fail to show any detectable ligand binding (N285I and K298stop) or coupling to a G-protein (D63N, D289A, and Y293A) were co-expressed with the WT receptor in Chinese hamster ovary and COS-7 cells. In this context, N285I and K298stop mutant receptors inhibited 3H-WEB2086 binding and surface expression. Co-transfection with D63N resulted in a constitutively active receptor phenotype. Platelet-activating factor-induced inositol phosphate production in cells transfected with a 1:1 ratio of WT:D63N was higher than with the WT cDNA alone but was abolished with a 1:3 ratio. We confirmed that these findings could be extended to other GPCRs by showing that co-expression of the WT C-C chemokine receptor 2b with a carboxyl-terminal deletion mutant (K311stop), resulted in a decreased affinity and responsiveness to MCP-1. A better understanding of this phenomenon could lead to important tools for the prevention or treatment of certain diseases. PMID:10212233

  11. A mutant chaperone converts a wild-type protein into a tumor-specific antigen.

    PubMed

    Schietinger, Andrea; Philip, Mary; Yoshida, Barbara A; Azadi, Parastoo; Liu, Hui; Meredith, Stephen C; Schreiber, Hans

    2006-10-13

    Monoclonal antibodies have become important therapeutic agents against certain cancers. Many tumor-specific antigens are mutant proteins that are predominantly intracellular and thus not readily accessible to monoclonal antibodies. We found that a wild-type transmembrane protein could be transformed into a tumor-specific antigen. A somatic mutation in the chaperone gene Cosmc abolished function of a glycosyltransferase, disrupting O-glycan Core 1 synthesis and creating a tumor-specific glycopeptidic neo-epitope consisting of a monosaccharide and a specific wild-type protein sequence. This epitope induced a high-affinity, highly specific, syngeneic monoclonal antibody with antitumor activity. Such tumor-specific glycopeptidic neo-epitopes represent potential targets for monoclonal antibody therapy.

  12. Exploiting Transient Protein States for the Design of Small-Molecule Stabilizers of Mutant p53

    PubMed Central

    Joerger, Andreas C.; Bauer, Matthias R.; Wilcken, Rainer; Baud, Matthias G.J.; Harbrecht, Hannes; Exner, Thomas E.; Boeckler, Frank M.; Spencer, John; Fersht, Alan R.

    2015-01-01

    Summary The destabilizing p53 cancer mutation Y220C creates an extended crevice on the surface of the protein that can be targeted by small-molecule stabilizers. Here, we identify different classes of small molecules that bind to this crevice and determine their binding modes by X-ray crystallography. These structures reveal two major conformational states of the pocket and a cryptic, transiently open hydrophobic subpocket that is modulated by Cys220. In one instance, specifically targeting this transient protein state by a pyrrole moiety resulted in a 40-fold increase in binding affinity. Molecular dynamics simulations showed that both open and closed states of this subsite were populated at comparable frequencies along the trajectories. Our data extend the framework for the design of high-affinity Y220C mutant binders for use in personalized anticancer therapy and, more generally, highlight the importance of implementing protein dynamics and hydration patterns in the drug-discovery process. PMID:26636255

  13. High throughput gene complementation screening permits identification of a mammalian mitochondrial protein synthesis (ρ(-)) mutant.

    PubMed

    Potluri, Prasanth; Procaccio, Vincent; Scheffler, Immo E; Wallace, Douglas C

    2016-08-01

    To identify nuclear DNA (nDNA) oxidative phosphorylation (OXPHOS) gene mutations using cultured cells, we have developed a complementation system based on retroviral transduction with a full length cDNA expression library and selection for OXHOS function by growth in galactose. We have used this system to transduce the Chinese hamster V79-G7 OXPHOS mutant cell line with a defect in mitochondrial protein synthesis. The complemented cells were found to have acquired the cDNA for the bS6m polypeptide of the small subunit of the mitochondrial ribosome. bS6m is a 14 kDa polypeptide located on the outside of the mitochondrial 28S ribosomal subunit and interacts with the rRNA. The V79-G7 mutant protein was found to harbor a methionine to threonine missense mutation at codon 13. The hamster bS6m null mutant could also be complemented by its orthologs from either mouse or human. bS6m protein tagged at its C-terminus by HA, His or GFP localized to the mitochondrion and was fully functional. Through site-directed mutagenesis we identified the probable RNA interacting residues of the bS6m peptide and tested the functional significance of mammalian specific C-terminal region. The N-terminus of the bS6m polypeptide functionally corresponds to that of the prokaryotic small ribosomal subunit, but deletion of C-terminal residues along with the zinc ion coordinating cysteine had no functional effect. Since mitochondrial diseases can result from hundreds to thousands of different nDNA gene mutations, this one step viral complementation cloning may facilitate the molecular diagnosis of a range of nDNA mitochondrial disease mutations. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26946086

  14. Accumulation of the F plasmid TraJ protein in cpx mutants of Escherichia coli.

    PubMed Central

    Silverman, P M; Tran, L; Harris, R; Gaudin, H M

    1993-01-01

    We report here studies of the cellular control of F plasmid TraJ protein levels, focusing on the effects of chromosomal cpx mutations. The principal conclusion from our results is that the cpx mutations impair accumulation of the TraJ protein, thereby reducing tra gene expression. We measured TraJ activity in vivo by expression of a traY'-'lacZ fusion gene and TraJ protein by immuno-overlay blot. In strains with normal TraJ levels, traY expression and donor-related functions were reduced in cells carrying any of four cpxA mutations. In the strain background used to isolate cpx mutants, these reductions were especially evident in cells grown to high density, when traY expression and donor activity both increased in cpx+ cells. In each of the four cpxA mutants tested, TraJ levels were lower than in the otherwise isogenic cpxA+ strain. In cells grown to high density, the differences ranged from 4-fold in the cpxA6 strain to > 10-fold in the cpxA2, cpxA5, and cpxA9 strains. The cpxA2 mutation had little or no effect on traY expression or on donor-related functions when TraJ was present in excess of its limiting level in F' or Hfr cells or on a mutant traY promoter whose expression in vivo was independent of TraJ. Images PMID:8432716

  15. Larger aggregates of mutant seipin in Celia's Encephalopathy, a new protein misfolding neurodegenerative disease.

    PubMed

    Ruiz-Riquelme, Alejandro; Sánchez-Iglesias, Sofía; Rábano, Alberto; Guillén-Navarro, Encarna; Domingo-Jiménez, Rosario; Ramos, Adriana; Rosa, Isaac; Senra, Ana; Nilsson, Peter; García, Ángel; Araújo-Vilar, David; Requena, Jesús R

    2015-11-01

    Celia's Encephalopathy (MIM #615924) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin (Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers.

  16. Larger aggregates of mutant seipin in Celia's Encephalopathy, a new protein misfolding neurodegenerative disease.

    PubMed

    Ruiz-Riquelme, Alejandro; Sánchez-Iglesias, Sofía; Rábano, Alberto; Guillén-Navarro, Encarna; Domingo-Jiménez, Rosario; Ramos, Adriana; Rosa, Isaac; Senra, Ana; Nilsson, Peter; García, Ángel; Araújo-Vilar, David; Requena, Jesús R

    2015-11-01

    Celia's Encephalopathy (MIM #615924) is a recently discovered fatal neurodegenerative syndrome associated with a new BSCL2 mutation (c.985C>T) that results in an aberrant isoform of seipin (Celia seipin). This mutation is lethal in both homozygosity and compounded heterozygosity with a lipodystrophic BSCL2 mutation, resulting in a progressive encephalopathy with fatal outcomes at ages 6-8. Strikingly, heterozygous carriers are asymptomatic, conflicting with the gain of toxic function attributed to this mutation. Here we report new key insights about the molecular pathogenic mechanism of this new syndrome. Intranuclear inclusions containing mutant seipin were found in brain tissue from a homozygous patient suggesting a pathogenic mechanism similar to other neurodegenerative diseases featuring brain accumulation of aggregated, misfolded proteins. Sucrose gradient distribution showed that mutant seipin forms much larger aggregates as compared with wild type (wt) seipin, indicating an impaired oligomerization. On the other hand, the interaction between wt and Celia seipin confirmed by coimmunoprecipitation (CoIP) assays, together with the identification of mixed oligomers in sucrose gradient fractionation experiments can explain the lack of symptoms in heterozygous carriers. We propose that the increased aggregation and subsequent impaired oligomerization of Celia seipin leads to cell death. In heterozygous carriers, wt seipin might prevent the damage caused by mutant seipin through its sequestration into harmless mixed oligomers. PMID:26282322

  17. Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes

    PubMed Central

    Basso, Manuela; Pozzi, Silvia; Tortarolo, Massimo; Fiordaliso, Fabio; Bisighini, Cinzia; Pasetto, Laura; Spaltro, Gabriella; Lidonnici, Dario; Gensano, Francesco; Battaglia, Elisa; Bendotti, Caterina; Bonetto, Valentina

    2013-01-01

    Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread. PMID:23592792

  18. RAN/TC4 mutants identify a common requirement for snRNP and protein import into the nucleus

    PubMed Central

    1996-01-01

    Kinetic competition experiments have demonstrated that at least some factors required for the nuclear import of proteins and U snRNPs are distinct. Both import processes require energy, and in the case of protein import, the energy requirement is known to be at least partly met by GTP hydrolysis by the Ran GTPase. We have compared the effects of nonhydrolyzable GTP analogues and two mutant Ran proteins on the nuclear import of proteins and U snRNPs in vitro. The mutant Ran proteins have different defects; Q69L (glutamine 69 changed to leucine) is defective in GTP hydrolysis while T24N (threonine 24 changed to asparagine) is defective in binding GTP. Both protein and snRNP import are sensitive either to the presence of the two mutant Ran proteins, which act as dominant negative inhibitors of nuclear import, or to incubation with nonhydrolyzable GTP analogues. This demonstrates that there is a requirement for a GTPase activity for the import of U snRNPs, as well as proteins, into the nucleus. The dominant negative effects of the two mutant Ran proteins indicate that the pathways of protein and snRNP import share at lease one common component. PMID:8636225

  19. Characterization of an immunoglobulin binding protein homolog in the maize floury-2 endosperm mutant.

    PubMed Central

    Fontes, E B; Shank, B B; Wrobel, R L; Moose, S P; OBrian, G R; Wurtzel, E T; Boston, R S

    1991-01-01

    The maize b-70 protein is an endoplasmic reticulum protein overproduced in the floury-2 (fl2) endosperm mutant. The increase in b-70 levels in fl2 plants occurs during seed maturation and is endosperm specific. We have used amino acid sequence homology to identify b-70 as a homolog of mammalian immunoglobulin binding protein (BiP). Purified b-70 fractions contain two 75-kilodalton polypeptides with pl values of 5.3 and 5.4. Both 75-kilodalton polypeptides share several properties with BiP, including the ability to bind ATP and localization within the lumen of the endoplasmic reticulum. In addition, both b-70 polypeptides can be induced in maize cell cultures with tunicamycin treatment. Like BiP, the pl 5.3 form of b-70 is post-translationally modified by phosphorylation and ADP-ribosylation. However, modification of the pl 5.4 species was not detected in vitro or in vivo. Although the b-70 gene is unlinked to fl2, b-70 overproduction is positively correlated with the fl2 gene and is regulated at the mRNA level. In contrast, the fl2 allele negatively affects the accumulation of the major endosperm storage proteins. The physical similarity of b-70 to BiP and its association with abnormal protein accumulation in fl2 endoplasmic reticulum may reflect a biological function to mediate protein folding and assembly in maize endosperm. PMID:1840923

  20. Virus-Specific Proteins Synthesized in Cells Infected with RNA+ Temperature-Sensitive Mutants of Sindbis Virus

    PubMed Central

    Scheele, Christina M.; Pfefferkorn, E. R.

    1970-01-01

    All Sindbis virus temperature-sensitive mutants defective in “late” functions were systematically surveyed by acrylamide-gel electrophoresis for similarities and differences in the intracellular pattern of virus-specific proteins synthesized at the permissive and nonpermissive temperatures. Only cells infected with mutants of complementation group C showed an altered pattern. At the nonpermissive temperature, these mutants failed to induce the synthesis of a polypeptide corresponding to the nucleocapsid protein and instead overproduced a protein of higher molecular weight than either viral structural protein. This defect was shown to be irreversible by the finding that 3H-leucine incorporated at 41.5 C specifically failed to appear in the nucleocapsid of virions subsequently released at 29 C. Attempts to demonstrate a precursor protein in wild-type infections were inconclusive. PMID:5461887

  1. Ultrafast Electronic and Vibrational Dynamics of Stabilized A State Mutants of the Green Fluorescent Protein (GFP): Snipping the Proton Wire

    PubMed Central

    Stoner-Ma, Deborah; Jaye, Andrew A.; Ronayne, Kate L.; Nappa, Jerome; Tonge, Peter J.; Meech, Stephen R.

    2008-01-01

    Two blue absorbing and emitting mutants (S65G/T203V/E222Q and S65T at pH 5.5) of the green fluorescent protein (GFP) have been investigated through ultrafast time resolved infra-red (TRIR) and fluorescence spectroscopy. In these mutants, in which the excited state proton transfer reaction observed in wild type GFP has been blocked, the photophysics are dominated by the neutral A state. It was found that the A* excited state lifetime is short, indicating that it is relatively less stabilised in the protein matrix than the anionic form. However, the lifetime of the A* state can be increased through modifications to the protein structure. The TRIR spectra show that a large shifts in protein vibrational modes on excitation of the A* state occurs in both these GFP mutants. This is ascribed to a change in H-bonding interactions between the protein matrix and the excited state. PMID:19554079

  2. Ultrafast electronic and vibrational dynamics of stabilized A state mutants of the green fluorescent protein (GFP): Snipping the proton wire

    NASA Astrophysics Data System (ADS)

    Stoner-Ma, Deborah; Jaye, Andrew A.; Ronayne, Kate L.; Nappa, Jérôme; Tonge, Peter J.; Meech, Stephen R.

    2008-06-01

    Two blue absorbing and emitting mutants (S65G/T203V/E222Q and S65T at pH 5.5) of the green fluorescent protein (GFP) have been investigated through ultrafast time resolved infra-red (TRIR) and fluorescence spectroscopy. In these mutants, in which the excited state proton transfer reaction observed in wild-type GFP has been blocked, the photophysics are dominated by the neutral A state. It was found that the A∗ excited state lifetime is short, indicating that it is relatively less stabilised in the protein matrix than the anionic form. However, the lifetime of the A state can be increased through modifications to the protein structure. The TRIR spectra show that a large shifts in protein vibrational modes on excitation of the A state occurs in both these GFP mutants. This is ascribed to a change in H-bonding interactions between the protein matrix and the excited state.

  3. Disrupting ER-associated protein degradation suppresses the abscission defect of a weak hae hsl2 mutant in Arabidopsis

    PubMed Central

    Baer, John; Taylor, Isaiah; Walker, John C.

    2016-01-01

    In Arabidopsis thaliana, the process of abscission, or the shedding of unwanted organs, is mediated by two genes, HAESA (HAE) and HAESA-LIKE 2 (HSL2), encoding receptor-like protein kinases (RLKs). The double loss-of-function mutant hae-3 hsl2-3 is completely deficient in floral abscission, but, interestingly, the hae-3 hsl2-9 mutant displays a less severe defect. This mutant was chosen for an ethyl methanesulfonate (EMS) screen to isolate enhancer and suppressor mutants, and two such suppressors are the focus of this study. Pooled DNA from the F2 generation of a parental backcross was analyzed by genome sequencing to reveal candidate genes, two of which complement the suppressor phenotype. These genes, EMS-MUTAGENIZED BRI1 SUPPRESSOR 3 (EBS3) and EBS4, both encode mannosyltransferases involved in endoplasmic reticulum (ER)-associated degradation (ERAD) of proteins. Further analysis of these suppressor lines revealed that suppressor mutations are acting solely on the partially functional hsl2-9 mutant receptor to modify the abscission phenotype. Expressing a hsl2-9–yellow fluorescent protein (YFP) transgene in ebs3 mutants yields a higher fluorescent signal than in EBS3/ebs3, suggesting that these mutants restore abscission by disrupting ERAD to allow accumulation of the hsl2-9 receptor, which probably escapes degradation to be trafficked to the plasma membrane to regain signaling. PMID:27566817

  4. Synthesis of outer membrane proteins in cpxA cpxB mutants of Escherichia coli K-12.

    PubMed Central

    McEwen, J; Sambucetti, L; Silverman, P M

    1983-01-01

    Two major proteins, the murein lipoprotein and the OmpF matrix porin, are deficient in the outer membrane of cpxA cpxB mutants of Escherichia coli K-12. We present evidence that the cpx mutations prevent or retard the translocation of these proteins to the outer membrane. The mutations had no effect on the rate of lipoprotein synthesis. Mutant cells labeled for 5 min with radioactive arginine accumulated as much lipoprotein as otherwise isogenic cpxA+ cpxB+ cells. This lipoprotein accumulated as such; no material synthesized in mutant cells and reactive with antilipoprotein antibodies had the electrophoretic mobility of prolipoprotein. Hence, the initial stages of prolipoprotein insertion into the inner membrane leading to its cleavage to lipoprotein appeared normal. However, after a long labeling interval, mutant cells were deficient in free lipoprotein and lacked lipoprotein covalently bound to peptidoglycan, suggesting that little if any of the lipoprotein synthesized in mutant cells reaches the outer membrane. Immunoreactive OmpF protein could also be detected in extracts of mutant cells labeled for 5 min, but the amount that accumulated was severalfold less in mutant cells than in cpxA+ cpxB+ cells. Analysis of beta-galactosidase synthesis from ompF-lacZ fusion genes showed this difference to be the result of a reduced rate of ompF transcription in mutant cells. Even so, little or none of the ompF protein synthesized in mutant cells was incorporated into the outer membrane. Images PMID:6339479

  5. In vitro protein phosphorylation in head preparations from normal and mutant Drosophila melanogaster.

    PubMed

    Buxbaum, J D; Dudai, Y

    1987-10-01

    We have characterized protein phosphorylation in vitro in subcellular fractions from Drosophila melanogaster heads. Optimal conditions for the incorporation of 32P into proteins, and its dependence on ATP, divalent cations, and cyclic nucleotides have been determined, as well as the effect of inhibitors of ATPase, protein phosphatase, and protein kinase on protein phosphorylation. Among these inhibitors, Zn2+ was found to affect the incorporation of 32P into specific bands and p-hydroxymercuribenzoate was found to be most suited for freezing the activity of both kinases and phosphatases. Cyclic AMP-dependent protein kinase (cAMP-dPK) activity was present in both supernatant (S2) and particulate (P2) fractions, with the majority (60-85%, depending on the homogenization medium) being associated with S2, as determined by phosphorylation of exogenous synapsin I. cAMP-dPK catalyzed the phosphorylation of at least 18 endogenous polypeptides in S2 and at least 10 endogenous polypeptides in P2. These proteins could be classified on the basis of the extent of stimulation of phosphorylation by cyclic nucleotides, dependence on cyclic nucleotide concentration, and rate of phosphorylation. A phosphoprotein of 51 kilodaltons (pp51) was a major component of the S2 and P2 fractions and displayed properties expected from the regulatory subunit of the cAMP-dPK, R-II. A phosphoprotein doublet of approximately 37 kilodaltons (pp37) was stimulated to the largest extent by cAMP in the P2 and S2 fractions. The phosphorylation of several proteins in both fractions was significantly lowered by the mammalian Walsh inhibitor of cAMP-dPK, whereas in some cases the stimulation of phosphorylation of the same proteins by exogeneous cAMP was relatively small. Phosphoproteins from two learning mutants known to be deficient in cAMP metabolism, dnc and rut, were analyzed for their extent of phosphorylation in the presence of a stable cAMP analogue; no significant differences from normal were

  6. Introduction of yeast artificial chromosomes containing mutant human amyloid precursor protein genes into transgenic mice

    SciTech Connect

    Call, L.M.; Lamb, B.T.; Boese, K.F.

    1994-09-01

    Several hypothetical mechanisms have been proposed for the generation and deposition of the amyloid beta (A{beta}) peptide in Alzheimer`s disease (AD). These include overexpression of the amyloid precursor protein (APP) gene, as suggested by Down Syndrome (DS, trisomy 21), and mutation of APP, as suggested by mutations associated with the presence of disease/amyloid deposition in some cases of familial AD (FAD). Although numerous in vitro studies have lead to certain insights into the molecular basis for amyloid deposition, the mechanisms(s) of amyloidogenesis in vivo remains poorly defined. To examine the effect of FAD mutations on amyloidogenesis in an animal model, we have focused on producing APP YAC transgenic mice containing the human APP gene with FAD mutations. These APP YAC transgenics are being produced by introduction of a 650 kb APP YAC through lipid-mediated transfection of ES cells. This strategy has two principal advantages: the APP genomic sequences contain transcriptional regulatory elements required for proper spatial and temporal expression and contain appropriate splice donor and acceptor sites needed to generate the entire spectrum of alternatively spliced APP transcripts. As a first step, we cloned the genomic regions surrounding APP exons 16 and 17 from an APP YAC sublibrary. Both the Swedish and the 717 mutations were then introduced into exons 16 and 17, respectively, by PCR mutagenesis, and subsequently transferred into the 650 kb APP YAC by a two step gene replacement in yeast. The mutant YACs have been introduced into ES cells, and we have determined that these cells are expressing human mutant APP mRNA and protein. These cells are being used to generate transgenic mice. This paradigm should provide the appropriate test of whether a mutant APP gene is capable of producing AD-like pathology in a mouse model.

  7. Expression of mutant amyloid precursor proteins decreases adhesion and delays differentiation of Hep-1 cells.

    PubMed

    Kusiak, J W; Lee, L L; Zhao, B

    2001-03-30

    The amyloid precursor protein (APP) is a type I integral membrane protein and is processed to generate several intra-cellular and secreted fragments. The physiological role of APP and its processed fragments is unclear. Several mutations have been discovered in APP, which are causative of early-onset, familial, neurological disease, including Alzheimer's disease (FAD). These mutations alter the processing of APP and lead to excess production and extra-cellular deposition of A-beta peptide (Abeta). We have examined the role of APP in a cell culture model of endothelial cell function. The endothelial cell line, Hep-1, was stably transfected with wild-type (wt) and FAD mutant forms of APP (mAPP). Secretion of sAPPalpha was reduced in cell lines over-expressing mAPP when these cells were grown on several different substrates. Levels of secreted Abeta were increased as measured by ELISA in the mutant cell lines. Cell adhesion to laminin-, fibronectin-, collagen I-, and collagen IV-coated culture flasks was reduced in all mAPP-expressing cell lines, while in lines over-expressing wt-APP, adhesiveness was slightly increased. Cell lines over-expressing mAPP differentiated more slowly into capillary network-like structures on Matrigel than those expressing wt-APP. No differences were detected among all cell lines in a migration/invasion assay. The results suggest that APP may have a role in cell adhesiveness and maturation of endothelial cells into capillary-like networks. The reduction in adhesion and differentiation in mutant cell lines may be due to reduced amounts of sAPPalpha released into the culture media or toxic effects of increased extracellular Abeta.

  8. Functional analysis of N-linked glycosylation mutants of the measles virus fusion protein synthesized by recombinant vaccinia virus vectors.

    PubMed Central

    Alkhatib, G; Shen, S H; Briedis, D; Richardson, C; Massie, B; Weinberg, R; Smith, D; Taylor, J; Paoletti, E; Roder, J

    1994-01-01

    The role of N-linked glycosylation in the biological activity of the measles virus (MV) fusion (F) protein was analyzed by expressing glycosylation mutants with recombinant vaccinia virus vectors. There are three potential N-linked glycosylation sites located on the F2 subunit polypeptide of MV F, at asparagine residues 29, 61, and 67. Each of the three potential glycosylation sites was mutated separately as well as in combination with the other sites. Expression of mutant proteins in mammalian cells showed that all three sites are used for the addition of N-linked oligosaccharides. Cell surface expression of mutant proteins was reduced by 50% relative to the wild-type level when glycosylation at either Asn-29 or Asn-61 was abolished. Despite the similar levels of cell surface expression, the Asn-29 and Asn-61 mutant proteins had different biological activities. While the Asn-61 mutant was capable of inducing syncytium formation, the Asn-29 mutant protein did not exhibit any significant cell fusion activity. Inactivation of the Asn-67 glycosylation site also reduced cell surface transport of mutant protein but had little effect on its ability to cause cell fusion. However, when the Asn-67 mutation was combined with mutations at either of the other two sites, cleavage-dependent activation, cell surface expression, and cell fusion activity were completely abolished. Our data show that the loss of N-linked oligosaccharides markedly impaired the proteolytic cleavage, stability, and biological activity of the MV F protein. The oligosaccharide side chains in MV F are thus essential for optimum conformation of the extracellular F2 subunit that is presumed to bind cellular membranes. Images PMID:8107215

  9. Mutant Cockayne syndrome group B protein inhibits repair of DNA topoisomerase I-DNA covalent complex.

    PubMed

    Horibata, Katsuyoshi; Saijo, Masafumi; Bay, Mui N; Lan, Li; Kuraoka, Isao; Brooks, Philip J; Honma, Masamitsu; Nohmi, Takehiko; Yasui, Akira; Tanaka, Kiyoji

    2011-01-01

    Two UV-sensitive syndrome patients who have mild photosensitivity without detectable somatic abnormalities lack detectable Cockayne syndrome group B (CSB) protein because of a homozygous null mutation in the CSB gene. In contrast, mutant CSB proteins are produced in CS-B patients with the severe somatic abnormalities of Cockayne syndrome and photosensitivity. It is known that the piggyBac transposable element derived 3 is integrated within the CSB intron 5, and that CSB-piggyBac transposable element derived 3 fusion (CPFP) mRNA is produced by alternative splicing. We found that CPFP or truncated CSB protein derived from CPFP mRNA was stably produced in CS-B patients, and that wild-type CSB, CPFP, and truncated CSB protein interacted with DNA topoisomerase I. We also found that CPFP inhibited repair of a camptothecin-induced topoisomerase I-DNA covalent complex. The inhibition was suppressed by the presence of wild-type CSB, consistent with the autosomal recessive inheritance of Cockayne syndrome. These results suggested that reduced repair of a DNA topoisomerase I-DNA covalent complex because of truncated CSB proteins is involved in the pathogenesis of CS-B. PMID:21143350

  10. v-mos proteins encoded by myeloproliferative sarcoma virus and its ts159 mutant.

    PubMed Central

    Singh, B; Stocking, C; Walker, R; Yang, Y D; Ostertag, W; Arlinghaus, R B

    1992-01-01

    The myeloproliferative sarcoma virus (MPSV) v-mos protein was predicted to be identical in size to p39c-mos because of an observed one-base deletion in the seventh codon of the env-mos open reading frame, which would allow translation to initiate at the methionine equivalent to codon 32 of the env-mos gene. On the basis of published results, p39c-mos is known to have greatly reduced in vitro protein kinase activity compared with p37env-mos encoded by Moloney murine sarcoma virus. Unexpectedly, the relative activity of the MPSV v-mos protein kinase was comparable to that of p37env-mos. Consistent with this finding, the size of MPSV v-mos protein was found to be similar to the size of p37env-mos. Moreover, the pattern and sizes of phosphorylated bands produced by autophosphorylation of the MPSV v-mos protein were similar to those of p37env-mos. These results were confirmed by in vitro transcription-translation of the MPSV v-mos gene. Resequencing portions of the MPSV mos gene failed to show the deletion within codon 7. Except for the codon 262 deletion, other mutations characteristic of MPSV and temperature-sensitive MPSV v-mos genes were confirmed. A glycine-to-arginine mutation at residue 338 of the MPSV env-mos sequence, previously shown to cause thermosensitivity of the mutant virus (termed ts159) transforming function, yielded a v-mos protein that had significantly reduced protein kinase activity in vitro. These findings indicate that MPSV, like other Moloney murine sarcoma virus strains, also encodes a functional env-mos protein. Images PMID:1309903

  11. A mutant of Escherichia coli defective in penicillin-binding protein 5 and lacking D-alanine carboxypeptidase IA.

    PubMed Central

    Nishimura, Y; Suzuki, H; Hirota, Y; Park, J T

    1980-01-01

    A mutant of Escherichia coli defective in penicillin-binding protein 5 activity was isolated. The mutation (pfv) was shown to be located at 14.0 min on the E. coli chromosome map. Loss of penicillin-binding protein 5 in the pfv mutant was associated with the loss of D-alanine carboxypeptidase IA activity and increased sensitivity to beta-lactam antibiotics. We conclude that penicillin-binding protein 5 catalyzes the major D-alanine carboxypeptidase IA activity and that the enzyme activity, in vivo, protects E. coli cells from killing by low inhibitory concentrations of beta-lactam antibiotics. PMID:6995448

  12. Variations in seed protein content of cotton (Gossypium hirsutum L.) mutant lines by in vivo and in vitro mutagenesis.

    PubMed

    Muthusamy, Annamalai; Jayabalan, Narayanasamy

    2013-01-01

    The present work describes the influence of gamma irradiation (GR), ethyl methane sulphonate (EMS) and sodium azide (SA) treatment on yield and protein content of selected mutant lines of cotton. Seeds of MCU 5 and MCU 11 were exposed to gamma rays (GR), ethyl methane sulphonate (EMS) and sodium azide (SA). Lower dose of gamma irradiation (100-500 Gy), 10-50 mM EMS and SA at lower concentration effectively influences in improving the yield and protein content. Significant increase in yield (258.9 g plant(-1)) and protein content (18.63 mg g(-1) d. wt.) as compared to parental lines was noted in M2 generations. During the subsequent field trials, number of mutant lines varied morphologically in terms of yield as well as biochemical characters such as protein. The selected mutant lines were bred true to their characters in M3 and M4 generations. The significant increase in protein content and profiles of the mutant lines with range of 10.21-18.63 mg g(-1). The SDS-PAGE analysis of mutant lines revealed 9 distinct bands of different intensities with range of 26-81 kDa. The difference in intensity of bands was more (41, 50 and 58 kDa) in the mutant lines obtained from in vitro mutation than in vivo mutation. Significance of such stimulation in protein content correlated with yielding ability of the mutant lines of cotton in terms of seed weight per plant. The results confirm that in cotton it is possible to enhance the both yield and biochemical characters by in vivo and in vitro mutagenic treatments.

  13. High level of antibiotic production in a double polyphosphate kinase and phosphate-binding protein mutant of Streptomyces lividans.

    PubMed

    Díaz, Margarita; Sevillano, Laura; Rico, Sergio; Lombo, Felipe; Braña, Alfredo F; Salas, Jose A; Mendez, Carmen; Santamaría, Ramón I

    2013-05-01

    Phosphate metabolism regulates most of the life processes of microorganisms. In the present work we obtained and studied a Streptomyces lividans ppk/pstS double mutant, which lacks polyphosphate kinase (PPK) and the high-affinity phosphate-binding protein (PstS), impairing at the same time the intracellular storage of polyphosphate and the intake of new inorganic phosphate from a phosphate-limited medium, respectively. In some of the aspects analyzed, the ppk/pstS double mutant was more similar to the wt strain than was the single pstS mutant. The double mutant was thus able to grow in phosphate-limited media, whereas the pstS mutant required the addition of 1 mM phosphate under the assay conditions used. The double mutant was able to incorporate more than one fourth of the inorganic phosphate incorporated by the wt strain, whereas phosphate incorporation was almost completely impaired in the pstS mutant. Noteworthy, under phosphate limitation conditions, the double ppk/pstS mutant showed a higher production of the endogenous antibiotic actinorhodin and the heterologous antitumor 8-demethyl-tetracenomycin (up to 10-fold with respect to the wt strain), opening new possibilities for the use of this strain in the heterologous expression of antibiotic pathways.

  14. Mutant Brucella abortus Membrane Fusogenic Protein Induces Protection against Challenge Infection in Mice

    PubMed Central

    de Souza Filho, Job Alves; Martins, Vicente de Paulo; Campos, Priscila Carneiro; Alves-Silva, Juliana; Santos, Nathalia V.; de Oliveira, Fernanda Souza; Menezes, Gustavo B.; Azevedo, Vasco; Cravero, Silvio Lorenzo

    2015-01-01

    Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies. PMID:25644010

  15. Mutant Brucella abortus membrane fusogenic protein induces protection against challenge infection in mice.

    PubMed

    de Souza Filho, Job Alves; de Paulo Martins, Vicente; Campos, Priscila Carneiro; Alves-Silva, Juliana; Santos, Nathalia V; de Oliveira, Fernanda Souza; Menezes, Gustavo B; Azevedo, Vasco; Cravero, Silvio Lorenzo; Oliveira, Sergio Costa

    2015-04-01

    Brucella species can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of the Brucella membrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed that B. abortus Δmfp::kan and Δomp19::kan deletion mutant strains have reduced persistence in vivo in C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kan or Δomp19::kan strain expressing green fluorescent protein (GFP) approximately 80% or 65% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments. B. abortus Δomp19::kan was attenuated in vivo but had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kan strain had a lower virulence in these same mouse models. Furthermore, Δmfp::kan and Δomp19::kan strains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kan strain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kan strain induced protection similar to that of RB51. Thus, these results demonstrate that Brucella Mfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kan mutant may serve as a potential vaccine candidate in future studies.

  16. Mutant p53 proteins bind DNA in a DNA structure-selective mode

    PubMed Central

    Göhler, Thomas; Jäger, Stefan; Warnecke, Gabriele; Yasuda, Hideyo; Kim, Ella; Deppert, Wolfgang

    2005-01-01

    Despite the loss of sequence-specific DNA binding, mutant p53 (mutp53) proteins can induce or repress transcription of mutp53-specific target genes. To date, the molecular basis for transcriptional modulation by mutp53 is not understood, but increasing evidence points to the possibility that specific interactions of mutp53 with DNA play an important role. So far, the lack of a common denominator for mutp53 DNA binding, i.e. the existence of common sequence elements, has hampered further characterization of mutp53 DNA binding. Emanating from our previous discovery that DNA structure is an important determinant of wild-type p53 (wtp53) DNA binding, we analyzed the binding of various mutp53 proteins to oligonucleotides mimicking non-B DNA structures. Using various DNA-binding assays we show that mutp53 proteins bind selectively and with high affinity to non-B DNA. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, mutp53 DNA binding to non-B DNA is solely dependent on the stereo-specific configuration of the DNA, and not on DNA sequence. We propose that DNA structure-selective binding of mutp53 proteins is the basis for the well-documented interaction of mutp53 with MAR elements and for transcriptional activities mediates by mutp53. PMID:15722483

  17. Hereditary protein C deficiency caused by compound heterozygous mutants in two independent Chinese families.

    PubMed

    Wu, Ying-Ting; Yue, Fei; Wang, Min; Lu, Ye-Ling; Dai, Jing; Ding, Qiu-Lan; Wang, Hong-Li; Chen, Hui-Fen; Wang, Xue-Feng

    2014-12-01

    We report two compound heterozygous mutants that caused severe type I protein C (PC) deficiency in two independent Chinese families.PC antigen was determined by enzyme-linked immunosorbent assay (ELISA), and PC activity was measured by chromogenic assay. Genetic mutations were screened with polymerase chain reaction (PCR) followed by direct sequencing. PC mutants were transiently expressed in COS-7 cells for the evaluation of PC secretory activity and function. The subcellular location was visualised by immunofluorescence assay. The structural analysis of mutation was performed as well.Compound heterozygous mutations of Arg178Trp and Asp255His with reduced PC activity and antigen levels were identified in Proband 1, a 28-year-old male with deep vein thrombosis (DVT) and pulmonary embolism. The other mutations of Leu-34Pro and Thr295Ile with reduced PC activity and antigen levels were identified in Proband 2, a 19-year-old male with DVT. The PC activities with Arg178Trp, Asp255His, Leu-34Pro and Thr295Ile mutations decreased significantly. Immunofluorescence assay demonstrated that only trace amount of PC with novel Thr295Ile mutation was transported to the Golgi apparatus. Subsequent structural analysis indicated severe impairments of intracellular folding and secretion.The two rare compound heterozygous mutations could cause type I PC deficiency via impairment of secretory activity of PC.

  18. Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins

    SciTech Connect

    Leung, Daisy W.; Borek, Dominika; Farahbakhsh, Mina; Ramanan, Parameshwaran; Nix, Jay C.; Wang, Tianjiao; Prins, Kathleen C.; Otwinowski, Zbyszek; Honzatko, Richard B.; Helgeson, Luke A.; Basler, Christopher F.; Amarasinghe, Gaya K.

    2010-06-21

    VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6{sub 1}22, P2{sub 1}2{sub 1}2{sub 1} and P2{sub 1}, respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source.

  19. Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein

    PubMed Central

    Picone, Delia

    2016-01-01

    MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques. PMID:27340829

  20. Impaired development of hippocampal mossy fibre synapses in mouse mutants for the presynaptic scaffold protein Bassoon.

    PubMed

    Lanore, Frederic; Blanchet, Christophe; Fejtova, Anna; Pinheiro, Paulo; Richter, Karin; Balschun, Detlef; Gundelfinger, Eckart; Mulle, Christophe

    2010-06-15

    Bassoon, a protein highly concentrated at the synaptic active zone, is thought to participate in the organization of the cytomatrix at the site of neurotransmitter release. Bassoon is amongst the first proteins to accumulate at newly formed synaptic junctions, raising the question of the functional role of this protein in the early stages of synaptic development. Here we show that the course of synaptic maturation of hippocampal mossy fibre (MF) synapses (glutamatergic synapses with multiple release sites) is markedly altered during the first 2 weeks of postnatal development in mutant mice lacking the central region of Bassoon (Bsn(-/-) mice). At postnatal day 7 (P7), Bsn(-/-) mice display large amplitude MF-EPSCs with decreased paired pulse ratios, an abnormality which may be linked to deficits in the organization of the presynaptic active zone. Surprisingly, 1 week later, decreased MF-EPSCs amplitude is observed in Bsn(-/-) mice, consistent with the inactivation of a subset of synaptic release sites. Finally, at more mature states a decreased posttetanic potentiation is observed at MF-synapses. These results support the notion that Bassoon is important for organizing the presynaptic active zone during the postnatal maturation of glutamatergic synapses.

  1. Vaccinia virus B1 kinase: phenotypic analysis of temperature-sensitive mutants and enzymatic characterization of recombinant proteins.

    PubMed Central

    Rempel, R E; Traktman, P

    1992-01-01

    The vaccinia virus B1 gene encodes a 34-kDa protein with homology to protein kinases. In L cells infected nonpermissively with mutants containing lesions in the B1 gene (ts2 and ts25), the infectious cycle arrests prior to DNA replication. In this report, we demonstrate that DNA synthesis ceases when cultures infected with these mutants at 32 degrees C are shifted to the nonpermissive temperature (39.5 degrees C) in the midst of DNA replication. We also show that B1 protein is synthesized transiently during the early phase of infection, even when the progression to later stages of gene expression is prevented. Although wild-type (wt) B1 is stable, the ts B1 proteins are markedly labile in both L and BSC40 cells at both permissive and nonpermissive temperatures. These results suggest that the ts phenotype of the mutants is complex and may in part reflect a temperature-dependent requirement for kinase activity, an induction of temperature sensitivity in B1 substrates under nonpermissive conditions, and/or ts complementation by host factors. To facilitate biochemical analyses, recombinant wt B1, ts2 B1, and ts25 B1 were produced in Escherichia coli. The wt protein was able to phosphorylate serine and threonine residues on several exogenous substrates in vitro. The activity of ts25 B1 was 3% that of the wt enzyme, and no detectable kinase activity was associated with ts2 B1. In light of the inactivity of the ts2 B1 protein in vitro and its extreme lability in vivo, we attempted to isolate a vaccinia virus B1 null mutant by targeted interruption of the B1 gene at 32 degrees C. No null mutants were isolated. These results indicate that the B1 protein kinase provides a vital function which cannot be supplied by the host or circumvented by incubation at 32 degrees C. Images PMID:1602551

  2. Thermodynamics of protein denaturation at temperatures over 100 °C: CutA1 mutant proteins substituted with hydrophobic and charged residues

    NASA Astrophysics Data System (ADS)

    Matsuura, Yoshinori; Takehira, Michiyo; Joti, Yasumasa; Ogasahara, Kyoko; Tanaka, Tomoyuki; Ono, Naoko; Kunishima, Naoki; Yutani, Katsuhide

    2015-10-01

    Although the thermodynamics of protein denaturation at temperatures over 100 °C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (Td) ranging from 101 to 113 °C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mutant protein (Td = 137 °C) by substituting six residues with charged ones. Thermodynamic analyses of these mutant proteins indicated that the hydrophobic mutants were stabilized by the accumulation of denaturation enthalpy (ΔH) with no entropic gain from hydrophobic solvation around 100 °C, and that the stabilization due to salt bridges resulted from both the increase in ΔH from ion-ion interactions and the entropic effect of the electrostatic solvation over 113 °C. This is the first experimental evidence that has successfully overcome the typical technical difficulties.

  3. Thermodynamics of protein denaturation at temperatures over 100 °C: CutA1 mutant proteins substituted with hydrophobic and charged residues.

    PubMed

    Matsuura, Yoshinori; Takehira, Michiyo; Joti, Yasumasa; Ogasahara, Kyoko; Tanaka, Tomoyuki; Ono, Naoko; Kunishima, Naoki; Yutani, Katsuhide

    2015-01-01

    Although the thermodynamics of protein denaturation at temperatures over 100 °C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (Td) ranging from 101 to 113 °C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mutant protein (Td = 137 °C) by substituting six residues with charged ones. Thermodynamic analyses of these mutant proteins indicated that the hydrophobic mutants were stabilized by the accumulation of denaturation enthalpy (ΔH) with no entropic gain from hydrophobic solvation around 100 °C, and that the stabilization due to salt bridges resulted from both the increase in ΔH from ion-ion interactions and the entropic effect of the electrostatic solvation over 113 °C. This is the first experimental evidence that has successfully overcome the typical technical difficulties.

  4. On the role of a Lipid-Transfer Protein. Arabidopsis ltp3 mutant is compromised in germination and seedling growth.

    PubMed

    Pagnussat, Luciana A; Oyarburo, Natalia; Cimmino, Carlos; Pinedo, Marcela L; de la Canal, Laura

    2015-01-01

    Plant Lipid-Transfer Proteins (LTPs) exhibit the ability to reversibly bind/transport lipids in vitro. LTPs have been involved in diverse physiological processes but conclusive evidence on their role has only been presented for a few members, none of them related to seed physiology. Arabidopsis seeds rely on storage oil breakdown to supply carbon skeletons and energy for seedling growth. Here, Arabidopsis ltp3 mutant was analyzed for its ability to germinate and for seedling establishment. Ltp3 showed delayed germination and reduced germination frequency. Seedling growth appeared reduced in the mutant but this growth restriction was rescued by the addition of an exogenous carbon supply, suggesting a defective oil mobilization. Lipid breakdown analysis during seedling growth revealed a differential profile in the mutant compared to the wild type. The involvement of LTP3 in germination and seedling growth and its relationship with the lipid transfer ability of this protein is discussed.

  5. On the role of a Lipid-Transfer Protein. Arabidopsis ltp3 mutant is compromised in germination and seedling growth.

    PubMed Central

    Pagnussat, Luciana A; Oyarburo, Natalia; Cimmino, Carlos; Pinedo, Marcela L; de la Canal, Laura

    2015-01-01

    Plant Lipid-Transfer Proteins (LTPs) exhibit the ability to reversibly bind/transport lipids in vitro. LTPs have been involved in diverse physiological processes but conclusive evidence on their role has only been presented for a few members, none of them related to seed physiology. Arabidopsis seeds rely on storage oil breakdown to supply carbon skeletons and energy for seedling growth. Here, Arabidopsis ltp3 mutant was analyzed for its ability to germinate and for seedling establishment. Ltp3 showed delayed germination and reduced germination frequency. Seedling growth appeared reduced in the mutant but this growth restriction was rescued by the addition of an exogenous carbon supply, suggesting a defective oil mobilization. Lipid breakdown analysis during seedling growth revealed a differential profile in the mutant compared to the wild type. The involvement of LTP3 in germination and seedling growth and its relationship with the lipid transfer ability of this protein is discussed. PMID:26479260

  6. Cancer therapeutic approach based on conformational stabilization of mutant p53 protein by small peptides

    PubMed Central

    Tal, Perry; Eizenberger, Shay; Cohen, Elad; Goldfinger, Naomi; Pietrokovski, Shmuel; Oren, Moshe; Rotter, Varda

    2016-01-01

    The p53 tumor suppressor serves as a major barrier against malignant transformation. Over 50% of tumors inactivate p53 by point mutations in its DNA binding domain. Most mutations destabilize p53 protein folding, causing its partial denaturation at physiological temperature. Thus a high proportion of human tumors overexpress a potential potent tumor suppressor in a non-functional, misfolded form. The equilibrium between the properly folded and misfolded states of p53 may be affected by molecules that interact with p53, stabilizing its native folding and restoring wild type p53 activity to cancer cells. To select for mutant p53 (mutp53) reactivating peptides, we adopted the phage display technology, allowing interactions between mutp53 and random peptide libraries presented on phages and enriching for phage that favor the correctly folded p53 conformation. We obtained a large database of potential reactivating peptides. Lead peptides were synthesized and analyzed for their ability to restore proper p53 folding and activity. Remarkably, many enriched peptides corresponded to known p53-binding proteins, including RAD9. Importantly, lead peptides elicited dramatic regression of aggressive tumors in mouse xenograft models. Such peptides might serve as novel agents for human cancer therapy. PMID:26943582

  7. Yeast phenotype classifies mammalian protein kinase C cDNA mutants.

    PubMed Central

    Riedel, H; Su, L; Hansen, H

    1993-01-01

    The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of eukaryotic cellular signals. Molecular dissection of their mechanisms of action has been limited in part by the lack of random mutagenesis approaches and by the complexity of signaling pathways in mammalian cells which involve multiple PKC isoforms. Here we present a rapid screen which permits the quantification of mammalian PKC activity phenotypically in the yeast Saccharomyces cerevisiae. Bovine PKC alpha cDNA is functionally expressed in S. cerevisiae. This results in a phorbol ester response: a fourfold increase in the cell doubling time and a substantial decrease in yeast colony size on agar plates. We have expressed pools of bovine PKC alpha cDNAs mutagenized by Bal 31 deletion of internal, amino-terminal, or carboxyl-terminal sequences and have identified three classes of mutants on the basis of their distinct yeast phenotypes. Representatives of each class were analyzed. An internal deletion of amino acids (aa) 172 to 225 displayed ligand-dependent but reduced catalytic activity, an amino-terminal truncation of aa 1 to 153 displayed elevated and ligand-independent activity, and a carboxyl-terminal 26-aa truncation (aa 647 to 672) lacked activity under any conditions. Additional mutations confirmed the distinct functional characteristics of these classes. Our data show that deletion of the V1 and C1 regions results in elevated basal catalytic activity which is still Ca2+ responsive. Internal deletions in the V2 and C2 regions do not abolish phorbol ester or Ca2+ regulation of PKC activity, suggesting that most of the C2 domain is not essential for phorbol ester stimulation and most of the regulatory domain is dispensable for Ca2+ regulation of PKC activity. These distinct activities od the PKC mutants correlate with a specific and proportional yeast phenotype and are quantified on agar plates by yeast colony size. This provides a phenotypic screen which is suitable

  8. Flat cells come full sphere: Are mutant cytoskeletal-related proteins oncoprotein-monsters or useful immunogens?

    PubMed

    Parry, Michele L; Blanck, George

    2016-01-01

    Osteogenesis imperfecta is inherited as a dominant disease because if one allele is mutated, it contributes a mutant, destructive subunit polypeptide to collagen, which requires many subunits to form normal, polymeric, collagenous structures. Recent cancer genome atlas (TCGA) data indicate that cytoskeletal-related proteins are among the most commonly mutated proteins in human cancers, in distinct mutation frequency groups, i.e., including low mutation frequency groups. Part of the explanation for this observation is likely to be the fact that many of the coding regions for these proteins are very large, and indeed, it is likely these coding regions are mutated in many cells that never become cancerous. However, it would not be surprising if mutations in cytoskeletal proteins, when combined with oncoprotein or tumor suppressor protein mutations, had significant impacts on cancer development, for a number of reasons, including results obtained almost 5 decades ago indicating that well-spread cells in tissue culture, with well-formed cytoskeletons, were less tumorigenic than spherical cells with disrupted cytoskeletons. This raises the question, are mutant cytoskeletal proteins, which would likely interfere with polymer formation, a new class of oncoproteins, in particular, dominant negative oncoproteins? If these proteins are so commonly mutant, could they be the bases for common cancer vaccines?

  9. Flat cells come full sphere: Are mutant cytoskeletal-related proteins oncoprotein-monsters or useful immunogens?

    PubMed Central

    Parry, Michele L; Blanck, George

    2016-01-01

    Osteogenesis imperfecta is inherited as a dominant disease because if one allele is mutated, it contributes a mutant, destructive subunit polypeptide to collagen, which requires many subunits to form normal, polymeric, collagenous structures. Recent cancer genome atlas (TCGA) data indicate that cytoskeletal-related proteins are among the most commonly mutated proteins in human cancers, in distinct mutation frequency groups, i.e., including low mutation frequency groups. Part of the explanation for this observation is likely to be the fact that many of the coding regions for these proteins are very large, and indeed, it is likely these coding regions are mutated in many cells that never become cancerous. However, it would not be surprising if mutations in cytoskeletal proteins, when combined with oncoprotein or tumor suppressor protein mutations, had significant impacts on cancer development, for a number of reasons, including results obtained almost 5 decades ago indicating that well-spread cells in tissue culture, with well-formed cytoskeletons, were less tumorigenic than spherical cells with disrupted cytoskeletons. This raises the question, are mutant cytoskeletal proteins, which would likely interfere with polymer formation, a new class of oncoproteins, in particular, dominant negative oncoproteins? If these proteins are so commonly mutant, could they be the bases for common cancer vaccines? PMID:26225584

  10. Influenza virus-specific RNA and protein syntheses in cells infected with temperature-sensitive mutants defective in the genome segment encoding nonstructural proteins.

    PubMed

    Wolstenholme, A J; Barrett, T; Nichol, S T; Mahy, B W

    1980-07-01

    Virus-specific protein and RNA syntheses have been analyzed in chicken embryo fibroblast cells infected with two group IV temperature-sensitive (ts) mutants of influenza A (fowl plague) virus in which the ts lesion maps in RNA segment 8 (J. W. Almond, D. McGeoch, and R. D. Barry, Virology 92:416-427, 1979), known to code to code for two nonstructural proteins, NS1 and NS2. Both mutants induced the synthesis of similar amounts of all the early virus-specific proteins (P1, P2, P3, NP, and NS1) at temperatures that were either permissive (34 degrees C) or nonpermissive (40.5 degrees C) for replication. However, the synthesis of M protein, which normally accumulates late in infection, was greatly reduced in ts mutant-infected cells at 40.5 degrees C compared to 34 degrees C. The NS2 protein was not detected at either temperature in cells infected with one mutant (mN3), and was detected only at the permissive temperature in cells infected with mutant ts47. There was no overall reduction in polyadenylated (A+) complementary RNA, which functions as mRNA, in cells infected with these mutants at 40.5 degrees C compared to 34 degrees C, nor was there any evidence of selective accumulation of this type of RNA within the nucleus at the nonpermissive temperature. No significant differences in ts mutant virion RNA transcriptase activity were detected by assays in vitro at 31 and 40.5 degrees C compared to wild-type virus. Virus-specific non-polyadenylated (A-) complementary RNA, which is believed to act as the template for new virion RNA production, accumulated normally in cells at both 34 and 40.5 degrees C, but at 40.5 degrees C accumulation of new virion RNA was reduced by greater than 90% when compared to accumulation at 34 degrees C.

  11. Interactions and stabilities of the UV RESISTANCE LOCUS8 (UVR8) protein dimer and its key mutants.

    PubMed

    Wu, Min; Strid, Ake; Eriksson, Leif A

    2013-07-22

    The dimeric UVR8 protein is an ultraviolet-B radiation (280-315 nm) photoreceptor responsible for the first step in UV-B regulation of gene expression in plants. Its action comprises the actual absorption of the UV quanta by a tryptophan array at the protein-protein interface, followed by monomerization and subsequent aggregation with downstream signaling components. A crystal structure of the Arabidopsis thaliana tryptophan-rich wild type UVR8 protein dimer was recently published, showing the presence of several salt bridges involving arginines R146, R286, R338, and R354. In this work, molecular dynamics simulations in conjunction with umbrella sampling were used to calculate the binding free energy for the wild type UVR8 dimer and three of its mutants (R286A, R338A, and R286A/R338A), in order to verify whether the key mutants are able to disrupt the dimeric structure as indicated experimentally.

  12. Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding.

    PubMed

    Granoff, Dan M; Giuntini, Serena; Gowans, Flor A; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T

    2016-01-01

    Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287

  13. Structural characterization of V57D and V57P mutants of human cystatin C, an amyloidogenic protein

    SciTech Connect

    Orlikowska, Marta; Szymańska, Aneta; Skowron, Piotr; Jankowska, Elżbieta

    2013-04-01

    Val57 point mutants of human cystatin C, which were designed to assess the influence of changes in the properties of the L1 loop on the dimerization propensity, were structurally characterized. Wild-type human cystatin C (hCC wt) is a low-molecular-mass protein (120 amino-acid residues, 13 343 Da) that is found in all nucleated cells. Physiologically, it functions as a potent regulator of cysteine protease activity. While the biologically active hCC wt is a monomeric protein, all crystallization efforts to date have resulted in a three-dimensional domain-swapped dimeric structure. In the recently published structure of a mutated hCC, the monomeric fold was preserved by a stabilization of the conformationally constrained loop L1 caused by a single amino-acid substitution: Val57Asn. Additional hCC mutants were obtained in order to elucidate the relationship between the stability of the L1 loop and the propensity of human cystatin C to dimerize. In one mutant Val57 was substituted by an aspartic acid residue, which is favoured in β-turns, and in the second mutant proline, a residue known for broadening turns, was substituted for the same Val57. Here, 2.26 and 3.0 Å resolution crystal structures of the V57D andV57P mutants of hCC are reported and their dimeric architecture is discussed in terms of the stabilization and destabilization effects of the introduced mutations.

  14. Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

    PubMed Central

    Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.

    2016-01-01

    Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens. PMID:27668287

  15. Enhanced protective antibody to a mutant meningococcal factor H-binding protein with low-factor H binding

    PubMed Central

    Granoff, Dan M.; Giuntini, Serena; Gowans, Flor A.; Lujan, Eduardo; Sharkey, Kelsey; Beernink, Peter T.

    2016-01-01

    Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens.

  16. Dystrophin Hot-Spot Mutants Leading to Becker Muscular Dystrophy Insert More Deeply into Membrane Models than the Native Protein.

    PubMed

    Ameziane-Le Hir, Sarah; Paboeuf, Gilles; Tascon, Christophe; Hubert, Jean-François; Le Rumeur, Elisabeth; Vié, Véronique; Raguénès-Nicol, Céline

    2016-07-26

    Dystrophin (DYS) is a membrane skeleton protein whose mutations lead to lethal Duchenne muscular dystrophy or to the milder Becker muscular dystrophy (BMD). One third of BMD "in-frame" exon deletions are located in the region that codes for spectrin-like repeats R16 to R21. We focused on four prevalent mutated proteins deleted in this area (called RΔ45-47, RΔ45-48, RΔ45-49, and RΔ45-51 according to the deleted exon numbers), analyzing protein/membrane interactions. Two of the mutants, RΔ45-48 and RΔ45-51, led to mild pathologies and displayed a similar triple coiled-coil structure as the full-length DYS R16-21, whereas the two others, RΔ45-47 and RΔ45-49, induced more severe pathologies and showed "fractional" structures unrelated to the normal one. To explore lipid packing, small unilamellar liposomes (SUVs) and planar monolayers were used at various initial surface pressures. The dissociation constants determined by microscale thermophoresis (MST) were much higher for the full-length DYS R161-21 than for the mutants; thus the wild type protein has weaker SUV binding. Comparing surface pressures after protein adsorption and analysis of atomic force microscopy images of mixed protein/lipid monolayers revealed that the mutants insert more into the lipid monolayer than the wild type does. In fact, in both models every deletion mutant showed more interactions with membranes than the full-length protein did. This means that mutations in the R16-21 part of dystrophin disturb the protein's molecular behavior as it relates to membranes, regardless of whether the accompanying pathology is mild or severe. PMID:27367833

  17. A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis

    PubMed Central

    1989-01-01

    A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F- actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development. PMID:2537840

  18. Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins.

    PubMed Central

    Blasina, A; Kittell, B L; Toukdarian, A E; Helinski, D R

    1996-01-01

    The broad host range plasmid RK2 replicates and regulates its copy number in a wide range of Gram-negative bacteria. The plasmid-encoded trans-acting replication protein TrfA and the origin of replication oriV are sufficient for controlled replication of the plasmid in all Gram-negative bacteria tested. The TrfA protein binds specifically to direct repeat sequences (iterons) at the origin of replication. A replication control model, designated handcuffing or coupling, has been proposed whereby the formation of coupled TrfA-oriV complexes between plasmid molecules results in hindrance of origin activity and, consequently, a shut-down of plasmid replication under conditions of higher than normal copy number. Therefore, according to this model, the coupling activity of an initiation protein is essential for copy number control and a copy-up initiation protein mutant should have reduced ability to form coupled complexes. To test this model for plasmid RK2, two previously characterized copy-up TrfA mutations, trfA-254D and trfA-267L, were combined and the resulting copy-up double mutant TFrfA protein TrfA-254D/267L was characterized. Despite initiating runaway (uncontrolled) replication in vivo, the copy-up double-mutant TrfA protein exhibited replication kinetics similar to the wild-type protein in vitro. Purified TrfA-254D, TrfA-267L, and TrfA-254D/267L proteins were then examined for binding to the iterons and for coupling activity using an in vitro ligase-catalyzed multimerization assay. It was found that both single and double TrfA mutant proteins exhibited substantially reduced (single mutants) or barely detectable (double mutant) levels of coupling activity while not being diminished in their capacity to bind to the origin of replication. These observations provide direct evidence in support of the coupling model of replication control. Images Fig. 1 Fig. 2 Fig. 4 PMID:8622975

  19. A poliovirus 2A(pro) mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects.

    PubMed Central

    Ventoso, I; Carrasco, L

    1995-01-01

    Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells. PMID:7666528

  20. Development of photochemical activity in relation to pigment and membrane protein accumulation in chloroplasts of barley and its virescens mutant.

    PubMed

    Kyle, D J; Zalik, S

    1982-06-01

    The development of photochemical activity in relation to pigment and membrane protein accumulation in chloroplasts of greening wild-type barley (Hordeum vulgare L. cv. Gateway) and its virescens mutant were studied. The rate of chlorophyll accumulation per plastid was faster in the wild-type than in the mutant seedlings upon illumination after 6 days of etiolation, but was not different after 8 days. Although the protein content per plastid did not vary during greening, there was a change in the sodium dodecyl sulfate-polyacrylamide gel polypeptide profiles. High molecular weight proteins of 96,000 and 66,000 decreased whereas those at 34,000, 27,000 and 22,000 increased in relative quantity as a function of greening. The fully greened mutant seedlings were not deficient in the light-harvesting chlorophyll protein complex (LHC) or the reaction centers of photosystem I and photosystem II. Photosystem I-associated photochemical activities appeared within the first hour of plastid development and photosystem II associated activities and O(2) evolution within the next 6 hours. In all cases, the developmental rates per unit protein were slower in the mutant following 6 days of etiolation, but no differences between the two genotypes could be seen after 8 days due to a decrease in the developmental rate of the wild-type chloroplasts. An increase in photosynthetic unit size associated with plastid morphogenesis was faster in the wild-type seedlings after 6 days, but again the difference was negligible after 8 days. It was concluded that no single measured photochemical parameter is affected by this mutation, but rather, all aspects of chloroplast development are affected similarly by an overall reduction in the rate of chloroplast morphogenesis. This mutant, therefore, undergoes the normal pattern of proplastid to chloroplast development, but at a markedly reduced rate.

  1. Metabolic and Phenotypic Differences between Mice Producing a Werner Syndrome Helicase Mutant Protein and Wrn Null Mice.

    PubMed

    Aumailley, Lucie; Garand, Chantal; Dubois, Marie Julie; Johnson, F Brad; Marette, André; Lebel, Michel

    2015-01-01

    Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter mean life span. In contrast, mice lacking the entire Wrn protein (i.e. Wrn null mice) do not exhibit a premature aging phenotype. In this study, we used a targeted mass spectrometry-based metabolomic approach to identify serum metabolites that are differentially altered in young Wrn helicase mutant and Wrn null mice. An antibody-based quantification of 43 serum cytokines and markers of cardiovascular disease risk complemented this study. We found that Wrn helicase mutants exhibited elevated and decreased levels, respectively, of the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine IL-18. Wrn helicase mutants also exhibited an increase in serum hydroxyproline and plasminogen activator inhibitor-1, markers of extracellular matrix remodeling of the vascular system and inflammation in aging. We also observed an abnormal increase in the ratio of very long chain to short chain lysophosphatidylcholines in the Wrn helicase mutants underlying a peroxisome perturbation in these mice. Remarkably, the Wrn mutant helicase protein was mislocalized to the endoplasmic reticulum and the peroxisomal fractions in liver tissues. Additional analyses with mouse embryonic fibroblasts indicated a severe defect of the autophagy flux in cells derived from Wrn helicase mutants compared to wild type and Wrn null animals. These results indicate that the deleterious effects of the helicase-deficient Wrn protein are mediated by the dysfunction of several cellular organelles. PMID:26447695

  2. Metabolic and Phenotypic Differences between Mice Producing a Werner Syndrome Helicase Mutant Protein and Wrn Null Mice

    PubMed Central

    Aumailley, Lucie; Garand, Chantal; Dubois, Marie Julie; Johnson, F. Brad; Marette, André; Lebel, Michel

    2015-01-01

    Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter mean life span. In contrast, mice lacking the entire Wrn protein (i.e. Wrn null mice) do not exhibit a premature aging phenotype. In this study, we used a targeted mass spectrometry-based metabolomic approach to identify serum metabolites that are differentially altered in young Wrn helicase mutant and Wrn null mice. An antibody-based quantification of 43 serum cytokines and markers of cardiovascular disease risk complemented this study. We found that Wrn helicase mutants exhibited elevated and decreased levels, respectively, of the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine IL-18. Wrn helicase mutants also exhibited an increase in serum hydroxyproline and plasminogen activator inhibitor-1, markers of extracellular matrix remodeling of the vascular system and inflammation in aging. We also observed an abnormal increase in the ratio of very long chain to short chain lysophosphatidylcholines in the Wrn helicase mutants underlying a peroxisome perturbation in these mice. Remarkably, the Wrn mutant helicase protein was mislocalized to the endoplasmic reticulum and the peroxisomal fractions in liver tissues. Additional analyses with mouse embryonic fibroblasts indicated a severe defect of the autophagy flux in cells derived from Wrn helicase mutants compared to wild type and Wrn null animals. These results indicate that the deleterious effects of the helicase-deficient Wrn protein are mediated by the dysfunction of several cellular organelles. PMID:26447695

  3. BRCA-associated protein 1 mutant cholangiocarcinoma: an aggressive disease subtype

    PubMed Central

    Al-Shamsi, Humaid O.; Anand, Deepa; Shroff, Rachna T.; Jain, Apurva; Zuo, Mingxin; Conrad, Claudius; Vauthey, Jean-Nicolas

    2016-01-01

    Background BRCA-associated protein 1, an enzyme encoded by the BAP1 gene, is commonly mutated in uveal melanoma, mesothelioma, and renal cancers. Tumors with BAP1 mutation follow an aggressive course. BAP1 mutations have also been observed in cholangiocarcinoma (CCA). The clinical phenotype of BAP1 mutant CCA may yield useful prognostic and therapeutic information but has not been defined. Methods The records of CCA patients who underwent next-generation sequencing (NGS) were reviewed, and data on clinical, histopathological, genetic, and radiological features; response to therapy; time to progression; and survival were analyzed. Results Twenty-two cases of BAP1-mutation associated CCA were diagnosed from January 1, 2009, to February 1, 2015, at our center. Twenty patients had intrahepatic CCA and two had extrahepatic CCA. Tumor sizes (largest dimension) ranged from 2 to 16 cm (mean, 8.5 cm). Twelve patients had tumors that were poorly differentiated. Majority of the patients had advanced disease at presentation and 13 had bone metastases. Thirteen patients (59%) experienced rapidly progressive disease following primary therapy (chemotherapy or surgical resection). The mean time to tumor progression was 3.8 months after the first line chemotherapy. Conclusions BAP1 mutation in CCA may be associated with aggressive disease and poor response to standard therapies. Therefore, BAP1-targeted therapies need to be investigated. PMID:27563445

  4. Molecular characterization of an acidic region deletion mutant of Cockayne syndrome group B protein.

    PubMed

    Sunesen, M; Selzer, R R; Brosh, R M; Balajee, A S; Stevnsner, T; Bohr, V A

    2000-08-15

    Cockayne syndrome (CS) is a human genetic disorder characterized by post-natal growth failure, neurological abnormalities and premature aging. CS cells exhibit high sensitivity to UV light, delayed RNA synthesis recovery after UV irradiation and defective transcription-coupled repair (TCR). Two genetic complementation groups of CS have been identified, designated CS-A and CS-B. The CSB gene encodes a helicase domain and a highly acidic region N-terminal to the helicase domain. This study describes the genetic characterization of a CSB mutant allele encoding a full deletion of the acidic region. We have tested its ability to complement the sensitivity of UV61, the hamster homolog of human CS-B cells, to UV and the genotoxic agent N-acetoxy-2-acetylaminofluorene (NA-AAF). Deleting 39 consecutive amino acids, of which approximately 60% are negatively charged, did not impact on the ability of the protein to complement the sensitive phenotype of UV61 cells to either UV or NA-AAF. Our data indicate that the highly acidic region of CSB is not essential for the TCR and general genome repair pathways of UV- and NA-AAF-induced DNA lesions. PMID:10931931

  5. Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein.

    PubMed

    Eckford, Paul D W; Li, Canhui; Bear, Christine E

    2015-03-09

    The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

  6. Kinetic Detection of Orthogonal Protein and Chemical Coordinates in Enzyme Catalysis: Double Mutants of Soybean Lipoxygenase.

    PubMed

    Sharma, Sudhir C; Klinman, Judith P

    2015-09-01

    Soybean lipoxygenase-1 (SLO-1) is a paradigmatic enzyme system for studying the contribution of hydrogen tunneling to enzymatic proton-coupled electron transfer processes. In this study, the impact of pairs of double mutants on the properties of SLO-1 is presented. Steady-state rates and their deuterium kinetic isotope effects (KIEs) have been measured for the bimolecular reaction of enzyme with free substrate (kcat/Km) and compared to the unimolecular rate constant, kcat. A key kinetic finding is that the competitive KIEs on the second-order rate constant (kcat/Km) are all reduced from (D)kcat and, despite large changes in rate and activation parameters, remain essentially unaltered under a variety of conditions. These data implicate a protein reaction coordinate that is orthogonal to the chemical reaction coordinate and controls the concentration of the active enzyme. This study introduces a new means to interrogate the alteration of conformational landscapes that can occur following site-specific mutagenesis.

  7. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function

    PubMed Central

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  8. Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders.

    PubMed

    Shibata, Norihito; Ohoka, Nobumichi; Sugaki, Yusuke; Onodera, Chiaki; Inoue, Mizuho; Sakuraba, Yoshiyuki; Takakura, Daisuke; Hashii, Noritaka; Kawasaki, Nana; Gondo, Yoichi; Naito, Mikihiko

    2015-11-20

    During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders. PMID:26442586

  9. Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30 S subunits.

    PubMed

    Fredrick, K; Dunny, G M; Noller, H F

    2000-05-01

    Ribosomal protein S7 nucleates folding of the 16 S rRNA 3' major domain, which ultimately forms the head of the 30 S ribosomal subunit. Recent crystal structures indicate that S7 lies on the interface side of the 30 S subunit, near the tRNA binding sites of the ribosome. To map the functional surface of S7, we have tagged the protein with a Protein Kinase A recognition site and engineered alanine substitutions that target each exposed, conserved residue. We have also deleted conserved features of S7, using its structure to guide our design. By radiolabeling the tag sequence using Protein Kinase A, we are able to track the partitioning of each mutant protein into 30 S, 70 S, and polyribosome fractions in vivo. Overexpression of S7 confers a growth defect, and we observe a striking correlation between this phenotype and proficiency in 30 S subunit assembly among our collection of mutants. We find that the side chain of K35 is required for efficient assembly of S7 into 30 S subunits in vivo, whereas those of at least 17 other conserved exposed residues are not required. In addition, an S7 derivative lacking the N-terminal 17 residues causes ribosomes to accumulate on mRNA to abnormally high levels, indicating that our approach can yield interesting mutant ribosomes.

  10. In Vitro and Molecular Modeling Analysis of Two Mutant Desert Hedgehog Proteins Associated with 46,XY Gonadal Dysgenesis

    PubMed Central

    Castro, Josué Joram; Méndez, Juan Pablo; Coral-Vázquez, Ramón Mauricio; Soriano-Ursúa, Marvin Antonio; Damian-Matsumura, Pablo; Benítez-Granados, Jesús; Rosas-Vargas, Haydee

    2013-01-01

    Mutations of Desert hedgehog (DHH) have been associated to 46,XY pure gonadal dysgenesis (PGD) and to mixed gonadal dysgenesis (MGD); however, there have been no functional studies of mutations described in DHH. To determine if mutations p.L162P and Δ1086delG yield functional impairment, we performed in vitro and in silico analysis of both DHH mutants. In complementary DNA of DHH, we performed site-directed mutagenesis, which was confirmed by DNA sequencing. Protein extracts were obtained from HEK293cells transfected with different constructs and analyzed by Western blot; besides, densitometric analysis of chemiluminescent signals was performed. In addition, the structure of the wt-DHH and its two mutant proteins was inferred using in silico protein molecular modeling. In the Western blot analysis, we observed the absence of signal for p.L162P in DHH-N and a diminished signal for Δ1086delG in DHH-C, when compared to wt-DHH. Protein modeling showed notable conformational changes for the side chains of p.L162P, while the secondary structure was drastically modified in Δ1086delG, when compared to wt-DHH. To our knowledge, this is the first study focused to determine by in vitro studies, the effect of two specific mutations in DHH associated with 46,XY PGD and MGD. Our results suggest that both mutations have a deleterious effect on the expression of the DHH mutant proteins. PMID:23786321

  11. In vitro and molecular modeling analysis of two mutant desert hedgehog proteins associated with 46,XY gonadal dysgenesis.

    PubMed

    Castro, Josué Joram; Méndez, Juan Pablo; Coral-Vázquez, Ramón Mauricio; Soriano-Ursúa, Marvin Antonio; Damian-Matsumura, Pablo; Benítez-Granados, Jesús; Rosas-Vargas, Haydee; Canto, Patricia

    2013-09-01

    Mutations of Desert hedgehog (DHH) have been associated to 46,XY pure gonadal dysgenesis (PGD) and to mixed gonadal dysgenesis (MGD); however, there have been no functional studies of mutations described in DHH. To determine if mutations p.L162P and Δ1086delG yield functional impairment, we performed in vitro and in silico analysis of both DHH mutants. In complementary DNA of DHH, we performed site-directed mutagenesis, which was confirmed by DNA sequencing. Protein extracts were obtained from HEK293cells transfected with different constructs and analyzed by Western blot; besides, densitometric analysis of chemiluminescent signals was performed. In addition, the structure of the wt-DHH and its two mutant proteins was inferred using in silico protein molecular modeling. In the Western blot analysis, we observed the absence of signal for p.L162P in DHH-N and a diminished signal for Δ1086delG in DHH-C, when compared to wt-DHH. Protein modeling showed notable conformational changes for the side chains of p.L162P, while the secondary structure was drastically modified in Δ1086delG, when compared to wt-DHH. To our knowledge, this is the first study focused to determine by in vitro studies, the effect of two specific mutations in DHH associated with 46,XY PGD and MGD. Our results suggest that both mutations have a deleterious effect on the expression of the DHH mutant proteins.

  12. [Physical-chemical properties of the mutant (protein) form of D-glucose/D-galactose-binding protein GGBP/H152C with an attached fluorescent dye BADAN].

    PubMed

    Fonin, A V; Stepanenko, O V; Povarova, O I; Volova, E A; Filippova, E M; Bublikov, G S; Kuznetsova, I M; Demchenko, A P; Turoverov, K K

    2013-01-01

    The influence of various factors on the physico-chemical characteristics and complexation of glucose with a mutant form of D-glucose/D-galactose-binding protein which can be regarded as a sensor of the glucometer, namely the protein GGBP/H152C with solvatochromic dye BADAN attached to the cysteine residue Cys 152, has been investigated. The point mutation His 152Cys and attaching BADAN reduced the affinity of the mutant form GGBP/H152C to glucose more than 8-fold compared to the wild type protein. This allows using this mutant for the determination of sugar content in biological fluids extracted by transdermal technologies. Sufficiently rapid complexation of GGBP/H152C with glucose (the time of protein-glucose complex formation is not more than three seconds even in solutions with a viscosity of 4 cP) provides timely monitoring changes in the concentration of sugar. The changes of ionic strength and pH within the physiological range of values of these variables do not have significant influence on fluorescent characteristics of GGBP/H152C-BADAN. At acidic pH, (see symbol) some of the molecules GGBP/H152C is in the unfolded state. It has been shown that mutant form GGBP/H152C has relatively low resistance to guanidine hydrochloride denaturing effects. This result indicates the need for more stable proteins to create a sensor for glucose biosensor system. PMID:25474908

  13. Immunogenicity and Cross-Protective Efficacy Induced by Outer Membrane Proteins from Salmonella Typhimurium Mutants with Truncated LPS in Mice

    PubMed Central

    Liu, Qiong; Liu, Qing; Zhao, Xinxin; Liu, Tian; Yi, Jie; Liang, Kang; Kong, Qingke

    2016-01-01

    Lipopolysaccharide (LPS) is a major virulence factor present in the outer membrane of Salmonella enterica serovar Typhimurium (S. Typhimurium). Outer membrane proteins (OMPs) from Salmonella show high immunogenicity and provide protection against Salmonella infection, and truncated LPS alters the outer membrane composition of the cell wall. In our previous study, we demonstrated that Salmonella mutants carrying truncated LPS failed to induce strong immune responses and cross-reaction to other enteric bacteria, due to their high attenuation and low colonization in the host. Therefore, we plan to investigate whether outer membrane proteins from Salmonella mutants with truncated LPS resulting from a series of nonpolar mutations, including ∆waaC12, ∆waaF15, ∆waaG42, ∆rfaH49, ∆waaI43, ∆waaJ44, ∆waaL46, ∆wbaP45 and ∆wzy-48, affect immunogenicity and provide protection against diverse Salmonella challenge. In this study, the immunogenicity and cross-protection efficiency of purified OMPs from all mutants were investigated to explore a potential OMP vaccine to protect against homologous or heterologous serotype Salmonella challenge. The results demonstrated that OMPs from three Salmonella mutants (∆waaC12, ∆waaJ44 and ∆waaL46) induced higher immune responses and provided good protection against homologous S. Typhimurium. The OMPs from these three mutants were also selected to determine the cross-protective efficacy against homologous and heterologous serotype Salmonella. Our results indicated that the mutant ∆waaC12 can elicit higher cross-reactivity and can provide good protection against S. Choleraesuis and S. Enteritidis infection and that the cross-reactivity may be ascribed to an antigen of approximately 18.4–30 kDa. PMID:27011167

  14. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse.

    PubMed

    Hashimoto, Masakazu; Yamashita, Yukiko; Takemoto, Tatsuya

    2016-10-01

    The CRISPR/Cas9 system is a powerful tool for elucidating the roles of genes in a wide variety of organisms including mice. To obtain genetically modified embryos or mice by this method, Cas9 mRNA and sgRNA are usually introduced into zygotes by microinjection or electroporation. However, most mutants generated with this method are genetically mosaic, composed of several types of cells carrying different mutations, which complicates phenotype analysis in founder embryos or mice. To simplify the analysis and to elucidate the roles of genes involved in developmental processes, a method for producing non-mosaic mutants is needed. Here, we established a method for generating non-mosaic mouse mutant embryos. We introduced Cas9 protein and sgRNA into in vitro fertilized (IVF) zygotes by electroporation, which enabled the genome editing to occur before the first replication of the mouse genome. As a result, all of the cells in the mutant carried the same set of mutations. This method solves the problem of mosaicism/allele complexity in founder mutant embryos or mice generated by the CRIPSR/Cas9 system.

  15. A phenotype survey of 36 mutant mouse strains with gene-targeted defects in glycosyltransferases or glycan-binding proteins

    PubMed Central

    Orr, Sally L; Le, Dzung; Long, Jeffrey M; Sobieszczuk, Peter; Ma, Bo; Tian, Hua; Fang, Xiaoqun; Paulson, James C; Marth, Jamey D; Varki, Nissi

    2013-01-01

    The consortium for functional glycomics (CFG) was a large research initiative providing networking and resources for investigators studying the role of glycans and glycan-binding proteins in health and disease. Starting in 2001, six scientific cores were established to generate data, materials and new technologies. By the end of funding in 2011, the mouse phenotype core (MPC) submitted data to a website from the phenotype screen of 36 mutant mouse strains deficient in a gene for either a glycan-binding protein (GBP) or glycosyltransferase (GT). Each mutant strain was allotted three months for analysis and screened by standard phenotype assays used in the fields of immunology, histology, hematology, coagulation, serum chemistry, metabolism and behavior. Twenty of the deficient mouse strains had been studied in other laboratories, and additional tests were performed on these strains to confirm previous observations and discover new data. The CFG constructed 16 new homozygous mutant mouse strains and completed the initial phenotype screen of the majority of these new mutant strains. In total, >300 phenotype changes were observed, but considering the over 100 assays performed on each strain, most of the phenotypes were unchanged. Phenotype differences include abnormal testis morphology in GlcNAcT9- and Siglec-H-deficient mice and lethality in Pomgnt1-deficient mice. The numerous altered phenotypes discovered, along with the consideration of the significant findings of normality, will provide a platform for future characterization to understand the important roles of glycans and GBPs in the mechanisms of health and disease. PMID:23118208

  16. Structural characterization of V57D and V57P mutants of human cystatin C, an amyloidogenic protein

    PubMed Central

    Orlikowska, Marta; Szymańska, Aneta; Borek, Dominika; Otwinowski, Zbyszek; Skowron, Piotr; Jankowska, Elżbieta

    2013-01-01

    Wild-type human cystatin C (hCC wt) is a low-molecular-mass protein (120 amino-acid residues, 13 343 Da) that is found in all nucleated cells. Physiologically, it functions as a potent regulator of cysteine protease activity. While the biologically active hCC wt is a monomeric protein, all crystallization efforts to date have resulted in a three-dimensional domain-swapped dimeric structure. In the recently published structure of a mutated hCC, the monomeric fold was preserved by a stabilization of the conformationally constrained loop L1 caused by a single amino-acid substitution: Val57Asn. Additional hCC mutants were obtained in order to elucidate the relationship between the stability of the L1 loop and the propensity of human cystatin C to dimerize. In one mutant Val57 was substituted by an aspartic acid residue, which is favoured in β-turns, and in the second mutant proline, a residue known for broadening turns, was substituted for the same Val57. Here, 2.26 and 3.0 Å resolution crystal structures of the V57D andV57P mutants of hCC are reported and their dimeric architecture is discussed in terms of the stabilization and destabilization effects of the introduced mutations. PMID:23519666

  17. Adapting protein solubility by glycosylation. N-glycosylation mutants of Coprinus cinereus peroxidase in salt and organic solutions.

    PubMed

    Tams, J W; Vind, J; Welinder, K G

    1999-07-13

    Protein solubility is a fundamental parameter in biology and biotechnology. In the present study we have constructed and analyzed five mutants of Coprinus cinereus peroxidase (CIP) with 0, 1, 2, 4 and 6 N-glycosylation sites. All mutants contain Man(x)(GlcNAc)(2) glycans. The peroxidase activity was the same for wild-type CIP and all the glycosylation mutants when measured with the large substrate 2,2'-azino-bis(-3-ethylbenzthiazoline-6-sulfonic acid). The solubility of the five CIP mutants showed a linear dependence on the number of carbohydrate residues attached to the protein in buffered solution of both ammonium sulfate (AMS) and acetone, increasing in AMS and decreasing in acetone. Moreover, the change in free energy of solvation appears to be a constant, though with opposite signs in these solvents, giving DeltaDeltaG degrees (sol)=-0.32+/-0.05 kJ/mol per carbohydrate residue in 2.0 M AMS, a value previously obtained comparing ordinary and deglycosylated horseradish peroxidase, and 0. 37+/-0.10 kJ/mol in 60 v/v% acetone.

  18. Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate

    NASA Technical Reports Server (NTRS)

    Shrestha, Suresh; Salins, Lyndon L E.; Mark Ensor, C.; Daunert, Sylvia

    2002-01-01

    Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the

  19. Control of grain protein contents through SEMIDWARF1 mutant alleles: sd1 increases the grain protein content in Dee-geo-woo-gen but not in Reimei.

    PubMed

    Terao, Tomio; Hirose, Tatsuro

    2015-06-01

    A new possibility for genetic control of the protein content of rice grains was suggested by the allele differences of the SEMIDWARF1 (SD1) mutation. Two quantitative trait loci-qPROT1 and qPROT12-were found on chromosomes 1 and 12, respectively, using backcrossed inbred lines of Sasanishiki/Habataki//Sasanishiki///Sasanishiki. One of them, qPROT1, increased almost all grain proteins instead of only certain proteins in the recessive Habataki allele. Fine mapping of qPROT1 revealed that two gene candidates-Os01g0883800 and Os01g0883900-were included in this region. Os01g0883800 encoded Gibberellin 20 oxidase 2 as well as SD1, the dwarf gene used in the so-called 'Green Revolution'. Mutant analyses as well as sequencing analysis using the semi-dwarf mutant cultivars Dee-geo-woo-gen and Calrose 76 revealed that the sd1 mutant showed significantly higher grain protein contents than their corresponding wild-type cultivars, strongly suggesting that the high protein contents were caused by sd1 mutation. However, the sd1 mutant Reimei did not have high grain protein contents. It is possible to control the grain protein content and column length separately by selecting for sd1 alleles. From this finding, the genetic control of grain protein content, as well as the column length of rice cultivars, might be possible. This ability might be useful to improve rice nutrition, particularly in areas where the introduction of semi-dwarf cultivars is not advanced.

  20. Immunohistochemical detection of the BRAF V600E mutant protein in colorectal neoplasms

    PubMed Central

    Vakiani, Efsevia; Yaeger, Rona; Brooke, Sylvester; Zhou, Yi; Klimstra, David S.; Shia, Jinru

    2016-01-01

    Reliable assessment of the BRAF mutation status is becoming increasingly important in the clinical management of colorectal carcinomas (CRC). The aim of this study was to investigate the use of a recently developed mutation-specific antibody (VE1, SpringBio, Pleasanton, CA) to detect the BRAF V600E protein in paraffin tissue. We analyzed by immunohistochemistry (IHC) 117 cases that had been evaluated for BRAF mutation using a MALDI-TOF mass-spectrometry based assay. IHC staining was evaluated without the knowledge of the genetic data and was considered positive when there was distinct homogeneous cytoplasmic staining in the tumor cells. The analyzed cases included 4 polyps, 63 primary and 50 metastatic CRC. Forty-five of the 46 (97.8%) cases that were positive by IHC had a BRAF V600E mutation by genetic analysis; the 1 discordant case was notably of signet ring cell type. Similarly, 66 of the 67 (98.5%) cases that were negative by IHC were also negative by genetic analysis. Four cases that showed weak cytoplasmic staining and/or nuclear staining in the tumor cells were considered to be IHC equivocal; by genetic analysis, 2 of the 4 were positive and 2 were negative. The overall sensitivity and specificity of IHC for the detection of a BRAF V600E mutant tumor was 93.7% and 95.6%, respectively. Our results support the use of VE1 IHC for identification of colorectal neoplasms harboring the BRAF V600E mutation. Difficulties in IHC interpretation may arise in a small number of cases, and in those cases molecular testing is required. PMID:25517872

  1. Human-to-mouse prion-like propagation of mutant huntingtin protein.

    PubMed

    Jeon, Iksoo; Cicchetti, Francesca; Cisbani, Giulia; Lee, Suji; Li, Endan; Bae, Jiwoo; Lee, Nayeon; Li, Ling; Im, Wooseok; Kim, Manho; Kim, Hyun Sook; Oh, Seung-Hun; Kim, Tae-Aug; Ko, Jung Jae; Aubé, Benoit; Oueslati, Abid; Kim, Yun Joong; Song, Jihwan

    2016-10-01

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder of the central nervous system (CNS) that is defined by a CAG expansion in exon 1 of the huntingtin gene leading to the production of mutant huntingtin (mHtt). To date, the disease pathophysiology has been thought to be primarily driven by cell-autonomous mechanisms, but, here, we demonstrate that fibroblasts derived from HD patients carrying either 72, 143 and 180 CAG repeats as well as induced pluripotent stem cells (iPSCs) also characterized by 143 CAG repeats can transmit protein aggregates to genetically unrelated and healthy host tissue following implantation into the cerebral ventricles of neonatal mice in a non-cell-autonomous fashion. Transmitted mHtt aggregates gave rise to both motor and cognitive impairments, loss of striatal medium spiny neurons, increased inflammation and gliosis in associated brain regions, thereby recapitulating the behavioural and pathological phenotypes which characterizes HD. In addition, both in vitro work using co-cultures of mouse neural stem cells with 143 CAG fibroblasts and the SH-SY5Y human neuroblastoma cell line as well as in vivo experiments conducted in newborn wild-type mice suggest that exosomes can cargo mHtt between cells triggering the manifestation of HD-related behaviour and pathology. This is the first evidence of human-to-mouse prion-like propagation of mHtt in the mammalian brain; a finding which will help unravel the molecular bases of HD pathology as well as to lead to the development of a whole new range of therapies for neurodegenerative diseases of the CNS. PMID:27221146

  2. Characterization of binding and structural properties of rat liver fatty-acid-binding protein using tryptophan mutants.

    PubMed Central

    Thumser, A E; Wilton, D C

    1994-01-01

    Rat liver fatty-acid-binding protein (FABP) does not contain tryptophan. Three mutant proteins have been produced in which a single tryptophan residue has been inserted by site-directed mutagenesis at positions 3 (F3W), 18 (F18W) and 69 (C69W). These tryptophans have been strategically located in order to provide fluorescent reporter groups to study the binding and structural characteristics of rat liver FABP. Two fluorescent fatty acid analogues, DAUDA (11-[(5-dimethylaminonaphthalene-1- sulphonyl)amino]undecanoic acid) and 3-[p-(6-phenyl)-hexa-1,3,5-trienyl]phenylpropionic acid, showed no significant difference in binding affinities for the different mutant proteins, although maximum fluorescence values were decreased for F3W and increased for C69W. These findings were confirmed by studies of DAUDA displacement by oleate. Protein-denaturation studies in the presence of urea indicated subtle differences for the three mutants which could be explained by multiple unfolding pathways. Fatty acid binding increased tryptophan fluorescence emission in the case of the F18W protein, but had no effect on the F3W and C69W proteins. Fluorescence quenching studies with 2-bromopalmitate showed that a fatty acid carboxylate is close to the tryptophan in the F18W protein. Energy-transfer studies showed that the fluorescent moiety of DAUDA is equidistant from the three mutated amino acids and is bound within the beta-clam solvent cavity of liver FABP. This interpretation of the fluorescence quenching and energy-transfer data supports the difference in ligand orientation between intestinal and liver FABP observed in previous studies. PMID:8010966

  3. Response to Comment on "Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism".

    PubMed

    Li, Fei; Liu, Jian; Zheng, Yi; Garavito, R Michael; Ferguson-Miller, Shelagh

    2015-10-30

    Wang comments that the diffraction data for the structure of the A139T mutant of translocator protein TSPO from Rhodobacter sphaeroides should be used to 1.65 instead of 1.8 angstroms and that the density interpreted as porphyrin and monoolein is better fitted as polyethylene glycol. Although different practices of data processing exist, in this case they do not substantially influence the final map. Additional data are presented supporting the fit of a porphyrin and monooleins. PMID:26516277

  4. A cyclic AMP receptor protein mutant that constitutively activates an Escherichia coli promoter disrupted by an IS5 insertion.

    PubMed

    Podolny, V; Lin, E C; Hochschild, A

    1999-12-01

    Previously an Escherichia coli mutant that had acquired the ability to grow on propanediol as the sole carbon and energy source was isolated. This phenotype is the result of the constitutive expression of the fucO gene (in the fucAO operon), which encodes one of the enzymes in the fucose metabolic pathway. The mutant was found to bear an IS5 insertion in the intergenic regulatory region between the divergently oriented fucAO and fucPIK operons. Though expression of the fucAO operon was constitutive, the fucPIK operon became noninducible such that the mutant could no longer grow on fucose. A fucose-positive revertant which was found to contain a suppressor mutation in the crp gene was selected. Here we identify this crp mutation, which results in a single amino acid substitution (K52N) that has been proposed previously to uncover a cryptic activating region in the cyclic AMP receptor protein (CRP). We show that the mutant CRP constitutively activates transcription from both the IS5-disrupted and the wild-type fucPIK promoters, and we identify the CRP-binding site that is required for this activity. Our results show that the fucPIK promoter, a complex promoter which ordinarily depends on both CRP and the fucose-specific regulator FucR for its activation, can be activated in the absence of FucR by a mutant CRP that uses three, rather than two, activating regions to contact RNA polymerase. For the IS5-disrupted promoter, which retains a single CRP-binding site, the additional activating region of the mutant CRP evidently compensates for the lack of upstream regulatory sequences. PMID:10601201

  5. Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases

    PubMed Central

    Niehues, Sven; Bussmann, Julia; Steffes, Georg; Erdmann, Ines; Köhrer, Caroline; Sun, Litao; Wagner, Marina; Schäfer, Kerstin; Wang, Guangxia; Koerdt, Sophia N.; Stum, Morgane; RajBhandary, Uttam L.; Thomas, Ulrich; Aberle, Hermann; Burgess, Robert W.; Yang, Xiang-Lei; Dieterich, Daniela; Storkebaum, Erik

    2015-01-01

    Dominant mutations in five tRNA synthetases cause Charcot–Marie–Tooth (CMT) neuropathy, suggesting that altered aminoacylation function underlies the disease. However, previous studies showed that loss of aminoacylation activity is not required to cause CMT. Here we present a Drosophila model for CMT with mutations in glycyl-tRNA synthetase (GARS). Expression of three CMT-mutant GARS proteins induces defects in motor performance and motor and sensory neuron morphology, and shortens lifespan. Mutant GARS proteins display normal subcellular localization but markedly reduce global protein synthesis in motor and sensory neurons, or when ubiquitously expressed in adults, as revealed by FUNCAT and BONCAT. Translational slowdown is not attributable to altered tRNAGly aminoacylation, and cannot be rescued by Drosophila Gars overexpression, indicating a gain-of-toxic-function mechanism. Expression of CMT-mutant tyrosyl-tRNA synthetase also impairs translation, suggesting a common pathogenic mechanism. Finally, genetic reduction of translation is sufficient to induce CMT-like phenotypes, indicating a causal contribution of translational slowdown to CMT. PMID:26138142

  6. New Mutants of Saccharomyces Cerevisiae Affected in the Transport of Proteins from the Endoplasmic Reticulum to the Golgi Complex

    PubMed Central

    Wuestehube, L. J.; Duden, R.; Eun, A.; Hamamoto, S.; Korn, P.; Ram, R.; Schekman, R.

    1996-01-01

    We have isolated new temperature-sensitive mutations in five complementation groups, sec31-sec35, that are defective in the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex. The sec31-sec35 mutants and additional alleles of previously identified sec and vacuolar protein sorting (vps) genes were isolated in a screen based on the detection of α-factor precursor in yeast colonies replicated to and lysed on nitrocellulose filters. Secretory protein precursors accumulated in sec31-sec35 mutants at the nonpermissive temperature were core-glycosylated but lacked outer chain carbohydrate, indicating that transport was blocked after translocation into the ER but before arrival in the Golgi complex. Electron microscopy revealed that the newly identified sec mutants accumulated vesicles and membrane structures reminiscent of secretory pathway organelles. Complementation analysis revealed that sec32-1 is an allele of BOS1, a gene implicated in vesicle targeting to the Golgi complex, and sec33-1 is an allele of RET1, a gene that encodes the α subunit of coatomer. PMID:8852839

  7. ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cell-derived motoneurons.

    PubMed

    Lenzi, Jessica; De Santis, Riccardo; de Turris, Valeria; Morlando, Mariangela; Laneve, Pietro; Calvo, Andrea; Caliendo, Virginia; Chiò, Adriano; Rosa, Alessandro; Bozzoni, Irene

    2015-07-01

    Patient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUS(R514S) and FUS(R521C) patient fibroblasts, whereas in the case of the severe FUS(P525L) mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis.

  8. Biochemical Characterization of Mutants in Chaperonin Proteins CCT4 and CCT5 Associated with Hereditary Sensory Neuropathy*

    PubMed Central

    Sergeeva, Oksana A.; Tran, Meme T.; Haase-Pettingell, Cameron; King, Jonathan A.

    2014-01-01

    Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the eukaryotic cytosolic chaperonin TRiC, a protein machine responsible for folding actin and tubulin in the cell. C450Y CCT4 was identified in a stock of Sprague-Dawley rats, whereas H147R CCT5 was found in a human Moroccan family. As with many genetically identified mutations associated with neuropathies, the underlying molecular basis of the mutants was not defined. We investigated the biochemical properties of these mutants using an expression system in Escherichia coli that produces homo-oligomeric rings of CCT4 and CCT5. Full-length versions of both mutant protein chains were expressed in E. coli at levels approaching that of the WT chains. Sucrose gradient centrifugation revealed chaperonin-sized complexes of both WT and mutant chaperonins, but with reduced recovery of C450Y CCT4 soluble subunits. Electron microscopy of negatively stained samples of C450Y CCT4 revealed few ring-shaped species, whereas WT CCT4, H147R CCT5, and WT CCT5 revealed similar ring structures. CCT5 complexes were assayed for their ability to suppress aggregation of and refold the model substrate γd-crystallin, suppress aggregation of mutant huntingtin, and refold the physiological substrate β-actin in vitro. H147R CCT5 was not as efficient in chaperoning these substrates as WT CCT5. The subtle effects of these mutations are consistent with the homozygous disease phenotype, in which most functions are carried out during development and adulthood, but some selective function is lost or reduced. PMID:25124038

  9. Identification of a 34 kDa protein altered in the LF-1 mutant as the herbicide-binding D1 protein of photosystem II

    SciTech Connect

    Metz, J.; Pakrasi, H.; Seibert, M.; Arntzen, C.

    1986-04-01

    The LF-1 mutant of Scenedesmus has a complete block on the oxidizing side of its PSII reaction center. However, the reaction center as well as the reducing side of PSII is fully functional in this mutant. Compared to the wildtype (WT) the only detected protein difference in the PSII complex of LF-1 is the change in mobility of a 34 kDa protein to 36 kDa. This protein has been implicated to have a major role in Mn-binding and water-oxidation. The authors have recently shown that photoaffinity labeling of thylakoids with azido-(/sup 14/C)-atrazine tags the 34 kDa protein in WT and the 36 kDa protein in LF-1. It has been shown that the azido-atrazine labeled protein, called D1, functions in herbicide binding and Q/sub A/ to Q/sub B/ electron transfer on the reducing side of PSII. Polyclonal antibodies directed against the D1 protein of Amaranthus hybridus (Ohad, et al., EMBOJ 1985) were found to recognize the Scenedesmus 34 kDa (WT) and 36 kDa (LF-1) proteins. The implied dual function for the D1 protein on the reducing as well as the oxidizing side of PSII reaction center will be discussed.

  10. [Construction and expression of six deletion mutants of human astrovirus C-terminal nsP1a/4 protein].

    PubMed

    Zhao, Wei; Niu, Ke; Zhao, Jian; Jin, Yi-ming; Sui, Ting-ting; Wang, Wen

    2013-09-01

    Human astrovirus (HAstV) is one of the leading causes of actue virual diarrhea in infants. HAstV-induced epithdlial cell apoptosis plays an important role in the pathogenesis of HAstV infection. Our previous study indicated that HAstV non-structural protein nsPla C-terminal protein nsPla/4 was the major apoptosis functional protein and probably contained the main apoptosis domains. In order to screen for astrovirus encoded apoptotic protien, nsPla/4 and six turncated proteins, which possessed nsPla/4 protein different function domain ,were cloned into green fluorescent protein (GFP) vector pEG-FP-N3. After 24-72 h transfection, the fusion protein expression in BHK21 cells, was analysis by fluorescence microscope and Western blot. The results indicated seven fusion proteins were observed successfully in BHK21 cell after transfected for 24 h. Western blot analysis showed that the level of fusion protein expressed in BHK21 cells was increased significantly at 72h compared to 48h in transfected cells. The successful expression of deletion mutants of nsPla/4 protein was an important foundation to gain further insights into the function of apoptosis domains of nsPla/4 protein and it would also provide research platform to further confirm the molecule pathogenic mechanism of human astrovirus.

  11. Soya bean Gα proteins with distinct biochemical properties exhibit differential ability to complement Saccharomyces cerevisiae gpa1 mutant.

    PubMed

    Roy Choudhury, Swarup; Wang, Yuqi; Pandey, Sona

    2014-07-01

    Signalling pathways mediated by heterotrimeric G-proteins are common to all eukaryotes. Plants have a limited number of each of the G-protein subunits, with the most elaborate G-protein network discovered so far in soya bean (Glycine max, also known as soybean) which has four Gα, four Gβ and ten Gγ proteins. Biochemical characterization of Gα proteins from plants suggests significant variation in their properties compared with the well-characterized non-plant proteins. Furthermore, the four soya bean Gα (GmGα) proteins exhibit distinct biochemical activities among themselves, but the extent to which such biochemical differences contribute to their in vivo function is also not known. We used the yeast gpa1 mutant which displays constitutive signalling and growth arrest in the pheromone-response pathway as an in vivo model to evaluate the effect of distinct biochemical activities of GmGα proteins. We showed that specific GmGα proteins can be activated during pheromone-dependent receptor-mediated signalling in yeast and they display different strengths towards complementation of yeast gpa1 phenotypes. We also identified amino acids that are responsible for differential complementation abilities of specific Gα proteins. These data establish that specific plant Gα proteins are functional in the receptor-mediated pheromone-response pathway in yeast and that the subtle biochemical differences in their activity are physiologically relevant.

  12. Mutant canine oral papillomavirus L1 capsid proteins which form virus-like particles but lack native conformational epitopes.

    PubMed

    Chen, Y; Ghim, S J; Jenson, A B; Schlegel, R

    1998-09-01

    Recently, the L1 capsid protein of canine oral papillomavirus (COPV) has been used as an effective systemic vaccine that prevents viral infections of the oral mucosa. The efficacy of this vaccine is critically dependent upon native L1 conformation and, when purified from Sf9 insect cells, the L1 protein not only displays type-specific, conformation-dependent epitopes but it also assembles spontaneously into virus-like particles (VLPs). To determine whether VLP formation was coupled to the expression of conformation-dependent epitopes, we generated a series of N- and C-terminal L1 deletion mutants and evaluated their ability to form VLPs (by electron microscopy) and to react with conformation-dependent antibodies (by immunofluorescence microscopy). We found that (a) deletion of the 26 C-terminal residues generated a mutant protein which formed VLPs efficiently and folded correctly both in the cytoplasm and in the nucleus; (b) further truncation of the L1 C terminus (67 amino acids) resulted in a capsid protein which formed VLPs but which failed to express conformational epitopes; (c) deletion of the first 25 N-terminal amino acids also abolished expression of conformational epitopes (without altering VLP formation) but the native conformation of this deletion mutant could be restored by the addition of the human papillomavirus type 11 N terminus. These results demonstrate that VLP formation and conformational epitope expression can be dissociated and that the L1 N terminus has a critical role in protein folding. In addition, it appears that correct L1 protein folding is not dependent upon the nucleoplasmic environment. PMID:9747722

  13. Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants

    NASA Astrophysics Data System (ADS)

    Schulz, G. E.; Dreyer, M.; Klein, C.; Kreusch, A.; Mittl, P.; Mu¨ller, C. W.; Mu¨ller-Dieckmann, J.; Muller, Y. A.; Proba, K.; Schlauderer, G.; Spu¨rgin, P.; Stehle, T.; Weiss, M. S.

    1992-08-01

    Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8A˚resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0A˚resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.

  14. The Maize High-Lysine Mutant opaque7 Is Defective in an Acyl-CoA Synthetase-Like Protein

    PubMed Central

    Miclaus, Mihai; Wu, Yongrui; Xu, Jian-Hong; Dooner, Hugo K.; Messing, Joachim

    2011-01-01

    Maize (Zea mays) has a large class of seed mutants with opaque or nonvitreous endosperms that could improve the nutritional quality of our food supply. The phenotype of some of them appears to be linked to the improper formation of protein bodies (PBs) where zein storage proteins are deposited. Although a number of genes affecting endosperm vitreousness have been isolated, it has been difficult to clone opaque7 (o7), mainly because of its low penetrance in many genetic backgrounds. The o7-reference (o7-ref) mutant arose spontaneously in a W22 inbred, but is poorly expressed in other lines. We report here the isolation of o7 with a combination of map-based cloning and transposon tagging. We first identified an o7 candidate gene by map-based cloning. The putative o7-ref allele has a 12-bp in-frame deletion of codons 350–353 in a 528-codon-long acyl-CoA synthetase-like gene (ACS). We then confirmed this candidate gene by generating another mutant allele from a transposon-tagging experiment using the Activator/Dissociation (Ac/Ds) system in a W22 background. The second allele, isolated from ∼1 million gametes, presented a 2-kb Ds insertion that resembles the single Ds component of double-Ds, McClintock’s original Dissociation element, at codon 496 of the ACS gene. PBs exhibited striking membrane invaginations in the o7-ref allele and a severe number reduction in the Ds-insertion mutant, respectively. We propose a model in which the ACS enzyme plays a key role in membrane biogenesis, by taking part in protein acylation, and that altered PBs render the seed nonvitreous. PMID:21926304

  15. The workflow for quantitative proteome analysis of chloroplast development and differentiation, chloroplast mutants, and protein interactions by spectral counting.

    PubMed

    Friso, Giulia; Olinares, Paul Dominic B; van Wijk, Klaas J

    2011-01-01

    This chapter outlines a quantitative proteomics workflow using a label-free spectral counting technique. The workflow has been tested on different aspects of chloroplast biology in maize and Arabidopsis, including chloroplast mutant analysis, cell-type specific chloroplast differentiation, and the proplastid-to-chloroplast transition. The workflow involves one-dimensional SDS-PAGE of the proteomes of leaves or chloroplast subfractions, tryptic digestions, online LC-MS/MS using a mass spectrometer with high mass accuracy and duty cycle, followed by semiautomatic data processing. The bioinformatics analysis can effectively select best gene models and deals with quantification of closely related proteins; the workflow avoids overidentification of proteins and results in more accurate protein quantification. The final output includes pairwise comparative quantitative analysis, as well as hierarchical clustering for discovery of temporal and spatial patterns of protein accumulation. A brief discussion about potential pitfalls, as well as the advantages and disadvantages of spectral counting, is provided.

  16. Subunit and amino acid interactions in the Escherichia coli mannitol permease: a functional complementation study of coexpressed mutant permease proteins.

    PubMed

    Saraceni-Richards, C A; Jacobson, G R

    1997-08-01

    Mannitol-specific enzyme II, or mannitol permease, of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system of Escherichia coli carries out the transport and phosphorylation of D-mannitol and is most active as a dimer in the membrane. We recently reported the importance of a glutamate residue at position 257 in the binding and transport of mannitol by this protein (C. Saraceni-Richards and G. R. Jacobson, J. Bacteriol. 179:1135-1142, 1997). Replacing Glu-257 with alanine (E257A) or glutamine (E257Q) eliminated detectable mannitol binding and transport by the permease. In contrast, an E257D mutant protein was able to bind and phosphorylate mannitol in a manner similar to that of the wild-type protein but was severely defective in mannitol uptake. In this study, we have coexpressed proteins containing mutations at position 257 with other inactive permeases containing mutations in each of the three domains of this protein. Activities of any active heterodimers resulting from this coexpression were measured. The results show that various inactive mutant permease proteins can complement proteins containing mutations at position 257. In addition, we show that both Glu at position 257 and His at position 195, both of which are in the membrane-bound C domain of the protein, must be on the same subunit of a permease dimer in order for efficient mannitol phosphorylation and uptake to occur. The results also suggest that mannitol bound to the opposite subunit within a permease heterodimer can be phosphorylated by the subunit containing the E257A mutation (which cannot bind mannitol) and support a model in which there are separate binding sites on each subunit within a permease dimer. Finally, we provide evidence from these studies that high-affinity mannitol binding is necessary for efficient transport by mannitol permease.

  17. Repressor mutant forms of the Azospirillum brasilense NtrC protein.

    PubMed

    Huergo, Luciano F; Assumpção, Marcelo C; Souza, Emanuel M; Steffens, M Berenice R; Yates, M Geoffrey; Chubatsu, Leda S; Pedrosa, Fábio O

    2004-10-01

    The Azospirillum brasilense mutant strains FP8 and FP9, after treatment with nitrosoguanidine, showed a null Nif phenotype and were unable to use nitrate as their sole nitrogen source. Sequencing of the ntrC genes revealed single nucleotide mutations in the NtrC nucleotide-binding site. The phenotypes of these strains are discussed in relation to their genotypes.

  18. Interactions of the alpha2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants.

    PubMed Central

    Wise, A; Watson-Koken, M A; Rees, S; Lee, M; Milligan, G

    1997-01-01

    The alpha2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of 'Gi-like' pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine alpha2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1alpha, Gi2alpha and Gi3alpha or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The alpha2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5'-[gamma-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein alpha-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1alpha, Gi2alpha and Gi3alpha resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys --> Gly mutations of Gi1alpha, Gi2alpha and Gi3alpha were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1alpha was correlated highly both with the level of expression of this G-protein and with the level of expression of the alpha2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase

  19. Functions that Protect Escherichia coli from Tightly Bound DNA-Protein Complexes Created by Mutant EcoRII Methyltransferase

    PubMed Central

    Henderson, Morgan L.; Kreuzer, Kenneth N

    2015-01-01

    Expression of mutant EcoRII methyltransferase protein (M.EcoRII-C186A) in Escherichia coli leads to tightly bound DNA-protein complexes (TBCs), located sporadically on the chromosome rather than in tandem arrays. The mechanisms behind the lethality induced by such sporadic TBCs are not well studied, nor is it clear whether very tight binding but non-covalent complexes are processed in the same way as covalent DNA-protein crosslinks (DPCs). Using 2D gel electrophoresis, we found that TBCs induced by M.EcoRII-C186A block replication forks in vivo. Specific bubble molecules were detected as spots on the 2D gel, only when M.EcoRII-C186A was induced, and a mutation that eliminates a specific EcoRII methylation site led to disappearance of the corresponding spot. We also performed a candidate gene screen for mutants that are hypersensitive to TBCs induced by M.EcoRII-C186A. We found several gene products necessary for protection against these TBCs that are known to also protect against DPCs induced with wild-type M.EcoRII (after 5-azacytidine incorporation): RecA, RecBC, RecG, RuvABC, UvrD, FtsK, XerCD and SsrA (tmRNA). In contrast, the RecFOR pathway and Rep helicase are needed for protection against TBCs but not DPCs induced by M.EcoRII. We propose that stalled fork processing by RecFOR and RecA promotes release of tightly bound (but non-covalent) blocking proteins, perhaps by licensing Rep helicase-driven dissociation of the blocking M.EcoRII-C186A. Our studies also argued against the involvement of several proteins that might be expected to protect against TBCs. We took the opportunity to directly compare the sensitivity of all tested mutants to two quinolone antibiotics, which target bacterial type II topoisomerases and induce a unique form of DPC. We uncovered rep, ftsK and xerCD as novel quinolone hypersensitive mutants, and also obtained evidence against the involvement of a number of functions that might be expected to protect against quinolones. PMID:25993347

  20. Rapid Proteasomal Degradation of Mutant Proteins Is the Primary Mechanism Leading to Tumorigenesis in Patients With Missense AIP Mutations

    PubMed Central

    Hernández-Ramírez, Laura C.; Martucci, Federico; Morgan, Rhodri M. L.; Trivellin, Giampaolo; Tilley, Daniel; Ramos-Guajardo, Nancy; Iacovazzo, Donato; D'Acquisto, Fulvio; Prodromou, Chrisostomos

    2016-01-01

    Context: The pathogenic effect of mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts. Objective: This study sought to analyze the mechanism/speed of protein turnover of wild-type and missense AIP variants, correlating protein half-life with clinical parameters. Design and Setting: Half-life and protein–protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed in a clinical academic research institution. Patients: Data were obtained from our cohort of pituitary adenoma patients and literature-reported cases. Interventions: Protein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. Glutathione-S-transferase pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by coimmunoprecipitation and gene knockdown. Relevant clinical data was collected. Main Outcome Measures: Half-life of wild-type and mutant AIP proteins and its correlation with clinical parameters. Results: Endogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7 h). AIP variants were divided into stable proteins (median, 77.7 h; interquartile range [IQR], 60.7–92.9 h), and those with short (median, 27 h; IQR, 21.6–28.7 h) or very short (median, 7.7 h; IQR, 5.6–10.5 h) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r = 0.411; P = .002). The FBXO3-containing SKP1–CUL1–F-box protein complex was identified as the E3 ubiquitin-ligase recognizing AIP. Conclusions: AIP is a stable protein, driven to ubiquitination by the SKP1–CUL1–F-box protein complex

  1. Mechanistic insights from functional characterization of an unnatural His37 mutant of the influenza A/M2 protein

    PubMed Central

    Polishchuk, Alexei L.; Cristian, Lidia; Pinto, Lawrence H.; Lear, James D.; DeGrado, William F.

    2014-01-01

    The influenza A/M2 protein is a homotetrameric single-pass integral membrane protein encoded by the influenza A viral genome. Its transmembrane domain represents both a crucial drug target and a minimalistic model system for transmembrane proton transport and charge stabilization. Recent structural and functional studies of M2 have suggested that the proton transport mechanism involves sequential extraviral protonation and intraviral deprotonation of a highly conserved His37 side chain by the transported proton, consistent with a pH-activated proton shuttle mechanism. Multiple tautomeric forms of His can be formed, and it is not known whether they contribute to the mechanism of proton shuttling. Here we present the thermodynamic and functional characterization of an unnatural amino acid mutant at His37, where the imidazole side chain is substituted with a 4-thiazolyl group that is unable to undergo tautomerization and has a significantly lower solution pKa. The mutant construct has a similar stability to the wild-type protein at pH 8 in bilayers and is virtually inactive at external pH 7.4 in a semiquantitative liposome flux assay as expected from its lower sidechain pKa. However when the external buffer pH is lowered to 4.9 and 2.4, the mutant shows increasing amantadine sensitive flux of a similar magnitude to that of the wild type construct at pH 7.4 and 4.9 respectively. These findings are in line with mechanistic hypotheses suggesting that proton flux through M2 is mediated by proton exchange from adjacent water molecules with the His37 sidechain, and that tautomerization is not required for proton translocation. PMID:24269540

  2. A Protein-Based Genetic Screening Uncovers Mutants Involved in Phytochrome Signaling in Arabidopsis

    PubMed Central

    Zhu, Ling; Xin, Ruijiao; Huq, Enamul

    2016-01-01

    Plants perceive red and far-red region of the light spectrum to regulate photomorphogenesis through a family of photoreceptors called phytochromes. Phytochromes transduce the light signals to trigger a cascade of downstream gene regulation in part via a subfamily of bHLH transcription factors called Phytochrome Interacting Factors (PIFs). As the repressors of light signaling pathways, most PIFs are phosphorylated and degraded through the ubiquitin/26S proteasome pathway in response to light. The mechanisms involved in the phosphorylation and degradation of PIFs have not been fully understood yet. Here we used an EMS mutagenesis and luminescent imaging system to identify mutants defective in the degradation of one of the PIFs, called PIF1. We identified five mutants named stable PIF (spf) that showed reduced degradation of PIF1 under light treatment in both luminescent imaging and immunoblot assays. The amounts of PIF1 in spf3, spf4, and spf5 were similar to a PIF1 missense mutant (PIF1–3M) that lacks interactions between PIF1 and phyA/phyB under light. The hypocotyl lengths of spf1 and spf2 were slightly longer under red light compared to the LUC-PIF1 control, while only spf1 displayed weak phenotype under far-red light conditions. Interestingly, the spf3, spf4, and spf5 displayed high abundance of PIF1, yet the hypocotyl lengths were similar to the wild type under these conditions. Cloning and characterization of these mutants will help identify key players in the light signaling pathways including, the light-regulated kinase(s) and the E3 ligase(s) necessary for the light-induced degradation of PIFs. PMID:27499759

  3. A Protein-Based Genetic Screening Uncovers Mutants Involved in Phytochrome Signaling in Arabidopsis.

    PubMed

    Zhu, Ling; Xin, Ruijiao; Huq, Enamul

    2016-01-01

    Plants perceive red and far-red region of the light spectrum to regulate photomorphogenesis through a family of photoreceptors called phytochromes. Phytochromes transduce the light signals to trigger a cascade of downstream gene regulation in part via a subfamily of bHLH transcription factors called Phytochrome Interacting Factors (PIFs). As the repressors of light signaling pathways, most PIFs are phosphorylated and degraded through the ubiquitin/26S proteasome pathway in response to light. The mechanisms involved in the phosphorylation and degradation of PIFs have not been fully understood yet. Here we used an EMS mutagenesis and luminescent imaging system to identify mutants defective in the degradation of one of the PIFs, called PIF1. We identified five mutants named stable PIF (spf) that showed reduced degradation of PIF1 under light treatment in both luminescent imaging and immunoblot assays. The amounts of PIF1 in spf3, spf4, and spf5 were similar to a PIF1 missense mutant (PIF1-3M) that lacks interactions between PIF1 and phyA/phyB under light. The hypocotyl lengths of spf1 and spf2 were slightly longer under red light compared to the LUC-PIF1 control, while only spf1 displayed weak phenotype under far-red light conditions. Interestingly, the spf3, spf4, and spf5 displayed high abundance of PIF1, yet the hypocotyl lengths were similar to the wild type under these conditions. Cloning and characterization of these mutants will help identify key players in the light signaling pathways including, the light-regulated kinase(s) and the E3 ligase(s) necessary for the light-induced degradation of PIFs. PMID:27499759

  4. High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis.

    PubMed

    Sridharan, Vinod; Heimiller, Joseph; Robida, Mark D; Singh, Ravinder

    2016-01-01

    The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5' ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis.

  5. High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis

    PubMed Central

    Sridharan, Vinod; Heimiller, Joseph; Robida, Mark D.; Singh, Ravinder

    2016-01-01

    The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5’ ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis. PMID:26942929

  6. An in silico algorithm for identifying stabilizing pockets in proteins: test case, the Y220C mutant of the p53 tumor suppressor protein.

    PubMed

    Bromley, Dennis; Bauer, Matthias R; Fersht, Alan R; Daggett, Valerie

    2016-09-01

    The p53 tumor suppressor protein performs a critical role in stimulating apoptosis and cell cycle arrest in response to oncogenic stress. The function of p53 can be compromised by mutation, leading to increased risk of cancer; approximately 50% of cancers are associated with mutations in the p53 gene, the majority of which are in the core DNA-binding domain. The Y220C mutation of p53, for example, destabilizes the core domain by 4 kcal/mol, leading to rapid denaturation and aggregation. The associated loss of tumor suppressor functionality is associated with approximately 75 000 new cancer cases every year. Destabilized p53 mutants can be 'rescued' and their function restored; binding of a small molecule into a pocket on the surface of mutant p53 can stabilize its wild-type structure and restore its function. Here, we describe an in silico algorithm for identifying potential rescue pockets, including the algorithm's integration with the Dynameomics molecular dynamics data warehouse and the DIVE visual analytics engine. We discuss the results of the application of the method to the Y220C p53 mutant, entailing finding a putative rescue pocket through MD simulations followed by an in silico search for stabilizing ligands that dock into the putative rescue pocket. The top three compounds from this search were tested experimentally and one of them bound in the pocket, as shown by nuclear magnetic resonance, and weakly stabilized the mutant. PMID:27503952

  7. An in silico algorithm for identifying stabilizing pockets in proteins: test case, the Y220C mutant of the p53 tumor suppressor protein.

    PubMed

    Bromley, Dennis; Bauer, Matthias R; Fersht, Alan R; Daggett, Valerie

    2016-09-01

    The p53 tumor suppressor protein performs a critical role in stimulating apoptosis and cell cycle arrest in response to oncogenic stress. The function of p53 can be compromised by mutation, leading to increased risk of cancer; approximately 50% of cancers are associated with mutations in the p53 gene, the majority of which are in the core DNA-binding domain. The Y220C mutation of p53, for example, destabilizes the core domain by 4 kcal/mol, leading to rapid denaturation and aggregation. The associated loss of tumor suppressor functionality is associated with approximately 75 000 new cancer cases every year. Destabilized p53 mutants can be 'rescued' and their function restored; binding of a small molecule into a pocket on the surface of mutant p53 can stabilize its wild-type structure and restore its function. Here, we describe an in silico algorithm for identifying potential rescue pockets, including the algorithm's integration with the Dynameomics molecular dynamics data warehouse and the DIVE visual analytics engine. We discuss the results of the application of the method to the Y220C p53 mutant, entailing finding a putative rescue pocket through MD simulations followed by an in silico search for stabilizing ligands that dock into the putative rescue pocket. The top three compounds from this search were tested experimentally and one of them bound in the pocket, as shown by nuclear magnetic resonance, and weakly stabilized the mutant.

  8. An autoactive mutant of the M flax rust resistance protein has a preference for binding ATP, whereas wild-type M protein binds ADP.

    PubMed

    Williams, Simon J; Sornaraj, Pradeep; deCourcy-Ireland, Emma; Menz, R Ian; Kobe, Bostjan; Ellis, Jeffrey G; Dodds, Peter N; Anderson, Peter A

    2011-08-01

    Resistance (R) proteins are key regulators of the plant innate immune system and are capable of pathogen detection and activation of the hypersensitive cell death immune response. To understand the molecular mechanism of R protein activation, we undertook a phenotypic and biochemical study of the flax nucleotide binding (NB)-ARC leucine-rich repeat protein, M. Using Agrobacterium-mediated transient expression in flax cotyledons, site-directed mutations of key residues within the P-loop, kinase 2, and MHD motifs within the NB-ARC domain of M were shown to affect R protein function. When purified using a yeast expression system and assayed for ATP and ADP, these mutated proteins exhibited marked differences in the quantity and identity of the bound nucleotide. ADP was bound to recombinant wild-type M protein, while the nonfunctional P-loop mutant did not have any nucleotides bound. In contrast, ATP was bound to an autoactive M protein mutated in the highly conserved MHD motif. These data provide direct evidence supporting a model of R protein function in which the "off" R protein binds ADP and activation of R protein defense signaling involves the exchange of ADP for ATP.

  9. Functional interaction of CCAAT/enhancer-binding-protein-α basic region mutants with E2F transcription factors and DNA.

    PubMed

    Kowenz-Leutz, Elisabeth; Schuetz, Anja; Liu, Qingbin; Knoblich, Maria; Heinemann, Udo; Leutz, Achim

    2016-07-01

    The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates cell cycle arrest and terminal differentiation of neutrophils and adipocytes. Mutations in the basic leucine zipper domain (bZip) of C/EBPα are associated with acute myeloid leukemia. A widely used murine transforming C/EBPα basic region mutant (BRM2) entails two bZip point mutations (I294A/R297A). BRM2 has been discordantly described as defective for DNA binding or defective for interaction with E2F. We have separated the two BRM2 mutations to shed light on the intertwined reciprocity between C/EBPα-E2F-DNA interactions. Both, C/EBPα I294A and R297A retain transactivation capacity and interaction with E2F-DP. The C/EBPα R297A mutation destabilized DNA binding, whereas the C/EBPα I294A mutation enhanced binding to DNA. The C/EBPα R297A mutant, like BRM2, displayed enhanced interaction with E2F-DP but failed to repress E2F-dependent transactivation although both mutants were readily suppressed by E2F1 for transcription through C/EBP cis-regulatory sites. In contrast, the DNA binding enhanced C/EBPα I294A mutant displayed increased repression of E2F-DP mediated transactivation and resisted E2F-DP mediated repression. Thus, the efficient repression of E2F dependent S-phase genes and the activation of differentiation genes reside in the balanced DNA binding capacity of C/EBPα.

  10. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

    PubMed

    He, Lihua; Kota, Pradeep; Aleksandrov, Andrei A; Cui, Liying; Jensen, Tim; Dokholyan, Nikolay V; Riordan, John R

    2013-02-01

    Most cystic fibrosis is caused by the deletion of a single amino acid (F508) from CFTR and the resulting misfolding and destabilization of the protein. Compounds identified by high-throughput screening to improve ΔF508 CFTR maturation have already entered clinical trials, and it is important to understand their mechanisms of action to further improve their efficacy. Here, we showed that several of these compounds, including the investigational drug VX-809, caused a much greater increase (5- to 10-fold) in maturation at 27 than at 37°C (<2-fold), and the mature product remained short-lived (T(1/2)∼4.5 h) and thermally unstable, even though its overall conformational state was similar to wild type, as judged by resistance to proteolysis and interdomain cross-linking. Consistent with its inability to restore thermodynamic stability, VX-809 stimulated maturation 2-5-fold beyond that caused by several different stabilizing modifications of NBD1 and the NBD1/CL4 interface. The compound also promoted maturation of several disease-associated processing mutants on the CL4 side of this interface. Although these effects may reflect an interaction of VX-809 with this interface, an interpretation supported by computational docking, it also rescued maturation of mutants in other cytoplasmic loops, either by allosteric effects or via additional sites of action. In addition to revealing the capabilities and some of the limitations of this important investigational drug, these findings clearly demonstrate that ΔF508 CFTR can be completely assembled and evade cellular quality control systems, while remaining thermodynamically unstable. He, L., Kota, P., Aleksandrov, A. A., Cui, L., Jensen, T., Dokholyan, N. V., Riordan, J. R. Correctors of ΔF508 CFTR restore global conformational maturation without thermally stabilizing the mutant protein.

  11. A Protein-Protein Interaction Assay FlimPIA Based on the Functional Complementation of Mutant Firefly Luciferases.

    PubMed

    Ohmuro-Matsuyama, Yuki; Ueda, Hiroshi

    2016-01-01

    There is a significant focus on detecting and assaying protein-protein interactions (PPIs) in biology and biotechnology. Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI by splitting the enzyme-coding or fluorescent protein-coding polypeptide, as well as Förster resonance energy transfer (FRET). Here, we describe a novel PPI assay FlimPIA (firefly luminescent intermediate-based protein-protein interaction assay) by a unique approach of splitting the two major catalytic steps (half reactions) of firefly luciferase (FLuc). PMID:27424900

  12. Analysis of parainfluenza virus-5 hemagglutinin-neuraminidase protein mutants that are blocked in internalization and degradation

    SciTech Connect

    Robach, Jessica G.; Lamb, Robert A.

    2010-10-25

    The PIV-5 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein with sialic acid binding, neuraminidase and fusion promotion activity. HN is internalized by clathrin-mediated endocytosis and degraded. HN lacks internalization signals in its cytoplasmic tail but a single glutamic acid present at residue 37 at the putative transmembrane/ectodomain boundary is critical. We rescued rPIV-5 with mutations E37D or E37K, which have been shown to impair or abolish HN internalization, respectively. These viruses exhibited growth properties similar to wild-type (wt) virus but are impaired for fitness in tissue culture. Biochemical analysis of HN activities showed differences between HN E37D and HN E37K in fusion promotion and incorporation of HN and F into virions. Furthermore, oligomeric analyses indicate that HN E37 mutants perturb the tetrameric organization of HN, probably by destabilizing the dimer-of-dimers interface.

  13. Mutant bacterial sodium channels as models for local anesthetic block of eukaryotic proteins.

    PubMed

    Smith, Natalie E; Corry, Ben

    2016-01-01

    Voltage gated sodium channels are the target of a range of local anesthetic, anti-epileptic and anti-arrhythmic compounds. But, gaining a molecular level understanding of their mode of action is difficult as we only have atomic resolution structures of bacterial sodium channels not their eukaryotic counterparts. In this study we used molecular dynamics simulations to demonstrate that the binding sites of both the local anesthetic benzocaine and the anti-epileptic phenytoin to the bacterial sodium channel NavAb can be altered significantly by the introduction of point mutations. Free energy techniques were applied to show that increased aromaticity in the pore of the channel, used to emulate the aromatic residues observed in eukaryotic Nav1.2, led to changes in the location of binding and dissociation constants of each drug relative to wild type NavAb. Further, binding locations and dissociation constants obtained for both benzocaine (660 μM) and phenytoin (1 μM) in the mutant channels were within the range expected from experimental values obtained from drug binding to eukaryotic sodium channels, indicating that these mutant NavAb may be a better model for drug binding to eukaryotic channels than the wild type.

  14. Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67

    PubMed Central

    Longo, Larissa V. G.; Nakayasu, Ernesto S.; Pires, Jhon H. S.; Gazos-Lopes, Felipe; Vallejo, Milene C.; Sobreira, Tiago J. P.; Almeida, Igor C.; Puccia, Rosana

    2015-01-01

    Cryptococcus neoformans is an opportunistic human pathogen that causes life-threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot SDS. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 non-covalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in extracellular vesicles. The possible role of the presently described structures in fungal-host relationship is discussed. PMID:25733123

  15. Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

    SciTech Connect

    Wang Qishan Bag, Jnanankur

    2008-05-23

    Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO{sub 4}, and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40, and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein.

  16. Accelerating the clearance of mutant huntingtin protein aggregates through autophagy induction by europium hydroxide nanorods.

    PubMed

    Wei, Peng-Fei; Zhang, Li; Nethi, Susheel Kumar; Barui, Ayan Kumar; Lin, Jun; Zhou, Wei; Shen, Yi; Man, Na; Zhang, Yun-Jiao; Xu, Jing; Patra, Chitta Ranjan; Wen, Long-Ping

    2014-01-01

    Autophagy is one of the well-known pathways to accelerate the clearance of protein aggregates, which contributes to the therapy of neurodegenerative diseases. Although there are numerous reports that demonstrate the induction of autophagy with small molecules including rapamycin, trehalose and lithium, however, there are few reports mentioning the clearance of aggregate-prone proteins through autophagy induction by nanoparticles. In the present article, we have demonstrated that europium hydroxide [Eu(III)(OH)3] nanorods can reduce huntingtin protein aggregation (EGFP-tagged huntingtin protein with 74 polyQ repeats), responsible for neurodegenerative diseases. Again, we have found that these nanorods induce authentic autophagy flux in different cell lines (Neuro 2a, PC12 and HeLa cells) through the expression of higher levels of characteristic autophagy marker protein LC3-II and degradation of selective autophagy substrate/cargo receptor p62/SQSTM1. Furthermore, depression of protein aggregation clearance through the autophagy blockade has also been observed by using specific inhibitors (wortmannin and chloroquine), indicating that autophagy is involved in the degradation of huntingtin protein aggregation. Since [Eu(III)(OH)3] nanorods can enhance the degradation of huntingtin protein aggregation via autophagy induction, we strongly believe that these nanorods would be useful for the development of therapeutic treatment strategies for various neurodegenerative diseases in near future using nanomedicine approach.

  17. Isolation and Characterization of β-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Mutants of Escherichia coli and Salmonella enterica Serovar Typhimurium

    PubMed Central

    Lai, Chiou-Yan; Cronan, John E.

    2004-01-01

    FabG, β-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The β-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature. PMID:14996818

  18. Identification of a new Pmp22 mouse mutant and trafficking analysis of a Pmp22 allelic series suggesting that protein aggregates may be protective in Pmp22-associated peripheral neuropathy.

    PubMed

    Isaacs, Adrian M; Jeans, Alexander; Oliver, Peter L; Vizor, Lucie; Brown, Steve D M; Hunter, A Jackie; Davies, Kay E

    2002-09-01

    We have identified and characterized a new peripheral myelin protein 22 (Pmp22) mouse mutant. The mutation results in a serine to threonine amino acid substitution at residue 72, which is a hot spot for mutation in human PMP22, leading to the peripheral neuropathy Dejerine-Sottas syndrome. We have previously described two other Pmp22 mutants, providing an allelic series for gene function analysis. Pmp22 mutations generally lead to abnormal intracellular trafficking of Pmp22, and we show that each mutant protein in the allelic series has a unique pattern of intracellular localization in transfected cell lines. The mutant protein from the less severely affected mutants occurs in large aggregates, while the mutant protein from the most severely affected mutant occurs in a diffuse perinuclear pattern that largely colocalizes with wild-type protein. This suggests that large Pmp22 aggregates may be protective in this form of peripheral neuropathy. PMID:12359155

  19. Characterization of Mutants of Human Small Heat Shock Protein HspB1 Carrying Replacements in the N-Terminal Domain and Associated with Hereditary Motor Neuron Diseases

    PubMed Central

    Muranova, Lydia K.; Weeks, Stephen D.; Strelkov, Sergei V.; Gusev, Nikolai B.

    2015-01-01

    Physico-chemical properties of the mutations G34R, P39L and E41K in the N-terminal domain of human heat shock protein B1 (HspB1), which have been associated with hereditary motor neuron neuropathy, were analyzed. Heat-induced aggregation of all mutants started at lower temperatures than for the wild type protein. All mutations decreased susceptibility of the N- and C-terminal parts of HspB1 to chymotrypsinolysis. All mutants formed stable homooligomers with a slightly larger apparent molecular weight compared to the wild type protein. All mutations analyzed decreased or completely prevented phosphorylation-induced dissociation of HspB1 oligomers. When mixed with HspB6 and heated, all mutants yielded heterooligomers with apparent molecular weights close to ~400 kDa. Finally, the three HspB1 mutants possessed lower chaperone-like activity towards model substrates (lysozyme, malate dehydrogenase and insulin) compared to the wild type protein, conversely the environmental probe bis-ANS yielded higher fluorescence with the mutants than with the wild type protein. Thus, in vitro the analyzed N-terminal mutations increase stability of large HspB1 homooligomers, prevent their phosphorylation-dependent dissociation, modulate their interaction with HspB6 and decrease their chaperoning capacity, preventing normal functioning of HspB1. PMID:25965061

  20. Genetic Analysis of Mps3 SUN Domain Mutants in Saccharomyces cerevisiae Reveals an Interaction with the SUN-Like Protein Slp1

    PubMed Central

    Friederichs, Jennifer M.; Gardner, Jennifer M.; Smoyer, Christine J.; Whetstine, Christine R.; Gogol, Madelaine; Slaughter, Brian D.; Jaspersen, Sue L.

    2012-01-01

    In virtually all eukaryotic cells, protein bridges formed by the conserved inner nuclear membrane SUN (for Sad1-UNC-84) domain-containing proteins and their outer nuclear membrane binding partners span the nuclear envelope (NE) to connect the nucleoplasm and cytoplasm. These linkages are important for chromosome movements within the nucleus during meiotic prophase and are essential for nuclear migration and centrosome attachment to the NE. In Saccharomyces cerevisiae, MPS3 encodes the sole SUN protein. Deletion of MPS3 or the conserved SUN domain is lethal in three different genetic backgrounds. Mutations in the SUN domain result in defects in duplication of the spindle pole body, the yeast centrosome-equivalent organelle. A genome-wide screen for mutants that exhibited synthetic fitness defects in combination with mps3 SUN domain mutants yielded a large number of hits in components of the spindle apparatus and the spindle checkpoint. Mutants in lipid metabolic processes and membrane organization also exacerbated the growth defects of mps3 SUN domain mutants, pointing to a role for Mps3 in nuclear membrane organization. Deletion of SLP1 or YER140W/EMP65 (for ER membrane protein of 65 kDa) aggravated growth of mps3 SUN domain mutants. Slp1 and Emp65 form an ER-membrane associated protein complex that is not required directly for spindle pole body duplication or spindle assembly. Rather, Slp1 is involved in Mps3 localization to the NE. PMID:23275891

  1. Oridonin induces NPM mutant protein translocation and apoptosis in NPM1c+ acute myeloid leukemia cells in vitro

    PubMed Central

    Li, Fei-fei; Yi, Sha; Wen, Lu; He, Jing; Yang, Li-jing; Zhao, Jie; Zhang, Ben-ping; Cui, Guo-hui; Chen, Yan

    2014-01-01

    Aim: Skewed cytoplasmic accumulation of NPM mutant protein (NPM1c+) is close related to leukemia pathogenesis. The aim of this study was to investigate whether oridonin, a diterpenoid isolated from the Chinese traditional medicine Rabdosia rubescens, was able to interfere with NPM1c+ protein trafficking and induce apoptosis in NPM1c+ acute myeloid leukemia cells in vitro. Methods: OCI-AML3 cell line harboring a NPM1 gene mutation was examined. Cell growth was detected by MTT assay. Cell apoptosis was evaluated using flow cytometry and Hoechst 33258 staining. The expression and subcellular localization of relevant proteins were detected by Western blot and immunofluorescent staining. The mRNA expression was detected by RT-PCR. Results: Oridonin (2–12 μmol/L) dose-dependently inhibited the viability of OCI-AML3 cells (the IC50 value was 3.27±0.23 μmol/L at 24 h). Moreover, oridonin induced OCI-AML3 cell apoptosis accompanied by activation of caspase-3 and nuclear translocation of NPM1c+ protein. Oridonin did not change the expression of Crm1 (the export receptor for nuclear export signal-containing proteins), but induced nuclear translocation of Crm1. Oridonin markedly increased the expression of nucleoporin98 (Nup98), which had an important role in Crm1-mediated nuclear protein export, and induced nuclear accumulation of Nup98. Furthermore, oridonin markedly increased the expression of p14arf and p53. Conclusion: In NPM1c+ leukemia cells, oridonin induces NPM1c+ protein translocation into the nucleus possibly via nuclear accumulation of Crm1; the compound markedly increases p53 and p14arf expression, which may contribute to cell apoptosis. PMID:24902788

  2. Alpha-Synuclein Proteins Promote Pro-Inflammatory Cascades in Microglia: Stronger Effects of the A53T Mutant

    PubMed Central

    Hoenen, Claire; Gustin, Audrey; Birck, Cindy; Kirchmeyer, Mélanie; Beaume, Nicolas; Felten, Paul; Grandbarbe, Luc; Heuschling, Paul; Heurtaux, Tony

    2016-01-01

    Parkinson’s disease (PD) is histologically described by the deposition of α-synuclein, whose accumulation in Lewy bodies causes dopaminergic neuronal death. Although most of PD cases are sporadic, point mutations of the gene encoding the α-synuclein protein cause inherited forms of PD. There are currently six known point mutations that result in familial PD. Oxidative stress and neuroinflammation have also been described as early events associated with dopaminergic neuronal degeneration in PD. Though it is known that microglia are activated by wild-type α-synuclein, little is known about its mutated forms and the signaling cascades responsible for this microglial activation. The present study was designed to investigate consequences of wild-type and mutant α-synuclein (A53T, A30P and E46K) exposure on microglial reactivity. Interestingly, we described that α-synuclein-induced microglial reactivity appeared to be peptide-dependent. Indeed, the A53T protein activated more strongly microglia than the wild-type α-synuclein and other mutants. This A53T-induced microglial reactivity mechanism was found to depend on phosphorylation mechanisms mediated by MAPKs and on successive NFkB/AP-1/Nrf2 pathways activation. These results suggest that the microgliosis intensity during PD might depend on the type of α-synuclein protein implicated. Indeed, mutated forms are more potent microglial stimulators than wild-type α-synuclein. Based on these data, anti-inflammatory and antioxidant therapeutic strategies may be valid in order to reduce microgliosis but also to subsequently slow down PD progression, especially in familial cases. PMID:27622765

  3. Alpha-Synuclein Proteins Promote Pro-Inflammatory Cascades in Microglia: Stronger Effects of the A53T Mutant.

    PubMed

    Hoenen, Claire; Gustin, Audrey; Birck, Cindy; Kirchmeyer, Mélanie; Beaume, Nicolas; Felten, Paul; Grandbarbe, Luc; Heuschling, Paul; Heurtaux, Tony

    2016-01-01

    Parkinson's disease (PD) is histologically described by the deposition of α-synuclein, whose accumulation in Lewy bodies causes dopaminergic neuronal death. Although most of PD cases are sporadic, point mutations of the gene encoding the α-synuclein protein cause inherited forms of PD. There are currently six known point mutations that result in familial PD. Oxidative stress and neuroinflammation have also been described as early events associated with dopaminergic neuronal degeneration in PD. Though it is known that microglia are activated by wild-type α-synuclein, little is known about its mutated forms and the signaling cascades responsible for this microglial activation. The present study was designed to investigate consequences of wild-type and mutant α-synuclein (A53T, A30P and E46K) exposure on microglial reactivity. Interestingly, we described that α-synuclein-induced microglial reactivity appeared to be peptide-dependent. Indeed, the A53T protein activated more strongly microglia than the wild-type α-synuclein and other mutants. This A53T-induced microglial reactivity mechanism was found to depend on phosphorylation mechanisms mediated by MAPKs and on successive NFkB/AP-1/Nrf2 pathways activation. These results suggest that the microgliosis intensity during PD might depend on the type of α-synuclein protein implicated. Indeed, mutated forms are more potent microglial stimulators than wild-type α-synuclein. Based on these data, anti-inflammatory and antioxidant therapeutic strategies may be valid in order to reduce microgliosis but also to subsequently slow down PD progression, especially in familial cases. PMID:27622765

  4. Cysteine shotgun–mass spectrometry (CS-MS) reveals dynamic sequence of protein structure changes within mutant and stressed cells

    PubMed Central

    Krieger, Christine C.; An, Xiuli; Tang, Hsin-Yao; Mohandas, Narla; Speicher, David W.; Discher, Dennis E.

    2011-01-01

    Questions of if and when protein structures change within cells pervade biology and include questions of how the cytoskeleton sustains stresses on cells—particularly in mutant versus normal cells. Cysteine shotgun labeling with fluorophores is analyzed here with mass spectrometry of the spectrin–actin membrane skeleton in sheared red blood cell ghosts from normal and diseased mice. Sheared samples are compared to static samples at 37 °C in terms of cell membrane intensity in fluorescence microscopy, separated protein fluorescence, and tryptic peptide modification in liquid chromatography–tandem mass spectrometry (LC-MS/MS). Spectrin labeling proves to be the most sensitive to shear, whereas binding partners ankyrin and actin exhibit shear thresholds in labeling and both the ankyrin-binding membrane protein band 3 and the spectrin–actin stabilizer 4.1R show minimal differential labeling. Cells from 4.1R-null mice differ significantly from normal in the shear-dependent labeling of spectrin, ankyrin, and band 3: Decreased labeling of spectrin reveals less stress on the mutant network as spectrin dissociates from actin. Mapping the stress-dependent labeling kinetics of α- and β-spectrin by LC-MS/MS identifies Cys in these antiparallel chains that are either force-enhanced or force-independent in labeling, with structural analyses indicating the force-enhanced sites are sequestered either in spectrin’s triple-helical domains or in interactions with actin or ankyrin. Shear-sensitive sites identified comprehensively here in both spectrin and ankyrin appear consistent with stress relief through forced unfolding followed by cytoskeletal disruption. PMID:21527722

  5. Photosystem II Repair and Plant Immunity: Lessons Learned from Arabidopsis Mutant Lacking the THYLAKOID LUMEN PROTEIN 18.3.

    PubMed

    Järvi, Sari; Isojärvi, Janne; Kangasjärvi, Saijaliisa; Salojärvi, Jarkko; Mamedov, Fikret; Suorsa, Marjaana; Aro, Eva-Mari

    2016-01-01

    Chloroplasts play an important role in the cellular sensing of abiotic and biotic stress. Signals originating from photosynthetic light reactions, in the form of redox and pH changes, accumulation of reactive oxygen and electrophile species or stromal metabolites are of key importance in chloroplast retrograde signaling. These signals initiate plant acclimation responses to both abiotic and biotic stresses. To reveal the molecular responses activated by rapid fluctuations in growth light intensity, gene expression analysis was performed with Arabidopsis thaliana wild type and the tlp18.3 mutant plants, the latter showing a stunted growth phenotype under fluctuating light conditions (Biochem. J, 406, 415-425). Expression pattern of genes encoding components of the photosynthetic electron transfer chain did not differ between fluctuating and constant light conditions, neither in wild type nor in tlp18.3 plants, and the composition of the thylakoid membrane protein complexes likewise remained unchanged. Nevertheless, the fluctuating light conditions repressed in wild-type plants a broad spectrum of genes involved in immune responses, which likely resulted from shade-avoidance responses and their intermixing with hormonal signaling. On the contrary, in the tlp18.3 mutant plants there was an imperfect repression of defense-related transcripts upon growth under fluctuating light, possibly by signals originating from minor malfunction of the photosystem II (PSII) repair cycle, which directly or indirectly modulated the transcript abundances of genes related to light perception via phytochromes. Consequently, a strong allocation of resources to defense reactions in the tlp18.3 mutant plants presumably results in the stunted growth phenotype under fluctuating light.

  6. Photosystem II Repair and Plant Immunity: Lessons Learned from Arabidopsis Mutant Lacking the THYLAKOID LUMEN PROTEIN 18.3

    PubMed Central

    Järvi, Sari; Isojärvi, Janne; Kangasjärvi, Saijaliisa; Salojärvi, Jarkko; Mamedov, Fikret; Suorsa, Marjaana; Aro, Eva-Mari

    2016-01-01

    Chloroplasts play an important role in the cellular sensing of abiotic and biotic stress. Signals originating from photosynthetic light reactions, in the form of redox and pH changes, accumulation of reactive oxygen and electrophile species or stromal metabolites are of key importance in chloroplast retrograde signaling. These signals initiate plant acclimation responses to both abiotic and biotic stresses. To reveal the molecular responses activated by rapid fluctuations in growth light intensity, gene expression analysis was performed with Arabidopsis thaliana wild type and the tlp18.3 mutant plants, the latter showing a stunted growth phenotype under fluctuating light conditions (Biochem. J, 406, 415–425). Expression pattern of genes encoding components of the photosynthetic electron transfer chain did not differ between fluctuating and constant light conditions, neither in wild type nor in tlp18.3 plants, and the composition of the thylakoid membrane protein complexes likewise remained unchanged. Nevertheless, the fluctuating light conditions repressed in wild-type plants a broad spectrum of genes involved in immune responses, which likely resulted from shade-avoidance responses and their intermixing with hormonal signaling. On the contrary, in the tlp18.3 mutant plants there was an imperfect repression of defense-related transcripts upon growth under fluctuating light, possibly by signals originating from minor malfunction of the photosystem II (PSII) repair cycle, which directly or indirectly modulated the transcript abundances of genes related to light perception via phytochromes. Consequently, a strong allocation of resources to defense reactions in the tlp18.3 mutant plants presumably results in the stunted growth phenotype under fluctuating light. PMID:27064270

  7. Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

    PubMed Central

    Brázdová, Marie; Navrátilová, Lucie; Tichý, Vlastimil; Němcová, Kateřina; Lexa, Matej; Hrstka, Roman; Pečinka, Petr; Adámik, Matej; Vojtesek, Borivoj; Paleček, Emil; Deppert, Wolfgang; Fojta, Miroslav

    2013-01-01

    Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed. PMID:23555710

  8. iTRAQ protein profile analysis of tomato green-ripe mutant reveals new aspects critical for fruit ripening.

    PubMed

    Pan, Xiaoqi; Zhu, Benzhong; Zhu, Hongliang; Chen, Yuexi; Tian, Huiqin; Luo, Yunbo; Fu, Daqi

    2014-04-01

    Green-ripe (Gr) tomato carries a dominant mutation and yields a nonripening fruit phenotype. The mutation results from a 334 bp deletion in a gene of unknown function at the Gr locus. The putative influence of Gr gene-deletion mutation on biochemical changes underlying the nonripening phenotype remains largely unknown. Respiration of Gr fruit was found to be reduced at mature green and breaker stage of ripening, while the fruit softening was dramatically prolonged. We studied the proteome of Gr mutant fruit using high-throughput iTRAQ and high-resolution mass spectrometry and identified 43 proteins representing 43 individual genes as potential influence-targets of Gr mutated fruit. The identified proteins are involved in several ripening-related pathways including cell-wall metabolism, photosynthesis, oxidative phosphorylation, carbohydrate and fatty acid metabolism, protein synthesis, and processing. Affected protein levels are correlated with the corresponding gene transcript levels. The modulation in the accumulation levels of PI(U1)P, PGIP, and PG2 supported the delayed softening phenotype of the Gr fruit. Further investigation in GR gene-silencing fruit ascertained the doubtless modulation of these targets by the deletion mutation of GR gene.

  9. A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

    PubMed Central

    Derman, A I; Puziss, J W; Bassford, P J; Beckwith, J

    1993-01-01

    The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all. Images PMID:8458344

  10. Structural properties of fibrillar proteins isolated from the cell surface and cytoplasm of Streptococcus salivarius (K+) cells and nonadhesive mutants.

    PubMed Central

    Weerkamp, A H; van der Mei, H C; Liem, R S

    1986-01-01

    Most Streptococcus salivarius (K+) cells contain two protein antigens with different adhesive functions. The subcellular distribution and some structural properties of purified proteins were studied. Antigen B (AgB), a protein involved in interbacterial coaggregation with gram-negative bacteria, was present in the cell wall fraction only of the wild-type strain and was absent from the cells of a nonadhesive mutant. Antigen C (AgC), a glycoprotein involved in host-associated adhesive functions, was predominantly associated with the cell wall of the wild-type strain (AgCw), but accumulated in high amounts in the cytoplasmic fraction (AgCin) of mutants lacking the wall-associated form. AgB, AgCw, and AgCin had molecular weights of 380,000, 250,000 to 320,000, and 488,000, respectively, upon gel electrophoresis under nondenaturing conditions. In the presence of sodium dodecyl sulfate and beta-mercaptoethanol the molecular weights were only slightly lower, suggesting that the free, isolated molecules exist as monomers under native conditions. AgCin readily stained with periodate-Schiff reagent, indicating a significant content of carbohydrate, similar to AgCw. Circular dichroism spectra showed that about 45% of the amino acids of AgCw were involved in alpha-helical coiled structures. AgB had a significantly lower proportion of ordered coiled structure. Electron microscopic observations of low-angle-shadowed preparations of purified antigens showed that they were flexible, thin rods with thickened or globular ends. Measurements corrected for shadow thickness showed lengths of 184 nm (AgB), 112 nm (AgCin), and 87 nm (AgCw). Treatment of AgCw with protease destroyed the fibrillar core, but seemed not to affect the globular ends. Comparison of the results with the localization of the antigens in wild-type and specific mutant strains suggested that each antigen molecule may represent a single, characteristic surface fibril with a specific adhesive capacity. Images PMID

  11. SPN1, a conserved gene identified by suppression of a postrecruitment-defective yeast TATA-binding protein mutant.

    PubMed Central

    Fischbeck, Julie A; Kraemer, Susan M; Stargell, Laurie A

    2002-01-01

    Little is known about TATA-binding protein (TBP) functions after recruitment to the TATA element, although several TBP mutants display postrecruitment defects. Here we describe a genetic screen for suppressors of a postrecruitment-defective TBP allele. Suppression was achieved by a single point mutation in a previously uncharacterized Saccharomyces cerevisiae gene, SPN1 (suppresses postrecruitment functions gene number 1). SPN1 is an essential yeast gene that is highly conserved throughout evolution. The suppressing mutation in SPN1 substitutes an asparagine for an invariant lysine at position 192 (spn1(K192N)). The spn1(K192N) strain is able to suppress additional alleles of TBP that possess postrecruitment defects, but not a TBP allele that is postrecruitment competent. In addition, Spn1p does not stably associate with TFIID in vivo. Cells containing the spn1(K192N) allele exhibit a temperature-sensitive phenotype and some defects in activated transcription, whereas constitutive transcription appears relatively robust in the mutant background. Consistent with an important role in postrecruitment functions, transcription from the CYC1 promoter, which has been shown to be regulated by postrecruitment mechanisms, is enhanced in spn1(K192N) cells. Moreover, we find that SPN1 is a member of the SPT gene family, further supporting a functional requirement for the SPN1 gene product in transcriptional processes. PMID:12524336

  12. Tailoring the surface properties of tobacco mosaic virions by the integration of bacterially expressed mutant coat protein.

    PubMed

    Eiben, Sabine; Stitz, Nina; Eber, Fabian; Wagner, Jerrit; Atanasova, Petia; Bill, Joachim; Wege, Christina; Jeske, Holger

    2014-02-13

    Due to its small dimensions and high stability, tobacco mosaic virus (TMV) is used as nano-scaffold frequently. Its surface can be engineered to meet specific needs for technical, medical or materials applications. However, not all technically desirable TMV coat protein (CP) mutants can be propagated in plants successfully, if they change the efficiency of virion assembly. In order to circumvent this problem, a novel wild type (wt) CP-assisted and RNA-directed assembly procedure was designed for a recalcitrant CP mutant: Although pure hexahistidine-tagged CP cannot form particles on its own with TMV RNA in vitro, it was integrated into full-length particles if blended with wt CP in different proportions. The resulting rods formed dense monolayers with short range alignment on silicon substrates, substantially different from the largely wavy patterns obtained with wt TMV. Since they also mediated efficient ZnO deposition under mild conditions, the approach has yielded a new class of biotemplates which are amenable to the formation of nanostructured hybrid materials with adjustable texture for various applications. PMID:24299619

  13. An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein.

    PubMed

    Irving, James A; Miranda, Elena; Haq, Imran; Perez, Juan; Kotov, Vadim R; Faull, Sarah V; Motamedi-Shad, Neda; Lomas, David A

    2015-05-15

    A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states.

  14. Deciphering the Dynamics of Non-Covalent Interactions Affecting Thermal Stability of a Protein: Molecular Dynamics Study on Point Mutant of Thermus thermophilus Isopropylmalate Dehydrogenase.

    PubMed

    Sharma, Reetu; Sastry, G Narahari

    2015-01-01

    Thermus thermophilius isopropylmalate dehydrogenase catalyzes oxidative decarboxylation and dehydrogenation of isopropylmalate. Substitution of leucine to alanine at position 172 enhances the thermal stability among the known point mutants. Exploring the dynamic properties of non-covalent interactions such as saltbridges, hydrogen bonds and hydrophobic interactions to explain thermal stability of a protein is interesting in its own right. In this study dynamic changes in the non-covalent interactions are studied to decipher the deterministic features of thermal stability of a protein considering a case study of a point mutant in Thermus thermophilus isopropylmalate dehydrogenase. A total of four molecular dynamic simulations of 0.2 μs were carried out on wild type and mutant's functional dimers at 300 K and 337 K. Higher thermal stability of the mutant as compared to wild type is revealed by root mean square deviation, root mean square fluctuations and Cα-Cα distance with an increase in temperature from 300 K to 337 K. Most of the regions of wild type fluctuate higher than the corresponding regions of mutant with an increase in temperature. Cα-Cα distance analysis suggests that long distance networks are significantly affected in wild type as compared to the mutant. Short lived contacts are higher in wild type, while long lived contacts are lost at 337 K. The mutant forms less hydrogen bonds with water as compared to wild type at 337 K. In contrast to wild type, the mutant shows significant increase in unique saltbridges, hydrogen bonds and hydrophobic contacts at 337 K. The current study indicates that there is a strong inter-dependence of thermal stability on the way in which non-covalent interactions reorganize, and it is rewarding to explore this connection in single mutant studies. PMID:26657745

  15. Deciphering the Dynamics of Non-Covalent Interactions Affecting Thermal Stability of a Protein: Molecular Dynamics Study on Point Mutant of Thermus thermophilus Isopropylmalate Dehydrogenase.

    PubMed

    Sharma, Reetu; Sastry, G Narahari

    2015-01-01

    Thermus thermophilius isopropylmalate dehydrogenase catalyzes oxidative decarboxylation and dehydrogenation of isopropylmalate. Substitution of leucine to alanine at position 172 enhances the thermal stability among the known point mutants. Exploring the dynamic properties of non-covalent interactions such as saltbridges, hydrogen bonds and hydrophobic interactions to explain thermal stability of a protein is interesting in its own right. In this study dynamic changes in the non-covalent interactions are studied to decipher the deterministic features of thermal stability of a protein considering a case study of a point mutant in Thermus thermophilus isopropylmalate dehydrogenase. A total of four molecular dynamic simulations of 0.2 μs were carried out on wild type and mutant's functional dimers at 300 K and 337 K. Higher thermal stability of the mutant as compared to wild type is revealed by root mean square deviation, root mean square fluctuations and Cα-Cα distance with an increase in temperature from 300 K to 337 K. Most of the regions of wild type fluctuate higher than the corresponding regions of mutant with an increase in temperature. Cα-Cα distance analysis suggests that long distance networks are significantly affected in wild type as compared to the mutant. Short lived contacts are higher in wild type, while long lived contacts are lost at 337 K. The mutant forms less hydrogen bonds with water as compared to wild type at 337 K. In contrast to wild type, the mutant shows significant increase in unique saltbridges, hydrogen bonds and hydrophobic contacts at 337 K. The current study indicates that there is a strong inter-dependence of thermal stability on the way in which non-covalent interactions reorganize, and it is rewarding to explore this connection in single mutant studies.

  16. Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein Dronpa

    PubMed Central

    Kaucikas, Marius; Fitzpatrick, Ann; Bryan, Elana; Struve, Abelone; Henning, Robert; Kosheleva, Irina; Srajer, Vukica; Groenhof, Gerrit; van Thor, Jasper J.

    2015-01-01

    The fluorescent protein Dronpa undergoes reversible photoswitching reactions between the bright ‘on’ and dark ‘off’ states via photoisomerisation and proton transfer reactions. We report the room temperature crystal structure of the fast switching Met159Thr mutant of Dronpa at 2.0 Å resolution in the bright on state. Structural differences with the wild type include shifted backbone positions of strand β8 containing Thr159 as well as an altered A-C dimer interface involving strands β7, β8, β10, and β11. The Met159Thr mutation increases the cavity volume for the p-hydroxybenzylidene-imidazolinone chromophore as a result of both the side chain difference and the backbone positional differences. PMID:25524427

  17. Modeling Alzheimer’s Disease in Mouse without Mutant Protein Overexpression: Cooperative and Independent Effects of Aβ and Tau

    PubMed Central

    Guo, Qinxi; Li, Hongmei; Cole, Allysa L.; Hur, Ji-Yeun; Li, Yueming; Zheng, Hui

    2013-01-01

    Background Alzheimer’s disease (AD), the most common cause of dementia in the elderly, has two pathological hallmarks: Aβ plaques and aggregation of hyperphosphorylated tau (p-tau). Aβ is a cleavage product of Amyloid Precursor Protein (APP). Presenilin 1 (PS1) and presenilin 2 (PS2) are the catalytic subunit of γ-secretase, which cleaves APP and mediates Aβ production. Genetic mutations in APP, PSEN1 or PSEN2 can lead to early onset of familial AD (FAD). Although mutations in the tau encoding gene MAPT leads to a subtype of frontotemporal dementia and these mutations have been used to model AD tauopathy, no MAPT mutations have been found to be associated with AD. Results To model AD pathophysiology in mice without the gross overexpression of mutant transgenes, we created a humanized AD mouse model by crossing the APP and PSEN1 FAD knock-in mice with the htau mice which express wildtype human MAPT genomic DNA on mouse MAPT null background (APP/PS1/htau). The APP/PS1/htau mice displayed mild, age-dependent, Aβ plaques and tau hyperphosphorylation, thus successfully recapitulating the late-onset AD pathological hallmarks. Selected biochemical analyses, including p-tau western blot, γ-secretase activity assay, and Aβ ELISA, were performed to study the interaction between Aβ and p-tau. Subsequent behavioral studies revealed that the APP/PS1/htau mice showed reduced mobility in old ages and exaggerated fear response. Genetic analysis suggested that the fear phenotype is due to a synergic interaction between Aβ and p-tau, and it can be completely abolished by tau deletion. Conclusion The APP/PS1/htau model represents a valuable and disease-relevant late-onset pre-clinical AD animal model because it incorporates human AD genetics without mutant protein overexpression. Analysis of the mice revealed both cooperative and independent effects of Aβ and p-tau. PMID:24278307

  18. Application of physical organic chemistry to engineered mutants of proteins: Hammond postulate behavior in the transition state of protein folding.

    PubMed Central

    Matouschek, A; Fersht, A R

    1993-01-01

    Transition states in protein folding may be analyzed by linear free-energy relationships (LFERs) analogous to the Brønsted equation for changes in reactivity with changes in structure. There is an additional source of LFERs in protein folding: the perturbation of the equilibrium and rate constants by denaturants. These LFERs give a measure of the position of the transition state along the reaction coordinate. The transition state for folding/unfolding of barnase has been analyzed by both types of LFERs: changing the structure by protein engineering and perturbation by denaturants. The combination has allowed the direct monitoring of Hammond postulate behavior of the transition state on the reaction pathway. Movement of the transition state has been found and analyzed to give further details of the order of events in protein folding. PMID:8356089

  19. Changes in 30K protein synthesis during delayed degeneration of the silk gland by a caspase-dependent pathway in a Bombyx (silkworm) mutant.

    PubMed

    Wang, Huan; Wang, Yulong; Wu, Chengjia; Tao, Hui; Chen, Xuedong; Yin, Weimin; Sima, Yanghu; Wang, Yujun; Xu, Shiqing

    2016-08-01

    The silk gland in silkworm (Bombyx mori) is a highly specialized organ that specifically synthesizes silk proteins. A function shift to the synthesis of large quantities of 30K proteins occurs in the degenerating silk gland cells during larval-pupal metamorphosis. The posterior silk gland developmental mutant model of silkworm was used in this study and changes in the programmed cell death (PCD) regulatory signals and 30K protein synthesis during silk gland degeneration were investigated. The results showed that PCD induced by 20-hydroxyecdysone was initiated early during larval-pupal metamorphosis in the mutant, but PCD proceeded slowly, resulting in the degeneration process of the silk gland being extended and took almost twice the time compared with the wild type. Caspase-dependent pathway signals regulated by Dronc in the silk gland cells of the mutant were significantly reduced, while the PCD initiation signal regulated by the Atg family was not delayed or reduced, and PCD-related epigenetic modification such as lysine methylation, acetylation, and succinylation, and tyrosine phosphorylation changed significantly. During the degeneration process in the mutant, 30K proteins were efficiently synthesized in the silk gland cells in stage PP1 even when no caspase protein was detected. Degeneration of the silk gland is a PCD process in which autophagy and apoptosis may participate. The degeneration process was regulated by a caspase-dependent pathway, while the synthesis of 30K proteins along with silk gland degeneration may not be entirely dependent on caspase signals.

  20. Temperature-Sensitive Mutants Blocked in the Folding or Subunit Assmbly of the Bacteriophage P22 Tailspike Protein. I. Fine-Structure Mapping

    PubMed Central

    Smith, Donna H.; Berget, Peter B.; King, Jonathan

    1980-01-01

    As part of a study of protein folding, we have constructed a fine-structure map of 9 existing and 29 newly isolated UV- and hydroxylamine-induced temperature-sensitive (ts) mutations in gene 9 of Salmonella bacteriophage P22. Gene 9 specifies the polypeptide chain of the multimeric tail spikes, six of which form the cell attachment organelle of the phage. The 38 ts mutants were mapped against deletion lysogens with endpoints in gene 9. They mapped in 10 of the 15 deletion intervals. Two- and three-factor crosses between mutants within each interval indicated that at least 31 ts sites are represented among the 38 mutants. To determine the distribution of ts sites within the physical map, we identified the protein fragments from infection of su- hosts with 10 gene 9 amber mutants. Their molecular weights, ranging from 13,900 to 55,000 daltons, were combined with the genetic data to yield a composite map of gene 9. The 31 ts sites were distributed through most of the gene, but were most densely clustered in the central third.—None of the ts mutant pairs tested exhibited intragenic complementation. Studies of the defective phenotypes of the ts mutants (Goldenberg and King 1981; Smith and King 1981) revealed that most do not affect the thermostability of the mature protein, but instead prevent the folding or subunit assembly of the mutant chains synthesized at restrictive temperature. Thus, many of thes ts mutations identify sites in the polypeptide chain that are critical for the folding or maturation of the tail-spike protein. PMID:7021307

  1. Prediction of ligand binding site by insilico approach in cold resistant protein isolated from cold resistant mutant of Pseudomonas fluorescens.

    PubMed

    Kumar, Amit; Khan, Mahejibin

    2012-09-01

    Cold shock proteins perform vital functions, such as mRNA masking, coupling of transcription to translation and developmental timing and regulation, which aids in survival of microbes in cold stress. Pseudomonas fluorescens is an ecologically important bacterium which helps in plant growth promotion. Since the cold tolerant mutant of the bacterium is able to grow at the temperature ranges from 30 to 4°C, it is of interest to study the process of its survival in the extreme temperatures. Therefore, this study is focused on the three dimensional structure and molecular modeling of cold resistant protein (CRP) from P. fluorescens to predict its molecular mechanism. Investigating the structure of CRP confirmed the presence of a conserved domain characteristic of the cold shock domain (CSD) family and a single nucleotide binding domain. When 3D structure of CRP was compared with the existing cold shock proteins, major deviations were found in the loop regions connecting the β2-β3, β3-β4 and β4-β5 sheets. Docking studies showed that CRP forms a significant clamp like structure at the substrate binding cleft which stabilizes the ligand. Therefore, it can be concluded that CRP has a strong affinity for the poly thymidine (poly T) stretch and can be considered a candidate for transcription regulation. PMID:23099776

  2. Crystallization and preliminary X-ray diffraction analysis of Val57 mutants of the amyloidogenic protein human cystatin C

    SciTech Connect

    Orlikowska, Marta; Jankowska, Elzbieta; Borek, Dominika; Otwinowski, Zbyszek; Skowron, Piotr; Szymanska, Aneta

    2012-03-15

    Human cystatin C (hCC) is a low-molecular-mass protein (120 amino-acid residues, 13 343 Da) found in all nucleated cells. Its main physiological role is regulation of the activity of cysteine proteases. Biologically active hCC is a monomeric protein, but all crystallization efforts have resulted in a dimeric domain-swapped structure. Recently, two monomeric structures were reported for cystatin C variants. In one of them stabilization was achieved by abolishing the possibility of domain swapping by the introduction of an additional disulfide bridge connecting the two protein domains (Cys47-Cys69). In the second structure, reported by this group, the monomeric hCC fold was preserved by stabilization of the conformationally constrained loop (L1) by a single-amino-acid substitution (V57N). To further assess the influence of changes in the sequence and properties of loop L1 on the dimerization propensity of cystatin C, two additional hCC mutants were obtained: one with a residue favoured in {beta}-turns (V57D) and another with proline (V57P), a residue that is known to be a structural element that can rigidify but also broaden turns. Here, the expression, purification and crystallization of V57D and V57P variants of recombinant human cystatin C are described. Crystals were grown by the vapour-diffusion method. Several diffraction data sets were collected using a synchrotron source at the Advanced Photon Source, Argonne National Laboratory, Chicago, USA.

  3. Dataset of differentially regulated proteins in HUVECs challenged with wild type and UGM1 mutant Aspergillus fumigatus strains.

    PubMed

    Neves, Gabriela Westerlund Peixoto; Curty, Nathália; Kubitschek-Barreira, Paula Helena; Fontaine, Thierry; Souza, Gustavo Henrique Martins Ferreira; Cunha, Marcel Lyra; Goldman, Gustavo H; Beauvais, Anne; Latgé, Jean-Paul; Lopes-Bezerra, Leila M

    2016-12-01

    Invasive aspergillosis is the primary opportunistic invasive fungal infection described in neutropenic hematologic patients, caused by the angioinvasive pathogen Aspergillus fumigatus. The molecular mechanisms associated with A. fumigatus infection in the vascular endothelium are poorly understood. In this context, we used a high-throughput proteomic approach to unveil the proteins modulated in HUVECs after interaction with a wild type strain and the UGM1 mutant (Δugm1) of A. fumigatus. The proteomic analysis was also performed in HUVECs challenged with a galactosaminogalactan (GAG) purified from A. fumigatus cell wall. The dataset presented here correspond to all proteins identified that fit a 2-fold change criteria (log 2 ratio ≥ 1 or ≤ -1), disregarding the statistical validation cut off, in order to supplement the research article entitled "Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates the HUVEC proteins associated with inflammatory and stress responses" (G.W.P. Neves, N.A. Curty, P.H. Kubitschek-Barreira, T. Fontaine, G.H.M.F. Souza, M. Lyra Cunha, G.H. Goldman, A. Beauvais, J.P. Latgé, L.M. Lopes-Bezerra, 2016) [1]. The mass spectrometry proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002823. PMID:27622208

  4. Dataset of differentially regulated proteins in HUVECs challenged with wild type and UGM1 mutant Aspergillus fumigatus strains.

    PubMed

    Neves, Gabriela Westerlund Peixoto; Curty, Nathália; Kubitschek-Barreira, Paula Helena; Fontaine, Thierry; Souza, Gustavo Henrique Martins Ferreira; Cunha, Marcel Lyra; Goldman, Gustavo H; Beauvais, Anne; Latgé, Jean-Paul; Lopes-Bezerra, Leila M

    2016-12-01

    Invasive aspergillosis is the primary opportunistic invasive fungal infection described in neutropenic hematologic patients, caused by the angioinvasive pathogen Aspergillus fumigatus. The molecular mechanisms associated with A. fumigatus infection in the vascular endothelium are poorly understood. In this context, we used a high-throughput proteomic approach to unveil the proteins modulated in HUVECs after interaction with a wild type strain and the UGM1 mutant (Δugm1) of A. fumigatus. The proteomic analysis was also performed in HUVECs challenged with a galactosaminogalactan (GAG) purified from A. fumigatus cell wall. The dataset presented here correspond to all proteins identified that fit a 2-fold change criteria (log 2 ratio ≥ 1 or ≤ -1), disregarding the statistical validation cut off, in order to supplement the research article entitled "Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates the HUVEC proteins associated with inflammatory and stress responses" (G.W.P. Neves, N.A. Curty, P.H. Kubitschek-Barreira, T. Fontaine, G.H.M.F. Souza, M. Lyra Cunha, G.H. Goldman, A. Beauvais, J.P. Latgé, L.M. Lopes-Bezerra, 2016) [1]. The mass spectrometry proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002823.

  5. Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein

    PubMed Central

    Perrot-Rechenmann, Catherine; Friml, Jiří

    2016-01-01

    The Auxin Binding Protein 1 (ABP1) is one of the most studied proteins in plants. Since decades ago, it has been the prime receptor candidate for the plant hormone auxin with a plethora of described functions in auxin signaling and development. The developmental importance of ABP1 has recently been questioned by identification of Arabidopsis thaliana abp1 knock-out alleles that show no obvious phenotypes under normal growth conditions. In this study, we examined the contradiction between the normal growth and development of the abp1 knock-outs and the strong morphological defects observed in three different ethanol-inducible abp1 knock-down mutants ( abp1-AS, SS12K, SS12S). By analyzing segregating populations of abp1 knock-out vs. abp1 knock-down crosses we show that the strong morphological defects that were believed to be the result of conditional down-regulation of ABP1 can be reproduced also in the absence of the functional ABP1 protein. This data suggests that the phenotypes in  abp1 knock-down lines are due to the off-target effects and asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles. PMID:26925228

  6. Crystallization and preliminary X-ray diffraction analysis of D53H mutant Escherichia coli cAMP receptor protein

    PubMed Central

    Huang, Jing; Wu, Tong; Guo, Zheng; Lou, Tiantian; Yu, Shaoning; Gong, Weimin; Ji, Chaoneng

    2013-01-01

    The Escherichia coli cyclic AMP receptor protein (CRP) is a prokaryotic global transcription activator protein that controls the expression of many different genes. Wild-type CRP can bind to special DNA sequences in the presence of cAMP. The substitution of Asp53 by His results in the CRP* phenotype, which does not require exogenous cAMP. In the present study, the D53H CRP mutant was overexpressed, purified and crystallized. cAMP-free D53H CRP crystals were obtained and diffracted to a resolution of 2.9 Å. Based on the systematic absences of the crystals, the space group is likely to be P212121, with unit-cell parameters a = 76.66, b = 152.14, c = 176.11 Å. The asymmetric unit was confirmed to contain four protein dimers, with a Matthews coefficient of 2.71 Å3 Da−1 and a solvent content of 54.68%. PMID:24316848

  7. Analysis of the chloroplast proteome in arc mutants and identification of novel protein components associated with FtsZ2.

    PubMed

    Gargano, Daniela; Maple-Grødem, Jodi; Reisinger, Veronika; Eichacker, Lutz Andreas; Møller, Simon Geir

    2013-02-01

    Chloroplasts are descendants of cyanobacteria and divide by binary fission. The number of chloroplasts is regulated in a cell type-specific manner to ensure that specialized cell types can perform their functions optimally. Several protein components of the chloroplast division apparatus have been identified in the past several years, but how this process is regulated in response to developmental status, environmental signals and stress is still unknown. To begin to address this we undertook a proteomic analysis of three accumulation and replication of chloroplasts mutants that show a spectrum of plastid division perturbations. We show that defects in the chloroplast division process results in changes in the abundance of proteins when compared to wild type, but that the profile of the native stromal and membrane complexes remains unchanged. Furthermore, by combining BN-PAGE with protein interaction assays we show that AtFtsZ2-1 and AtFtsZ2-2 assemble together with rpl12A and EF-Tu into a novel chloroplast membrane complex. PMID:23225155

  8. trans-dominant interference with virus infection at two different stages by a mutant envelope protein of Friend murine leukemia virus.

    PubMed Central

    Matano, T; Odawara, T; Ohshima, M; Yoshikura, H; Iwamoto, A

    1993-01-01

    A dominant negative mutant Friend murine leukemia virus (FMLV) env gene was cloned from an immunoselected Friend erythroleukemia cell. The mutant env had a point mutation which resulted in a Cys-to-Arg substitution at the 361st amino acid in the FMLV envelope protein (Env). The mutant Env was retained in the endoplasmic reticulum (ER) and accumulated because of its slow degradation. The NIH 3T3 cells expressing the mutant env were resistant to ecotropic Moloney MLV (MoMLV) penetration, suggesting that the mutant Env traps the ecotropic MLV receptors in the ER. When the mutant env gene was transfected into and expressed in the cells persistently infected with MoMLV, the wild-type Env was trapped in the ER, and the MoMLV production was suppressed. Thus, the mutant Env accumulating in the ER trans-dominantly and efficiently interfered with the ecotropic MLV infection at both the early and the late stages. Images PMID:8445721

  9. Most mutant OccR proteins that are defective in positive control hold operator DNA in a locked high-angle bend.

    PubMed

    Tsai, Ching-Sung; Chen, Chia-Sui; Winans, Stephen C

    2011-10-01

    OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of the occ genes occurs in response to octopine, a nutrient released from crown gall tumors. OccR also functions as an autorepressor in the presence or absence of octopine. OccR binds to a site between occQ and occR in the presence or absence of octopine, although octopine triggers a conformational change that shortens the DNA footprint and relaxes a DNA bend. In order to determine the roles of this conformational change in transcriptional activation, we isolated 11 OccR mutants that were defective in activation of the occQ promoter but were still capable of autorepression. The mutations in these mutants spanned most of the length of the protein. Two additional positive-control mutants were isolated using site-directed mutagenesis. Twelve mutant proteins displayed a high-angle DNA bend in the presence or absence of octopine. One mutant, the L26A mutant, showed ligand-responsive DNA binding similar to that of wild-type OccR and therefore must be impaired in a subsequent step in activation. PMID:21804007

  10. Mutant MCP-1 Protein Delivery from Layer-by-Layer Coatings on Orthopaedic Implants to Modulate Inflammatory Response

    PubMed Central

    Keeney, Michael; Waters, Heather; Barcay, Katie; Jiang, Xinyi; Yao, Zhenyu; Pajarinen, Jukka; Egashira, Kensuke; Goodman, Stuart; Yang, Fan

    2013-01-01

    Total joint replacement (TJR) is a common and effective surgical procedure for hip or knee joint reconstruction. However, the production of wear particles is inevitable for all TJRs, which activates macrophages and initiates an inflammatory cascade often resulting in bone loss, prosthetic loosening and eventual TJR failure. Macrophage Chemoattractant Protein-1 (MCP-1) is one of the most potent cytokines responsible for macrophage cell recruitment, and previous studies suggest that mutant MCP-1 proteins such as 7ND may be used as a decoy drug to block the receptor and reduce inflammatory cell recruitment. Here we report the development of a biodegradable, layer-by-layer (LBL) coating platform that allows efficient loading and controlled release of 7ND proteins from the surface of orthopaedic implants using as few as 14 layers. Scanning electron microscopy and fluorescence imaging confirmed effective coating using the LBL procedure on titanium rods. 7ND protein loading concentration and release kinetics can be modulated by varying the polyelectrolytes of choice, the polymer chemistry, the pH of the polyelectrolyte solution, and the degradation rate of the LBL assembly. The released 7ND from LBL coating retained its bioactivity and effectively reduced macrophage migration towards MCP-1. Finally, the LBL coating remained intact following a femoral rod implantation procedure as determined by immunostaining of the 7ND coating. The LBL platform reported herein may be applied for in situ controlled release of 7ND protein from orthopaedic implants, to reduce wear particle-induced inflammatory responses in an effort to prolong the lifetime of implants. PMID:24075408

  11. Deciphering the Dynamics of Non-Covalent Interactions Affecting Thermal Stability of a Protein: Molecular Dynamics Study on Point Mutant of Thermus thermophilus Isopropylmalate Dehydrogenase

    PubMed Central

    Sharma, Reetu; Sastry, G. Narahari

    2015-01-01

    Thermus thermophilius isopropylmalate dehydrogenase catalyzes oxidative decarboxylation and dehydrogenation of isopropylmalate. Substitution of leucine to alanine at position 172 enhances the thermal stability among the known point mutants. Exploring the dynamic properties of non-covalent interactions such as saltbridges, hydrogen bonds and hydrophobic interactions to explain thermal stability of a protein is interesting in its own right. In this study dynamic changes in the non-covalent interactions are studied to decipher the deterministic features of thermal stability of a protein considering a case study of a point mutant in Thermus thermophilus isopropylmalate dehydrogenase. A total of four molecular dynamic simulations of 0.2 μs were carried out on wild type and mutant’s functional dimers at 300 K and 337 K. Higher thermal stability of the mutant as compared to wild type is revealed by root mean square deviation, root mean square fluctuations and Cα-Cα distance with an increase in temperature from 300 K to 337 K. Most of the regions of wild type fluctuate higher than the corresponding regions of mutant with an increase in temperature. Cα-Cα distance analysis suggests that long distance networks are significantly affected in wild type as compared to the mutant. Short lived contacts are higher in wild type, while long lived contacts are lost at 337 K. The mutant forms less hydrogen bonds with water as compared to wild type at 337 K. In contrast to wild type, the mutant shows significant increase in unique saltbridges, hydrogen bonds and hydrophobic contacts at 337 K. The current study indicates that there is a strong inter-dependence of thermal stability on the way in which non-covalent interactions reorganize, and it is rewarding to explore this connection in single mutant studies. PMID:26657745

  12. Active photosynthesis in cyanobacterial mutants with directed modifications in the ligands for two iron-sulfur clusters on the PsaC protein of photosystem I.

    PubMed Central

    Mannan, R M; He, W Z; Metzger, S U; Whitmarsh, J; Malkin, R; Pakrasi, H B

    1996-01-01

    The PsaC protein of the Photosystem I (PSI) complex in thylakoid membranes coordinates two [4Fe-4S] clusters, FA and FB. Although it is known that PsaC participates in electron transfer to ferredoxin, the pathway of electrons through this protein is unknown. To elucidate the roles of FA and FB, we created two site-directed mutant strains of the cyanobacterium Anabaena variabilis ATCC 29413. In one mutant, cysteine 13, a ligand for FB was replaced by an aspartic acid (C13D); in the other mutant, cysteine 50, a ligand for FA was modified similarly (C50D). Low-temperature electron paramagnetic resonance studies demonstrated that the C50D mutant has a normal FB center and a modified FA center. In contrast, the C13D strain has normal FA, but failed to reveal any signal from FB. Room-temperature optical studies showed that C13D has only one functional electron acceptor in PsaC, whereas two such acceptors are functional in the C50D and wild-type strains. Although both mutants grow under photoautotrophic conditions, the rate of PSI-mediated electron transfer in C13D under low light levels is about half that of C50D or wild type. These data show that (i) FB is not essential for the assembly of the PsaC protein in PSI and (ii) FB is not absolutely required for electron transfer from the PSI reaction center to ferredoxin. PMID:8617228

  13. Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation.

    PubMed

    Zhang, Zhi-Hui; Liu, Xiao-Qian; Zhang, Cheng; He, Wei; Wang, Hua; Chen, Yuan-Hua; Liu, Xiao-Jing; Chen, Xi; Xu, De-Xiang

    2016-01-01

    Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. PMID:27627966

  14. Tlr4-mutant mice are resistant to acute alcohol-induced sterol-regulatory element binding protein activation and hepatic lipid accumulation

    PubMed Central

    Zhang, Zhi-Hui; Liu, Xiao-Qian; Zhang, Cheng; He, Wei; Wang, Hua; Chen, Yuan-Hua; Liu, Xiao-Jing; Chen, Xi; Xu, De-Xiang

    2016-01-01

    Previous studies demonstrated that acute alcohol intoxication caused hepatic lipid accumulation. The present study showed that acute alcohol intoxication caused hepatic lipid accumulation in Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic sterol-regulatory element binding protein (SREBP)-1, a transcription factor regulating fatty acid and triglyceride (TG) synthesis, was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic Fas, Acc, Scd-1 and Dgat-2, the key genes for fatty acid and TG synthesis, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Additional experiment showed that hepatic MyD88 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic NF-κB was activated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Moreover, hepatic GSH content was reduced and hepatic MDA level was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic CYP2E1 was elevated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Hepatic p67phox and gp91phox, two NADPH oxidase subunits, were up-regulated in alcohol-treated Tlr4-wild-type mice but not in Tlr4-mutant mice. Alpha-phenyl-N-t-butylnitrone (PBN), a free radical spin-trapping agent, protected against alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. In conclusion, Tlr4-mutant mice are resistant to acute alcohol-induced hepatic SREBP-1 activation and hepatic lipid accumulation. PMID:27627966

  15. Curcumin treatment abrogates endoplasmic reticulum retention and aggregation-induced apoptosis associated with neuropathy-causing myelin protein zero-truncating mutants.

    PubMed

    Khajavi, Mehrdad; Inoue, Ken; Wiszniewski, Wojciech; Ohyama, Tomoko; Snipes, G Jackson; Lupski, James R

    2005-11-01

    Mutations in MPZ, the gene encoding myelin protein zero (MPZ), the major protein constituent of peripheral myelin, can cause the adult-onset, inherited neuropathy Charcot-Marie-Tooth disease, as well as the more severe, childhood-onset Dejerine-Sottas neuropathy and congenital hypomyelinating neuropathy. Most MPZ-truncating mutations associated with severe forms of peripheral neuropathy result in premature termination codons within the terminal or penultimate exons that are not subject to nonsense-mediated decay and are stably translated into mutant proteins with potential dominant-negative activity. However, some truncating mutations at the 3' end of MPZ escape the nonsense-mediated decay pathway and cause a mild peripheral neuropathy phenotype. We examined the functional properties of MPZ-truncating proteins that escaped nonsense-mediated decay, and we found that frameshift mutations associated with severe disease cause an intracellular accumulation of mutant proteins, primarily within the endoplasmic reticulum (ER), which induces apoptosis. Curcumin, a chemical compound derived from the curry spice tumeric, releases the ER-retained MPZ mutants into the cytoplasm accompanied by a lower number of apoptotic cells. Our findings suggest that curcumin treatment is sufficient to relieve the toxic effect of mutant aggregation-induced apoptosis and may potentially have a therapeutic role in treating selected forms of inherited peripheral neuropathies. PMID:16252242

  16. The role of cavities in protein dynamics: crystal structure of a photolytic intermediate of a mutant myoglobin.

    PubMed

    Brunori, M; Vallone, B; Cutruzzola, F; Travaglini-Allocatelli, C; Berendzen, J; Chu, K; Sweet, R M; Schlichting, I

    2000-02-29

    We determined the structure of the photolytic intermediate of a sperm whale myoglobin (Mb) mutant called Mb-YQR [Leu-(B10)-->Tyr; His(E7)-->Gln; Thr(E10)-->Arg] to 1.4-A resolution by ultra-low temperature (20 K) x-ray diffraction. Starting with the CO complex, illumination leads to photolysis of the Fe-CO bond, and migration of the photolyzed carbon monoxide (CO*) to a niche in the protein 8.1 A from the heme iron; this cavity corresponds to that hosting an atom of Xe when the crystal is equilibrated with xenon gas at 7 atmospheres [Tilton, R. F., Jr., Kuntz, I. D. & Petsko, G. A. (1984) Biochemistry 23, 2849-2857]. The site occupied by CO* corresponds to that predicted by molecular dynamics simulations previously carried out to account for the NO geminate rebinding of Mb-YQR observed in laser photolysis experiments at room temperature. This secondary docking site differs from the primary docking site identified by previous crystallographic studies on the photolyzed intermediate of wild-type sperm whale Mb performed at cryogenic temperatures [Teng et al. (1994) Nat. Struct. Biol. 1, 701-705] and room temperature [Srajer et al. (1996) Science 274, 1726-1729]. Our experiment shows that the pathway of a small molecule in its trajectory through a protein may be modified by site-directed mutagenesis, and that migration within the protein matrix to the active site involves a limited number of pre-existing cavities identified in the interior space of the protein. PMID:10681426

  17. Overexpression of mutant amyloid-β protein precursor and presenilin 1 modulates enteric nervous system.

    PubMed

    Puig, Kendra L; Lutz, Brianna M; Urquhart, Siri A; Rebel, Andrew A; Zhou, Xudong; Manocha, Gunjan D; Sens, MaryAnn; Tuteja, Ashok K; Foster, Norman L; Combs, Colin K

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder histologically characterized by amyloid-β (Aβ) protein accumulation and activation of associated microglia. Although these features are well described in the central nervous system, the process and consequences of Aβ accumulation in the enteric nervous system have not been extensively studied. We hypothesized that Aβ also may accumulate in the enteric nervous system and lead to immune cell activation and neuronal dysfunction in the digestive tract not unlike that observed in diseased brain. To test this hypothesis, ileums of the small intestine of thirteen month old AβPP/PS1 and C57BL/6 (wild type) mice were collected and analyzed using immunohistochemistry, western blot analysis, cytokine arrays, and ELISA. AβPP/PS1 mice demonstrated no differences in intestinal motility or water absorption but elevated luminal IgA levels compared to wild type mice. They also had increased protein levels of AβPP and the proteolytic enzyme, BACE, corresponding to an increase in Aβ1-40 in the intestinal lysate as well as an increase in both Aβ1-40 and Aβ1-42 in the stool. This correlated with increased protein markers of proinflammatory and immune cell activation. Histologic analysis localized AβPP within enteric neurons but also intestinal epithelial cells with elevated Aβ immunoreactivity in the AβPP/PS1 mice. The presence of AβPP, Aβ, and CD68 immunoreactivity in the intestines of some patients with neuropathologically-confirmed AD are consistent with the findings in this mouse model. These data support the hypothesis that in AD the intestine, much like the brain, may develop proinflammatory and immune changes related to AβPP and Aβ.

  18. Mutant monocyte chemoattractant protein 1 protein attenuates migration of and inflammatory cytokine release by macrophages exposed to orthopedic implant wear particles

    PubMed Central

    Yao, Zhenyu; Keeney, Michael; Lin, Tzu-Hua; Pajarinen, Jukka; Barcay, Katherine; Waters, Heather; Egashira, Kensuke; Yang, Fan; Goodman, Stuart

    2014-01-01

    Wear particles generated from total joint replacements can stimulate macrophages to release chemokines, such as monocyte chemoattractant protein 1 (MCP-1), which is the most important chemokine regulating systemic and local cell trafficking and infiltration of monocyte/macrophages in chronic inflammation. One possible strategy to curtail the adverse events associated with wear particles is to mitigate migration and activation of monocyte/macrophages. The purpose of this study is to modulate the adverse effects of particulate biomaterials and inflammatory stimuli such as endotoxin by interfering with the biological effects of the chemokine MCP-1. In the current study, the function of MCP-1 was inhibited by the mutant MCP-1 protein called 7ND, which blocks its receptor, the C–C chemokine receptor type 2 (CCR2) on macrophages. Addition of 7ND decreased MCP-1-induced migration of THP-1 cells in cell migration experiments in a dose-dependent manner. Conditioned media from murine macrophages exposed to clinically relevant polymethylmethacrylate (PMMA) particles with/without endotoxin [lipopolysaccharide (LPS)] had a chemotactic effect on human macrophages, which was decreased dramatically by 7ND. 7ND demonstrated no adverse effects on the viability of macrophages, and the capability of mesenchymal stem cells (MSCs) to form bone at the doses tested. Finally, proinflammatory cytokine production was mitigated when macrophages were exposed to PMMA particles with/without LPS in the presence of 7ND. Our studies confirm that the MCP-1 mutant protein 7ND can decrease macrophage migration and inflammatory cytokine release without adverse effects at the doses tested. Local delivery of 7ND at the implant site may provide a therapeutic strategy to diminish particle-associated periprosthetic inflammation and osteolysis. PMID:24123855

  19. Abscisic acid-responsive guard cell metabolomes of Arabidopsis wild-type and gpa1 G-protein mutants.

    PubMed

    Jin, Xiaofen; Wang, Rui-Sheng; Zhu, Mengmeng; Jeon, Byeong Wook; Albert, Reka; Chen, Sixue; Assmann, Sarah M

    2013-12-01

    Individual metabolites have been implicated in abscisic acid (ABA) signaling in guard cells, but a metabolite profile of this specialized cell type is lacking. We used liquid chromatography-multiple reaction monitoring mass spectrometry for targeted analysis of 85 signaling-related metabolites in Arabidopsis thaliana guard cell protoplasts over a time course of ABA treatment. The analysis utilized ∼ 350 million guard cell protoplasts from ∼ 30,000 plants of the Arabidopsis Columbia accession (Col) wild type and the heterotrimeric G-protein α subunit mutant, gpa1, which has ABA-hyposensitive stomata. These metabolomes revealed coordinated regulation of signaling metabolites in unrelated biochemical pathways. Metabolites clustered into different temporal modules in Col versus gpa1, with fewer metabolites showing ABA-altered profiles in gpa1. Ca(2+)-mobilizing agents sphingosine-1-phosphate and cyclic adenosine diphosphate ribose exhibited weaker ABA-stimulated increases in gpa1. Hormone metabolites were responsive to ABA, with generally greater responsiveness in Col than in gpa1. Most hormones also showed different ABA responses in guard cell versus mesophyll cell metabolomes. These findings suggest that ABA functions upstream to regulate other hormones, and are also consistent with G proteins modulating multiple hormonal signaling pathways. In particular, indole-3-acetic acid levels declined after ABA treatment in Col but not gpa1 guard cells. Consistent with this observation, the auxin antagonist α-(phenyl ethyl-2-one)-indole-3-acetic acid enhanced ABA-regulated stomatal movement and restored partial ABA sensitivity to gpa1.

  20. Cryoprotective effect of an insect antifreeze protein MpAFP 698 and its mutants from the desert beetle Microdera punctipennis.

    PubMed

    Jiang, M; Ma, J; Qiu, L M

    2011-01-01

    An insect antifreeze protein, MpAFP698, from the desert beetle Microdera punctipennis, shares 77% similarity with TmTHP (YL-3) from Tenebrio molitor. The predicted tertiary structure for MpAFP698 was modeled as a regular A-helix with TCT motifs arrayed to form the putative ice-binding surface. This model was validated via site-directed mutagenesis. The results suggest that the desert beetle in central Asia has evolved antifreeze proteins similar to those from North America continent. In an additional study, the recombinant MpAFP698 and its mutants were expressed in Escherichia coli strain BL21 (DE3). The thermal hysteresis activity of MpAFP698 was 1.73 degree C at 1.0 mg/ml. MpAFP698 was also tested by in vitro antifreeze activity assay to evaluate its cryoprotective effect at -20 degree C. MpAFP698 displays high cryoprotective effect on bacteria cells at freezing temperatures. PMID:22020466

  1. Structural insights into Noonan/LEOPARD syndrome-related mutants of protein-tyrosine phosphatase SHP2 (PTPN11)

    PubMed Central

    2014-01-01

    Background The ubiquitous non-receptor protein tyrosine phosphatase SHP2 (encoded by PTPN11) plays a key role in RAS/ERK signaling downstream of most, if not all growth factors, cytokines and integrins, although its major substrates remain controversial. Mutations in PTPN11 lead to several distinct human diseases. Germ-line PTPN11 mutations cause about 50% of Noonan Syndrome (NS), which is among the most common autosomal dominant disorders. LEOPARD Syndrome (LS) is an acronym for its major syndromic manifestations: multiple Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of genitalia, Retardation of growth, and sensorineural Deafness. Frequently, LS patients have hypertrophic cardiomyopathy, and they might also have an increased risk of neuroblastoma (NS) and acute myeloid leukemia (AML). Consistent with the distinct pathogenesis of NS and LS, different types of PTPN11 mutations cause these disorders. Results Although multiple studies have reported the biochemical and biological consequences of NS- and LS-associated PTPN11 mutations, their structural consequences have not been analyzed fully. Here we report the crystal structures of WT SHP2 and five NS/LS-associated SHP2 mutants. These findings enable direct structural comparisons of the local conformational changes caused by each mutation. Conclusions Our structural analysis agrees with, and provides additional mechanistic insight into, the previously reported catalytic properties of these mutants. The results of our research provide new information regarding the structure-function relationship of this medically important target, and should serve as a solid foundation for structure-based drug discovery programs. PMID:24628801

  2. Catalytically impaired hMYH and NEIL1 mutant proteins identified in patients with primary sclerosing cholangitis and cholangiocarcinoma

    PubMed Central

    Forsbring, Monika; Vik, Erik S.; Dalhus, Bjørn; Karlsen, Tom H.; Bergquist, Annika; Schrumpf, Erik; Bjørås, Magnar; Boberg, Kirsten M.; Alseth, Ingrun

    2009-01-01

    The human hMYH and NEIL1 genes encode DNA glycosylases involved in repair of oxidative base damage and mutations in these genes are associated with certain cancers. Primary sclerosing cholangitis (PSC), a chronic cholestatic liver disease characterized by inflammatory destruction of the biliary tree, is often complicated by the development of cholangiocarcinoma (CCA). Here, we aimed to investigate the influence of genetic variations in the hMYH and NEIL1 genes on risk of CCA in PSC patients. The hMYH and NEIL1 gene loci in addition to the DNA repair genes hOGG1, NTHL1 and NUDT1 were analyzed in 66 PSC patients (37 with CCA and 29 without cancer) by complete genomic sequencing of exons and adjacent intronic regions. Several single-nucleotide polymorphisms and mutations were identified and severe impairment of protein function was observed for three non-synonymous variants. The NEIL1 G83D mutant was dysfunctional for the major oxidation products 7,8-dihydro-8-oxoguanine (8oxoG), thymine glycol and dihydrothymine in duplex DNA, and the ability to perform δ-elimination at abasic sites was significantly reduced. The hMYH R260Q mutant had severe defect in adenine DNA glycosylase activity, whereas hMYH H434D could excise adenines from A:8oxoG pairs but not from A:G mispairs. We found no overall associations between the 18 identified variants and susceptibility to CCA in PSC patients; however, the impaired variants may be of significance for carcinogenesis in general. Our findings demonstrate the importance of complete resequencing of selected candidate genes in order to identify rare genetic variants and their possible contribution to individual susceptibility to cancer development. PMID:19443904

  3. Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

    PubMed Central

    Jin, Hongping; Li, Dongsheng; Sivakumaran, Haran; Lor, Mary; Rustanti, Lina; Cloonan, Nicole; Wani, Shivangi

    2016-01-01

    ABSTRACT Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA) and JQ1 had no effect, while suberanilohydroxamic acid (SAHA) modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA), but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII) and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells. PMID:27381288

  4. Familial amyloid precursor protein mutants cause caspase-6-dependent but amyloid β-peptide-independent neuronal degeneration in primary human neuron cultures.

    PubMed Central

    Sivananthan, S N; Lee, A W; Goodyer, C G; LeBlanc, A C

    2010-01-01

    Although familial Alzheimer disease (AD)-associated autosomal dominant mutants have been extensively studied, little is known about the underlying molecular mechanisms of neurodegeneration induced by these mutants in AD. Wild-type, Swedish or London amyloid precursor protein (APP) transfection in primary human neurons induced neuritic beading, in which several co-expressed proteins, such as enhanced green fluorescent protein, red fluorescent protein (RFP)-tau and RFP-ubiquitin, accumulated. APP-induced neuritic beading was dependent on caspase-6 (Casp6), because it was inhibited with 5 μM z-VEID-fmk or with dominant-negative Casp6. Neuritic beading was independent from APP-mediated amyloid β-peptide (Aβ) production, because the APPM596V (APPMV) mutant, which cannot generate Aβ, still induced Casp6-dependent neuritic beading. However, the beaded neurons underwent Casp6- and Aβ-dependent cell death. These results indicate that overexpression of wild-type or mutant APP causes Casp6-dependent but Aβ-independent neuritic degeneration in human neurons. Because Casp6 is activated early in AD and is involved in axonal degeneration, these results suggest that the inhibition of Casp6 may represent an efficient early intervention against familial forms of AD. Furthermore, these results indicate that removing Aβ without inhibiting Casp6 may have little effect in preventing the progressive dementia associated with sporadic or familial AD. PMID:21368865

  5. The cold sensitivity of a mutant of Saccharomyces cerevisiae lacking a mitochondrial heat shock protein 70 is suppressed by loss of mitochondrial DNA

    PubMed Central

    1996-01-01

    SSH1, a newly identified member of the heat shock protein (hsp70) multigene family of the budding yeast Saccharomyces cerevisiae, encodes a protein localized to the mitochondrial matrix. Deletion of the SSH1 gene results in extremely slow growth at 23 degrees C or 30 degrees C, but nearly wild-type growth at 37 degrees C. The matrix of the mitochondria contains another hsp70, Ssc1, which is essential for growth and required for translocation of proteins into mitochondria. Unlike SSC1 mutants, an SSH1 mutant showed no detectable defects in import of several proteins from the cytosol to the matrix compared to wild type. Increased expression of Ssc1 partially suppressed the cold- sensitive growth defect of the SSH1 mutant, suggesting that when present in increased amounts, Ssc1 can at least partially carry out the normal functions of Ssh1. Spontaneous suppressors of the cold-sensitive phenotype of an SSH1 null mutant were obtained at a high frequency at 23 degrees C, and were all found to be respiration deficient. 15 of 16 suppressors that were analyzed lacked mitochondrial DNA, while the 16th had reduced amounts. We suggest that Ssh1 is required for normal mitochondrial DNA replication, and that disruption of this process in ssh1 cells results in a defect in mitochondrial function at low temperatures. PMID:8707841

  6. Alterations in Gene Expression in Mutant Amyloid Precursor Protein Transgenic Mice Lacking Niemann-Pick Type C1 Protein

    PubMed Central

    Maulik, Mahua; Thinakaran, Gopal; Kar, Satyabrata

    2013-01-01

    Niemann-Pick type C (NPC) disease, a rare autosomal recessive disorder caused mostly by mutation in NPC1 gene, is pathologically characterized by the accumulation of free cholesterol in brain and other tissues. This is accompanied by gliosis and loss of neurons in selected brain regions, including the cerebellum. Recent studies have shown that NPC disease exhibits intriguing parallels with Alzheimer’s disease, including the presence of neurofibrillary tangles and increased levels of amyloid precursor protein (APP)-derived β-amyloid (Aβ) peptides in vulnerable brain neurons. To evaluate the role of Aβ in NPC disease, we determined the gene expression profile in selected brain regions of our recently developed bigenic ANPC mice, generated by crossing APP transgenic (Tg) mice with heterozygous Npc1-deficient mice. The ANPC mice exhibited exacerbated neuronal and glial pathology compared to other genotypes [i.e., APP-Tg, double heterozygous (Dhet), Npc1-null and wild-type mice]. Analysis of expression profiles of 86 selected genes using real-time RT-PCR arrays showed a wide-spectrum of alterations in the four genotypes compared to wild-type controls. The changes observed in APP-Tg and Dhet mice are limited to only few genes involved mostly in the regulation of cholesterol metabolism, whereas Npc1-null and ANPC mice showed alterations in the expression profiles of a number of genes regulating cholesterol homeostasis, APP metabolism, vesicular trafficking and cell death mechanism in both hippocampus and cerebellum compared to wild-type mice. Intriguingly, ANPC and Npc1-null mice, with some exceptions, exhibited similar changes, although more genes were differentially expressed in the affected cerebellum than the relatively spared hippocampus. The altered gene profiles were found to match with the corresponding protein levels. These results suggest that lack of Npc1 protein can alter the expression profile of selected transcripts as well as proteins, and APP

  7. Evolution of the adhE gene product of Escherichia coli from a functional reductase to a dehydrogenase. Genetic and biochemical studies of the mutant proteins.

    PubMed

    Membrillo-Hernandez, J; Echave, P; Cabiscol, E; Tamarit, J; Ros, J; Lin, E C

    2000-10-27

    The multifunctional AdhE protein of Escherichia coli (encoded by the adhE gene) physiologically catalyzes the sequential reduction of acetyl-CoA to acetaldehyde and then to ethanol under fermentative conditions. The NH(2)-terminal region of the AdhE protein is highly homologous to aldehyde:NAD(+) oxidoreductases, whereas the COOH-terminal region is homologous to a family of Fe(2+)-dependent ethanol:NAD(+) oxidoreductases. This fusion protein also functions as a pyruvate formate lyase deactivase. E. coli cannot grow aerobically on ethanol as the sole carbon and energy source because of inadequate rate of adhE transcription and the vulnerability of the AdhE protein to metal-catalyzed oxidation. In this study, we characterized 16 independent two-step mutants with acquired and improved aerobic growth ability on ethanol. The AdhE proteins in these mutants catalyzed the sequential oxidation of ethanol to acetaldehyde and to acetyl-CoA. All first stage mutants grew on ethanol with a doubling time of about 240 min. Sequence analysis of a randomly chosen mutant revealed an Ala-267 --> Thr substitution in the acetaldehyde:NAD(+) oxidoreductase domain of AdhE. All second stage mutants grew on ethanol with a doubling time of about 90 min, and all of them produced an AdhE(A267T/E568K). Purified AdhE(A267T) and AdhE(A267T/E568K) showed highly elevated acetaldehyde dehydrogenase activities. It therefore appears that when AdhE catalyzes the two sequential reactions in the counter-physiological direction, acetaldehyde dehydrogenation is the rate-limiting step. Both mutant proteins were more thermosensitive than the wild-type protein, but AdhE(A267T/E568K) was more thermal stable than AdhE(A267T). Since both mutant enzymes exhibited similar kinetic properties, the second mutation probably conferred an increased growth rate on ethanol by stabilizing AdhE(A267T). PMID:10922373

  8. Differentiation of the shutoff of protein synthesis by virion host shutoff and mutant gamma (1)34.5 genes of herpes simplex virus 1.

    PubMed

    Poon, A P; Roizman, B

    1997-03-01

    vhs protein is the product of the UL41 open reading frame of herpes simplex virus 1. The protein, made late in infection, is packaged into virions and, in newly infected cells, shuts off host protein synthesis by degrading mRNA. gamma (1)34.5 gene encodes a protein which precludes total shutoff of protein synthesis after the onset of viral DNA synthesis in infected cells of human derivation. The experiments reported here were designed to test the hypothesis that in cells infected with gamma (1)34.5- mutant the total shutoff of protein synthesis reflects the failure to alter the function of vhs made late in infection. Hence, double mutants, vhs- and gamma (1)34.5 should not cause total shutoff of protein synthesis. The mutants constructed to test the hypothesis were (i) viruses lacking 1 kbp from the coding domain of gamma (1)34.5 and carrying lacZ inserted into the coding domain of UI41, (ii) viruses with deletions in gamma (1)34.5 genes, (iii) viruses with lacZ inserted into UL41, and (iv) viruses in which the sequences of the deleted or interrupted genes were restored. We report that viruses with wild-type UL41 gene shut off the synthesis of actin, whereas viruses with interrupted genes made amounts of actin comparable to those of mock-infected cells. However, late in infection, protein synthesis in human neuroblastoma cells infected with the gamma (1)34.5- mutants was shut off irrespective of the status of the UL41 gene. Conversely, the phenotype of UI41 viruses with wild-type gamma (1)34.5 gene could not be differentiated from those of wild-type virus in the same assays. These studies indicate that the functions of the UL41 and gamma (1)34.5 genes and their products are independent of each other.

  9. Altered synaptic plasticity in the mossy fibre pathway of transgenic mice expressing mutant amyloid precursor protein

    PubMed Central

    2010-01-01

    Aβ peptides derived from the cleavage of amyloid precursor protein are widely believed to play an important role in the pathophysiology of Alzheimer's disease. A common way to study the impact of these molecules on CNS function is to compare the physiology of transgenic mice that overproduce Aβ with non-transgenic animals. In the hippocampus, this approach has been frequently applied to the investigation of synaptic transmission and plasticity in the perforant and Schaffer collateral commissural pathways, the first and third components of the classical hippocampal trisynaptic circuit, respectively. Similar studies however have not been carried out on the remaining component of the trisynaptic circuit, the mossy fibre pathway. Using transverse hippocampal slices prepared from ~2 year old animals we have compared mossy fibre synaptic function in wild-type mice and their Tg2576 littermates which age-dependently overproduce Aβ. Input-output curves were not altered in slices from Tg2576 mice, but these animals exhibited a significant loss of the prominent frequency-facilitation expressed by the mossy fibre pathway. In addition to this change in short term synaptic plasticity, high frequency stimulation-induced, NMDA-receptor-independent LTP was absent in slices from the transgenic mice. These data represent the first description of functional deficits in the mossy fibre pathway of Aβ-overproducing transgenic mice. PMID:21040543

  10. Rapamycin reverses cellular phenotypes and enhances mutant protein clearance in Hutchinson-Gilford progeria syndrome cells.

    PubMed

    Cao, Kan; Graziotto, John J; Blair, Cecilia D; Mazzulli, Joseph R; Erdos, Michael R; Krainc, Dimitri; Collins, Francis S

    2011-06-29

    Hutchinson-Gilford progeria syndrome (HGPS) is a lethal genetic disorder characterized by premature aging. HGPS is most commonly caused by a de novo single-nucleotide substitution in the lamin A/C gene (LMNA) that partially activates a cryptic splice donor site in exon 11, producing an abnormal lamin A protein termed progerin. Accumulation of progerin in dividing cells adversely affects the integrity of the nuclear scaffold and leads to nuclear blebbing in cultured cells. Progerin is also produced in normal cells, increasing in abundance as senescence approaches. Here, we report the effect of rapamycin, a macrolide antibiotic that has been implicated in slowing cellular and organismal aging, on the cellular phenotypes of HGPS fibroblasts. Treatment with rapamycin abolished nuclear blebbing, delayed the onset of cellular senescence, and enhanced the degradation of progerin in HGPS cells. Rapamycin also decreased the formation of insoluble progerin aggregates and induced clearance through autophagic mechanisms in normal fibroblasts. Our findings suggest an additional mechanism for the beneficial effects of rapamycin on longevity and encourage the hypothesis that rapamycin treatment could provide clinical benefit for children with HGPS.

  11. A MUTANT PRION PROTEIN SENSITIZES NEURONS TO GLUTAMATE-INDUCED EXCITOTOXICITY

    PubMed Central

    Biasini, Emiliano; Unterberger, Ursula; Solomon, Isaac H.; Massignan, Tania; Senatore, Assunta; Bian, Hejiao; Voigtlaender, Till; Bowman, Frederick P.; Bonetto, Valentina; Chiesa, Roberto; Luebke, Jennifer; Toselli, Paul; Harris, David A.

    2013-01-01

    Growing evidence suggests that a physiological activity of the cellular prion protein (PrPC) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer’s diseases. However, how the functional activity of PrPC is subverted to deliver neurotoxic signals remains uncertain. Transgenic mice expressing PrP with a deletion of residues 105–125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose-dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we have tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices, and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders due to toxic, β-rich oligomers that bind to PrPC. PMID:23392670

  12. The Chloroplast Function Database II: a comprehensive collection of homozygous mutants and their phenotypic/genotypic traits for nuclear-encoded chloroplast proteins.

    PubMed

    Myouga, Fumiyoshi; Akiyama, Kenji; Tomonaga, Yumi; Kato, Aya; Sato, Yuka; Kobayashi, Megumi; Nagata, Noriko; Sakurai, Tetsuya; Shinozaki, Kazuo

    2013-02-01

    The Chloroplast Function Database has so far offered phenotype information on mutants of the nuclear-encoded chloroplast proteins in Arabidopsis that pertains to >200 phenotypic data sets that were obtained from 1,722 transposon- or T-DNA-tagged lines. Here, we present the development of the second version of the database, which is named the Chloroplast Function Database II and was redesigned to increase the number of mutant characters and new user-friendly tools for data mining and integration. The upgraded database offers information on genome-wide mutant screens for any visible phenotype against 2,495 tagged lines to create a comprehensive homozygous mutant collection. The collection consists of 147 lines with seedling phenotypes and 185 lines for which we could not obtain homozygotes, as well as 1,740 homozygotes with wild-type phenotypes. Besides providing basic information about primer lists that were used for the PCR genotyping of T-DNA-tagged lines and explanations about the preparation of homozygous mutants and phenotype screening, the database includes access to a link between the gene locus and existing publicly available databases. This gives users access to a combined pool of data, enabling them to gain valuable insights into biological processes. In addition, high-resolution images of plastid morphologies of mutants with seedling-specific chloroplast defects as observed with transmission electron microscopy (TEM) are available in the current database. This database is used to compare the phenotypes of visually identifiable mutants with their plastid ultrastructures and to evaluate their potential significance from characteristic patterns of plastid morphology in vivo. Thus, the Chloroplast Function Database II is a useful and comprehensive information resource that can help researchers to connect individual Arabidopsis genes to plastid functions on the basis of phenotype analysis of our tagged mutant collection. It can be freely accessed at http://rarge.psc.riken.jp/chloroplast/.

  13. Reduced Sweetness of a Monellin (MNEI) Mutant Results from Increased Protein Flexibility and Disruption of a Distant Poly-(L-Proline) II Helix

    PubMed Central

    Templeton, Catherine M.; Ostovar pour, Saeideh; Hobbs, Jeanette R.; Blanch, Ewan W.; Munger, Steven D.

    2011-01-01

    Monellin is a highly potent sweet-tasting protein but relatively little is known about how it interacts with the sweet taste receptor. We determined X-ray crystal structures of 3 single-chain monellin (MNEI) proteins with alterations at 2 core residues (G16A, V37A, and G16A/V37A) that induce 2- to 10-fold reductions in sweetness relative to the wild-type protein. Surprisingly, no changes were observed in the global protein fold or the positions of surface amino acids important for MNEI sweetness that could explain these differences in protein activity. Differential scanning calorimetry showed that while the thermal stability of each mutant MNEI was reduced, the least sweet mutant, G16A-MNEI, was not the least stable protein. In contrast, solution spectroscopic measurements revealed that changes in protein flexibility and the C-terminal structure correlate directly with protein activity. G16A mutation-induced disorder in the protein core is propagated via changes to hydrophobic interactions that disrupt the formation and/or position of a critical C-terminal poly-(L-proline) II helix. These findings suggest that MNEI interaction with the sweet taste receptor is highly sensitive to the relative positions of key residues across its protein surface and that loss of sweetness in G16A-MNEI may result from an increased entropic cost of binding. PMID:21343241

  14. Prevalence of Staphylococcus aureus protein A (spa) mutants in the community and hospitals in Oxfordshire

    PubMed Central

    2014-01-01

    Background Staphylococcal protein A (spa) is an important virulence factor which enables Staphylococcus aureus to evade host immune responses. Genotypes known as “spa-types”, based on highly variable Xr region sequences of the spa-gene, are frequently used to classify strains. A weakness of current spa-typing primers is that rearrangements in the IgG-binding region of the gene cause 1-2% of strains to be designated as “non-typeable”. Results We developed an improved primer which enabled sequencing of all strains, containing any type of genetic rearrangement, in a large study among community carriers and hospital inpatients in Oxfordshire, UK (6110 isolates). We identified eight novel spa-gene variants, plus one previously described. Three of these rearrangements would be designated “non-typeable” using current spa-typing methods; they occurred in 1.8% (72/3905) asymptomatically carried and 0.6% (14/2205) inpatient S. aureus strains. Some individuals were simultaneously colonized by both formerly non-typeable and typeable strains; previously such patients would have been identified as carrying only currently typeable strains, underestimating mixed carriage prevalence and diversity. Formerly non-typeable strains were found in more spa-types associated with multilocus sequence type ST398 (35%), common among livestock, compared to other groups with any non-typeable strains (1-4%), suggesting particular spa-types may have been under-represented in previous human studies. Conclusions This improved method allows us to spa-type previously non-typeable strains with rearrangements in the spa-gene and to resolve cases of mixed colonization with deletions in one or more strains, thus accounting for hidden diversity of S. aureus in both community and hospital environments. PMID:24621342

  15. Abnormal behaviors and developmental disorder of hippocampus in zinc finger protein 521 (ZFP521) mutant mice.

    PubMed

    Ohkubo, Nobutaka; Matsubara, Etsuko; Yamanouchi, Jun; Akazawa, Rie; Aoto, Mamoru; Suzuki, Yoji; Sakai, Ikuya; Abe, Takaya; Kiyonari, Hiroshi; Matsuda, Seiji; Yasukawa, Masaki; Mitsuda, Noriaki

    2014-01-01

    Zinc finger protein 521 (ZFP521) regulates a number of cellular processes in a wide range of tissues, such as osteoblast formation and adipose commitment and differentiation. In the field of neurobiology, it is reported to be an essential factor for transition of epiblast stem cells into neural progenitors in vitro. However, the role of ZFP521 in the brain in vivo still remains elusive. To elucidate the role of ZFP521 in the mouse brain, we generated mice lacking exon 4 of the ZFP521 gene. The birth ratio of our ZFP521Δ/Δ mice was consistent with Mendel's laws. Although ZFP521Δ/Δ pups had no apparent defect in the body and were indistinguishable from ZFP521+/+ and ZFP521+/Δ littermates at the time of birth, ZFP521Δ/Δ mice displayed significant weight reduction as they grew, and most of them died before 10 weeks of age. They displayed abnormal behavior, such as hyper-locomotion, lower anxiety and impaired learning, which correspond to the symptoms of schizophrenia. The border of the granular cell layer of the dentate gyrus in the hippocampus of the mice was indistinct and granular neurons were reduced in number. Furthermore, Sox1-positive neural progenitor cells in the dentate gyrus and cerebellum were significantly reduced in number. Taken together, these findings indicate that ZFP521 directly or indirectly affects the formation of the neuronal cell layers of the dentate gyrus in the hippocampus, and thus ZFP521Δ/Δ mice displayed schizophrenia-relevant symptoms. ZFP521Δ/Δ mice may be a useful research tool as an animal model of schizophrenia.

  16. Resolution of fluorescence intensity decays of the two tryptophan residues in glutamine-binding protein from Escherichia coli using single tryptophan mutants.

    PubMed Central

    Axelsen, P H; Bajzer, Z; Prendergast, F G; Cottam, P F; Ho, C

    1991-01-01

    Time correlated single photon counting measurements of tryptophan (Trp) fluorescence intensity decay and other spectroscopic studies were performed on glutamine-binding protein (GlnBP) from Escherichia coli. Using site-specifically mutated forms of the protein in which tyrosine (Tyr) and phenylalanine (Phe) substitute for the Trp residues at positions 32 and 220, we have examined whether wild-type (Wtyp) intensity decay components may be assigned to specific Trp residues. Results indicate that: (a) two exponential intensity decay components are recovered from the Wtyp protein (6.16 ns, 0.46 ns); (b) the long decay component arises from Trp-220 and comprises greater than 90% of the total fluorescence emission; (c) the short component arises from Trp-32 and is highly quenched; (d) all four single-Trp mutants exhibit multiexponential intensity decays, yet equimolar mixtures of two single-Trp mutants yield only two decay components which are virtually indistinguishable from the Wtyp protein; (e) the recovery of additional components in protein mixtures is obscured by statistical noise inherent in the technique of photon counting; (f) various spectroscopic measurements suggest that Trp-Trp interactions occur in the Wtyp protein, but the Wtyp intensity decay may be closely approximated by a linear combination of intensity decays from single-Trp mutants; and (g) inferences derived independently from fluorescence and NMR spectroscopy which pertain to the presence of Trp-Trp interactions and the relative solvent exposure of the two Trp residues are in agreement. PMID:1932553

  17. Conservative Tryptophan Mutants of the Protein Tyrosine Phosphatase YopH Exhibit Impaired WPD-Loop Function and Crystallize with Divanadate Esters in Their Active Sites

    PubMed Central

    Moise, Gwendolyn; Gallup, Nathan M.; Alexandrova, Anastassia N.; Hengge, Alvan C.; Johnson, Sean J.

    2016-01-01

    Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution. PMID:26445170

  18. Conservative tryptophan mutants of the protein tyrosine phosphatase YopH exhibit impaired WPD-loop function and crystallize with divanadate esters in their active sites.

    PubMed

    Moise, Gwendolyn; Gallup, Nathan M; Alexandrova, Anastassia N; Hengge, Alvan C; Johnson, Sean J

    2015-10-27

    Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution. PMID:26445170

  19. Ectopic expression of a polyalanine expansion mutant of poly(A)-binding protein N1 in muscle cells in culture inhibits myogenesis.

    PubMed

    Wang, Qishan; Bag, Jnanankur

    2006-02-17

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset dominant genetic disease caused by the expansion of a GCG trinucleotide repeat that encodes the polyalanine tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1). Presence of intranuclear inclusions (INIs) containing PABPN1 aggregates in the skeletal muscles is the hallmark of OPMD. Here, we show that ectopic expression of the mutant PABPN1 produced INIs in a muscle cell culture model and reduced expression of several muscle-specific proteins including alpha-actin, slow troponin C, muscle creatine kinase, and two myogenic transcription factors, myogenin and MyoD. However, the levels of two upstream regulators of the MyoD gene, the Myf-5 and Pax3/7, were not affected, but both proteins co-localized with the PABPN1 aggregates in the mutant PABPN1 overexpressing cells. In these cells, although myogenin and MyoD levels were reduced, these two transcription factors did not co-localize with the mutant PABPN1 aggregates. Therefore, sequestration of Myf5 and Pax3/7 by the mutant PABPN1 aggregates was a specific effect on these factors. Our results suggest that trapping of these two important myogenic determinants may interfere with an early step in myogenesis.

  20. Ectopic expression of a polyalanine expansion mutant of poly(A)-binding protein N1 in muscle cells in culture inhibits myogenesis

    SciTech Connect

    Wang Qishan; Bag, Jnanankur . E-mail: jbag@uoguelph.ca

    2006-02-17

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset dominant genetic disease caused by the expansion of a GCG trinucleotide repeat that encodes the polyalanine tract at the N-terminus of the nuclear poly(A)-binding protein (PABPN1). Presence of intranuclear inclusions (INIs) containing PABPN1 aggregates in the skeletal muscles is the hallmark of OPMD. Here, we show that ectopic expression of the mutant PABPN1 produced INIs in a muscle cell culture model and reduced expression of several muscle-specific proteins including {alpha}-actin, slow troponin C, muscle creatine kinase, and two myogenic transcription factors, myogenin and MyoD. However, the levels of two upstream regulators of the MyoD gene, the Myf-5 and Pax3/7, were not affected, but both proteins co-localized with the PABPN1 aggregates in the mutant PABPN1 overexpressing cells. In these cells, although myogenin and MyoD levels were reduced, these two transcription factors did not co-localize with the mutant PABPN1 aggregates. Therefore, sequestration of Myf5 and Pax3/7 by the mutant PABPN1 aggregates was a specific effect on these factors. Our results suggest that trapping of these two important myogenic determinants may interfere with an early step in myogenesis.

  1. Local delivery of mutant CCL2 protein-reduced orthopaedic implant wear particle-induced osteolysis and inflammation in vivo.

    PubMed

    Jiang, Xinyi; Sato, Taishi; Yao, Zhenyu; Keeney, Michael; Pajarinen, Jukka; Lin, Tzu-Hua; Loi, Florence; Egashira, Kensuke; Goodman, Stuart; Yang, Fan

    2016-01-01

    Total joint replacement (TJR) has been widely used as a standard treatment for late-stage arthritis. One challenge for long-term efficacy of TJR is the generation of ultra-high molecular weight polyethylene wear particles from the implant surface that activates an inflammatory cascade which may lead to bone loss, prosthetic loosening and eventual failure of the procedure. Here, we investigate the efficacy of local administration of mutant CCL2 proteins, such as 7ND, on reducing wear particle-induced inflammation and osteolysis in vivo using a mouse calvarial model. Mice were treated with local injection of 7ND or phosphate buffered saline (PBS) every other day for up to 14 days. Wear particle-induced osteolysis and the effects of 7ND treatment were evaluated using micro-CT, histology, and immunofluorescence staining. Compared with the PBS control, 7ND treatment significantly decreased wear particle-induced osteolysis, which led to a higher bone volume fraction and bone mineral density. Furthermore, immunofluorescence staining showed 7ND treatment decreased the number of recruited inflammatory cells and osteoclasts. Together, our results support the feasibility of local delivery of 7ND for mitigating wear particle-induced inflammation and osteolysis, which may offer a promising strategy for extending the life time of TJRs. PMID:26174978

  2. Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons

    PubMed Central

    Trushina, Eugenia; Rana, Sandeep; McMurray, Cynthia T; Hua, Duy H

    2009-01-01

    Background Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion mutation in the coding region of a novel gene. The mechanism of HD is unknown. Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt) contributes to the pathology. However, recent studies have identified early cellular dysfunctions that preclude aggregate formation. Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches. Previously, we demonstrated that tricyclic pyrone (TP) compounds efficiently inhibited formation of amyloid-β (Aβ) aggregates in cell and mouse models representing Alzheimer's Disease (AD). In the present study, we aimed to determine whether TP compounds could prevent aggregation and restore early cellular defects in primary embryonic striatal neurons from animal model representing HD. Results TP compounds effectively inhibit aggregation caused by mhtt in neurons and glial cells. Treatment with TP compounds also alleviated cholesterol accumulation and restored clathrin-independent endocytosis in HD neurons. Conclusion We have found that TP compounds not only blocked mhtt-induced aggregation, but also alleviated early cellular dysfunctions that preclude aggregate formation. Our data suggest TP molecules may be used as lead compounds for prevention or treatment of multiple neurodegenerative diseases including HD and AD. PMID:19586540

  3. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

    PubMed

    Rodriguez-Fernandez, Imilce A; Dell'Angelica, Esteban C

    2015-01-01

    The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in

  4. Properties of a Streptococcus salivarius spontaneous mutant in which the methionine at position 48 in the protein HPr has been replaced by a valine.

    PubMed Central

    Vadeboncoeur, C; Gauthier, L; Gagnon, G; Leduc, A; Brochu, D; Lapointe, R; Desjardins, B; Frenette, M

    1994-01-01

    HPr is a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) that participates in the concomitant transport and phosphorylation of sugars in bacteria. In gram-positive bacteria, HPr is also reversibly phosphorylated at a seryl residue at position 46 (Ser-46) by a metabolite-activated ATP-dependent kinase and a Pi-dependent HPr(Ser-P) phosphatase. We report in this article the isolation of a spontaneous mutant (mutant A66) from a streptococcus (Streptococcus salivarius) in which the methionine at position 48 (Met-48) in the protein HPr has been replaced by a valine (Val). The mutation inhibited the phosphorylation of HPr on Ser-46 by the ATP-dependent kinase but did not prevent phosphorylation of HPr by enzyme I or the phosphorylation of enzyme II complexes by HPr(His-P). The results, however, suggested that replacement of Met-48 by Val decreased the affinity of enzyme I for HPr or the affinity of enzyme II proteins for HPr(His-P) or both. Characterization of mutant A66 demonstrated that it has pleiotropic properties, including the lack of IIILman, a specific protein of the mannose PTS; decreased levels of HPr; derepression of some cytoplasmic proteins; reduced growth on PTS as well as on non-PTS sugars; and aberrant growth in medium containing a mixture of sugars. Images PMID:8288549

  5. Wolbachia Protein TomO Targets nanos mRNA and Restores Germ Stem Cells in Drosophila Sex-lethal Mutants.

    PubMed

    Ote, Manabu; Ueyama, Morio; Yamamoto, Daisuke

    2016-09-12

    Wolbachia, endosymbiotic bacteria prevalent in invertebrates, manipulate their hosts in a variety of ways: they induce cytoplasmic incompatibility, male lethality, male-to-female transformation, and parthenogenesis. However, little is known about the molecular basis for host manipulation by these bacteria. In Drosophila melanogaster, Wolbachia infection makes otherwise sterile Sex-lethal (Sxl) mutant females capable of producing mature eggs. Through a functional genomic screen for Wolbachia genes with growth-inhibitory effects when expressed in cultured Drosophila cells, we identified the gene WD1278 encoding a novel protein we call toxic manipulator of oogenesis (TomO), which phenocopies some of the Wolbachia effects in Sxl mutant D. melanogaster females. We demonstrate that TomO enhances the maintenance of germ stem cells (GSCs) by elevating Nanos (Nos) expression via its interaction with nos mRNA, ultimately leading to the restoration of germ cell production in Sxl mutant females that are otherwise without GSCs. PMID:27498563

  6. Induction of lytic pathways in T cell clones derived from wild-type or protein tyrosine kinase Fyn mutant mice.

    PubMed

    Lancki, D W; Fields, P; Qian, D; Fitch, F W

    1995-08-01

    The OVA-reactive CD4+ Th1 clones and alloreactive CD8+ clones derived from wild-type or fyn-/- mice serve as model systems which have allowed us to investigate several aspects of the molecular events associated with T cell-mediated cytotoxicity, including 1) the differential utilization of two distinct cytolytic pathways by CD4+ Th1 clones and CD8+ CTL, 2) a comparison of the pathways of lysis induced by stimulation of the TCR or by alternative stimuli, 3) the requirement of Fyn for derivation of antigen-specific T-cell clones having properties of CD4+ Th1 and CD8+ CTL cells 4) the differential requirement of Fyn in the induction of responses by TCR and the alternative stimuli. Stimulation through the TCR, either by APC bearing relevant antigen or by immobilized anti-CD3 mAb, resulted in comparable levels of target cell lysis by clones from both wild-type and fyn-/- mice. These clones also utilize the Fas pathway to lyse target cells. Thus, Fyn does not appear to be required for expression of the Fas pathway when triggered through the TCR. In contrast, lysis of target cells by T-cell clones lacking Fyn was deficient when stimulated through Thy-1 or Ly-6C (using mAb) or with Con A or phorbol ester as compared to clones derived from wild-type mice. The basis for the defect in response to stimulation through the GPI-linked molecules appears to be a signaling defect which affects all of the functional responses we measured, while the defect in response to Con A stimulation appears to affect lysis but not lymphokine production. Thus, Fyn expression is selectively required for efficient activation of the Fas pathway of lysis through Thy-1, Ly-6C, and by Con A or phorbol ester in these T-cell clones. CD8+ clones derived from fyn-/- mutant mice, like clones derived from wild-type mice, display antigen-specific lysis, and appear to express perforin message and perforin protein. A Ca(++)-dependent (presumably perforin/exocytosis) component and Fas component of lysis was

  7. Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier–dependent pathway

    PubMed Central

    Ahner, Annette; Gong, Xiaoyan; Schmidt, Bela Z.; Peters, Kathryn W.; Rabeh, Wael M.; Thibodeau, Patrick H.; Lukacs, Gergely L.; Frizzell, Raymond A.

    2013-01-01

    Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27’s ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4’s impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin–proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein. PMID:23155000

  8. Characterization of mutants of a highly cross-reactive calcium-binding protein from Brassica pollen for allergen-specific immunotherapy.

    PubMed

    Garmatiuk, Tetiana; Swoboda, Ines; Twardosz-Kropfmüller, Anna; Dall'antonia, Fabio; Keller, Walter; Singh, Mohan B; Bhalla, Prem L; Okada, Takashi; Toriyama, Kinya; Weber, Milena; Ghannadan, Minoo; Sperr, Wolfgang R; Blatt, Katharina; Valent, Peter; Klein, Brigitte; Niederberger, Verena; Curin, Mirela; Balic, Nadja; Spitzauer, Susanne; Valenta, Rudolf

    2013-09-01

    The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy. Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calcium-binding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE. Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens. PMID:23790497

  9. Characterization of mutants of a highly cross-reactive calcium-binding protein from Brassica pollen for allergen-specific immunotherapy.

    PubMed

    Garmatiuk, Tetiana; Swoboda, Ines; Twardosz-Kropfmüller, Anna; Dall'antonia, Fabio; Keller, Walter; Singh, Mohan B; Bhalla, Prem L; Okada, Takashi; Toriyama, Kinya; Weber, Milena; Ghannadan, Minoo; Sperr, Wolfgang R; Blatt, Katharina; Valent, Peter; Klein, Brigitte; Niederberger, Verena; Curin, Mirela; Balic, Nadja; Spitzauer, Susanne; Valenta, Rudolf

    2013-09-01

    The major turnip (Brassica rapa) pollen allergen, belongs to a family of calcium-binding proteins (i.e., two EF-hand proteins), which occur as highly cross-reactive allergens in pollen of weeds, grasses and trees. In this study, the IgE binding capacity and allergenic activity of three recombinant allergen variants containing mutations in their calcium-binding sites were analyzed in sensitized patients with the aim to identify the most suitable hypoallergenic molecule for specific immunotherapy. Analysis of the wildtype allergen and the mutants regarding IgE reactivity and activation of basophils in allergic patients indicated that the allergen derivative mutated in both calcium-binding domains had the lowest allergenic activity. Gel filtration and circular dichroism experiments showed that both, the wildtype and the double mutant, occurred as dimers in solution and assumed alpha-helical fold, respectively. However, both fold and thermal stability were considerably reduced in the double mutant. The use of bioinformatic tools for evaluation of the solvent accessibility and charge distribution suggested that the reduced IgE reactivity and different structural properties of the double mutant may be due to a loss of negatively charged amino acids on the surface. Interestingly, immunization of rabbits showed that only the double mutant but not the wildtype allergen induced IgG antibodies which recognized the allergen and blocked binding of allergic patients IgE. Due to the extensive structural similarity and cross-reactivity between calcium-binding pollen allergens the hypoallergenic double mutant may be useful not only for immunotherapy of turnip pollen allergy, but also for the treatment of allergies to other two EF-hand pollen allergens.

  10. Reduced function of the RNA-binding protein FPA rescues a T-DNA insertion mutant in the Arabidopsis ZHOUPI gene by promoting transcriptional read-through.

    PubMed

    Zhang, Yaohua; Li, Xin; Goodrich, Justin; Wu, Chunxia; Wei, Haichao; Yang, Suxin; Feng, Xianzhong

    2016-07-01

    T-DNA insertion mutants have been widely used to investigate plant gene functions. Unexpectedly, in several reported cases, the phenotype of T-DNA insertion mutations can be suppressed because of trans T-DNA interactions associated with epigenetic modification, which indicates that caution is needed when T-DNA mutants are used. In the present study, we characterized a novel process suppressing a T-DNA mutation. The spz2 (suppressor of zou 2) mutant was isolated as a suppressor of the phenotype of the zou-4 mutant caused by a T-DNA insertion in the first intron. The spz2 mutation partially recovered the native ZOU gene expression in the zou-4 background, but not in two other zou alleles, zou-2 and zou-3, with T-DNAs inserted in the exon and intron, respectively. The suppressed phenotype was inherited in a Mendelian fashion and is not associated with epigenetic modification. The recovery of the native ZOU gene expression in the spz2 zou-4 double mutant is caused by transcriptional read-through of the intronic T-DNA as a result of decreased proximal polyadenylation. SPZ2 encodes an RNA-binding protein, FPA, which is known to regulate polyadenylation site selection. This is the first example of FPA rescuing a T-DNA insertion mutation by affecting the polyadenylation site selection. PMID:27164978

  11. Opacity proteins of neisseria gonorrhoeae in lipooligosaccharide mutants lost ability to interact with neutrophil-restricted CEACAM3 (CD66d).

    PubMed

    Zhang, Song; Tu, Ya-Ting; Cai, Hua-Hua; Ding, Hong-Hui; Li, Qiao; He, Ying-Xia; Liu, Xin-Xin; Wang, Xin; Hu, Feng; Chen, Tie; Chen, Hong-Xiang

    2016-06-01

    Lipooligosacharide (LOS) of Neisseria gonorrhoeae (gonococci, GC) is involved in the interaction of GC with host cells. Deletion of the alpha-oligosaccharide (alpha-OS) moiety of LOS (lgtF mutant) significantly impairs invasion of GC into epithelial cell lines. GC opacity (Opa) proteins, such as OpaI, mediate phagocytosis and stimulate chemiluminescence responses in neutrophils in part through interaction with members of the carcinoembryonic antigen (CEA) family, which includes CEACAM3 (CD66d), a human neutrophil specific receptor for phagocytosis of bacteria. In the present work, we examined the effects of OpaI-expressing lgtF mutant on phagocytosis by HeLa-CEACAM3 cells and chemiluminescence responses in neutrophils. The results showed that lgtF mutant even expressing OpaI completely lost the ability to promote either phagocytosis mediated by CEACAM3 interaction in HeLa cells or chemiluminescence responses in neutrophils. These data indicated that Opa proteins in the lgtF mutant, which might result from the conformational change, cannot be functional. PMID:27376801

  12. Structures of the N47A and E109Q mutant proteins of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii

    SciTech Connect

    Soriano, Erika V.; McCloskey, Diane E.; Kinsland, Cynthia; Pegg, Anthony E.; Ealick, Steven E.

    2008-04-01

    The crystal structures of two arginine decarboxylase mutant proteins provide insights into the mechanisms of pyruvoyl-group formation and the decarboxylation reaction. Pyruvoyl-dependent arginine decarboxylase (PvlArgDC) catalyzes the first step of the polyamine-biosynthetic pathway in plants and some archaebacteria. The pyruvoyl group of PvlArgDC is generated by an internal autoserinolysis reaction at an absolutely conserved serine residue in the proenzyme, resulting in two polypeptide chains. Based on the native structure of PvlArgDC from Methanococcus jannaschii, the conserved residues Asn47 and Glu109 were proposed to be involved in the decarboxylation and autoprocessing reactions. N47A and E109Q mutant proteins were prepared and the three-dimensional structure of each protein was determined at 2.0 Å resolution. The N47A and E109Q mutant proteins showed reduced decarboxylation activity compared with the wild-type PvlArgDC. These residues may also be important for the autoprocessing reaction, which utilizes a mechanism similar to that of the decarboxylation reaction.

  13. Structures of KaiC Circadian Clock Mutant Proteins: A New Phosphorylation Site at T426 and Mechanisms of Kinase, ATPase and Phosphatase

    SciTech Connect

    Pattanayek, Rekha; Mori, Tetsuya; Xu, Yao; Pattanayek, Sabuj; Johnson, Carl H.; Egli, Martin

    2010-09-02

    The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by three proteins, KaiA, KaiB and KaiC. Homo-hexameric KaiC displays kinase, phosphatase and ATPase activities; KaiA enhances KaiC phosphorylation and KaiB antagonizes KaiA. Phosphorylation and dephosphorylation of the two known sites in the C-terminal half of KaiC subunits, T432 and S431, follow a strict order (TS {yields} pTS {yields} pTpS {yields} TpS {yields} TS) over the daily cycle, the origin of which is not understood. To address this void and to analyze the roles of KaiC active site residues, in particular T426, we determined structures of single and double P-site mutants of S. elongatus KaiC. The conformations of the loop region harboring P-site residues T432 and S431 in the crystal structures of six KaiC mutant proteins exhibit subtle differences that result in various distances between Thr (or Ala/Asn/Glu) and Ser (or Ala/Asp) residues and the ATP {gamma}-phosphate. T432 is phosphorylated first because it lies consistently closer to P{gamma}. The structures of the S431A and T432E/S431A mutants reveal phosphorylation at T426. The environments of the latter residue in the structures and functional data for T426 mutants in vitro and in vivo imply a role in dephosphorylation. We provide evidence for a third phosphorylation site in KaiC at T426. T426 and S431 are closely spaced and a KaiC subunit cannot carry phosphates at both sites simultaneously. Fewer subunits are phosphorylated at T426 in the two KaiC mutants compared to phosphorylated T432 and/or S431 residues in the structures of wt and other mutant KaiCs, suggesting that T426 phosphorylation may be labile. The structures combined with functional data for a host of KaiC mutant proteins help rationalize why S431 trails T432 in the loss of its phosphate and shed light on the mechanisms of the KaiC kinase, ATPase and phosphatase activities.

  14. Structures of KaiC Circadian Clock Mutant Proteins: A New Phosphorylation Site at T426 and Mechanisms of Kinase, ATPase and Phosphatase

    PubMed Central

    Pattanayek, Rekha; Mori, Tetsuya; Xu, Yao; Pattanayek, Sabuj; Johnson, Carl H.; Egli, Martin

    2009-01-01

    Background The circadian clock of the cyanobacterium Synechococcus elongatus can be reconstituted in vitro by three proteins, KaiA, KaiB and KaiC. Homo-hexameric KaiC displays kinase, phosphatase and ATPase activities; KaiA enhances KaiC phosphorylation and KaiB antagonizes KaiA. Phosphorylation and dephosphorylation of the two known sites in the C-terminal half of KaiC subunits, T432 and S431, follow a strict order (TS→pTS→pTpS→TpS→TS) over the daily cycle, the origin of which is not understood. To address this void and to analyze the roles of KaiC active site residues, in particular T426, we determined structures of single and double P-site mutants of S. elongatus KaiC. Methodology and Principal Findings The conformations of the loop region harboring P-site residues T432 and S431 in the crystal structures of six KaiC mutant proteins exhibit subtle differences that result in various distances between Thr (or Ala/Asn/Glu) and Ser (or Ala/Asp) residues and the ATP γ-phosphate. T432 is phosphorylated first because it lies consistently closer to Pγ. The structures of the S431A and T432E/S431A mutants reveal phosphorylation at T426. The environments of the latter residue in the structures and functional data for T426 mutants in vitro and in vivo imply a role in dephosphorylation. Conclusions and Significance We provide evidence for a third phosphorylation site in KaiC at T426. T426 and S431 are closely spaced and a KaiC subunit cannot carry phosphates at both sites simultaneously. Fewer subunits are phosphorylated at T426 in the two KaiC mutants compared to phosphorylated T432 and/or S431 residues in the structures of wt and other mutant KaiCs, suggesting that T426 phosphorylation may be labile. The structures combined with functional data for a host of KaiC mutant proteins help rationalize why S431 trails T432 in the loss of its phosphate and shed light on the mechanisms of the KaiC kinase, ATPase and phosphatase activities. PMID:19956664

  15. Stimulation of the Replication of ICP0-Null Mutant Herpes Simplex Virus 1 and pp71-Deficient Human Cytomegalovirus by Epstein-Barr Virus Tegument Protein BNRF1

    PubMed Central

    Lu, Yongxu; Orr, Anne

    2016-01-01

    ABSTRACT It is now well established that several cellular proteins that are components of promyelocytic leukemia nuclear bodies (PML NBs, also known as ND10) have restrictive effects on herpesvirus infections that are countered by viral proteins that are either present in the virion particle or are expressed during the earliest stages of infection. For example, herpes simplex virus 1 (HSV-1) immediate early (IE) protein ICP0 overcomes the restrictive effects of PML-NB components PML, Sp100, hDaxx, and ATRX while human cytomegalovirus (HCMV) IE protein IE1 targets PML and Sp100, and its tegument protein pp71 targets hDaxx and ATRX. The functions of these viral regulatory proteins are in part interchangeable; thus, both IE1 and pp71 stimulate the replication of ICP0-null mutant HSV-1, while ICP0 increases plaque formation by pp71-deficient HCMV. Here, we extend these studies by examining proteins that are expressed by Epstein-Barr virus (EBV). We report that EBV tegument protein BNRF1, discovered by other investigators to target the hDaxx/ATRX complex, increases the replication of both ICP0-null mutant HSV-1 and pp71-deficient HCMV. In addition, EBV protein EBNA-LP, which targets Sp100, also augments ICP0-null mutant HSV-1 replication. The combination of these two EBV regulatory proteins had a greater effect than each one individually. These findings reinforce the concept that disruption of the functions of PML-NB proteins is important for efficient herpesvirus infections. IMPORTANCE Whether a herpesvirus initiates a lytic infection in a host cell or establishes quiescence or latency is influenced by events that occur soon after the viral genome has entered the host cell nucleus. Certain cellular proteins respond in a restrictive manner to the invading pathogen's DNA, while viral functions are expressed that counteract the cell-mediated repression. One aspect of cellular restriction of herpesvirus infections is mediated by components of nuclear structures known as

  16. Structural Stability and Local Dynamics in Disease-Causing Mutants of Human Apolipoprotein A-I: What Makes the Protein Amyloidogenic?

    PubMed

    Das, Madhurima; Wilson, Christopher J; Mei, Xiaohu; Wales, Thomas E; Engen, John R; Gursky, Olga

    2016-01-29

    ApoA-I, the major protein of plasma high-density lipoprotein, removes cellular cholesterol and protects against atherosclerosis. ApoA-I mutations can cause familial amyloidosis, a life-threatening disease wherein N-terminal protein fragments form fibrils in vital organs. To unveil the protein misfolding mechanism and to understand why some mutations cause amyloidosis while others do not, we analyzed the structure, stability, and lipid-binding properties of naturally occurring mutants of full-length human apoA-I causing either amyloidosis (G26R, W50R, F71Y, and L170P) or aberrant lipid metabolism (L159R). Global and local protein conformation and dynamics in solution were assessed by circular dichroism, fluorescence, and hydrogen-deuterium exchange mass spectrometry. All mutants showed increased deuteration in residues 14-22, supporting our hypothesis that decreased protection of this major amyloid "hot spot" can trigger protein misfolding. In addition, L159R showed local helical unfolding near the mutation site, consistent with cleavage of this mutant in plasma to generate the labile 1-159 fragment. Together, the results suggest that reduced protection of the major amyloid "hot spot", combined with the structural integrity of the native helix bundle conformation, shifts the balance from protein clearance to β-aggregation. A delicate balance between the overall structural integrity of a globular protein and the local destabilization of its amyloidogenic segments may be a fundamental determinant of this and other amyloid diseases. Furthermore, mutation-induced conformational changes observed in the helix bundle, which comprises the N-terminal 75% of apoA-I, and its flexible C-terminal tail suggest the propagation of structural perturbations to distant sites via an unexpected template-induced ensemble-based mechanism, challenging the classical structure-based view.

  17. Mutant prion protein expression is associated with an alteration of the Rab GDP dissociation inhibitor alpha (GDI)/Rab11 pathway.

    PubMed

    Massignan, Tania; Biasini, Emiliano; Lauranzano, Eliana; Veglianese, Pietro; Pignataro, Mauro; Fioriti, Luana; Harris, David A; Salmona, Mario; Chiesa, Roberto; Bonetto, Valentina

    2010-04-01

    The prion protein (PrP) is a glycosylphosphatidylinositol-anchored membrane glycoprotein that plays a vital role in prion diseases, a class of fatal neurodegenerative disorders of humans and animals. Approximately 20% of human prion diseases display autosomal dominant inheritance and are linked to mutations in the PrP gene on chromosome 20. PrP mutations are thought to favor the conformational conversion of PrP into a misfolded isoform that causes disease by an unknown mechanism. The PrP mutation D178N/Met-129 is linked to fatal familial insomnia, which causes severe sleep abnormalities and autonomic dysfunction. We showed by immunoelectron microscopy that this mutant PrP accumulates abnormally in the endoplasmic reticulum and Golgi of transfected neuroblastoma N2a cells. To investigate the impact of intracellular PrP accumulation on cellular homeostasis, we did a two-dimensional gel-based differential proteomics analysis. We used wide range immobilized pH gradient strips, pH 4-7 and 6-11, to analyze a large number of proteins. We found changes in proteins involved in energy metabolism, redox regulation, and vesicular transport. Rab GDP dissociation inhibitor alpha (GDI) was one of the proteins that changed most. GDI regulates vesicular protein trafficking by acting on the activity of several Rab proteins. We found a specific reduction in the level of functional Rab11 in mutant PrP-expressing cells associated with impaired post-Golgi trafficking. Our data are consistent with a model by which mutant PrP induces overexpression of GDI, activating a cytotoxic feedback loop that leads to protein accumulation in the secretory pathway.

  18. Regulation of Cl- transport in T84 cell clones expressing a mutant regulatory subunit of cAMP-dependent protein kinase.

    PubMed Central

    Rogers, K V; Goldman, P S; Frizzell, R A; McKnight, G S

    1990-01-01

    Cl- channels in the apical membranes of salt-secreting epithelia are activated by both cAMP and Ca2+ second-messenger systems, and dysfunctions in their hormonal regulation have been demonstrated in patients with cystic fibrosis. We have transfected the epithelial cell line T84 with an expression vector containing a mutant form of the regulatory subunit of the cAMP-dependent protein kinase. Stable transformants that express this construct have reduced basal cAMP-dependent protein kinase activity and do not increase kinase activity beyond the basal level of control cells in response to cAMP. Forskolin, vasoactive intestinal peptide, and prostaglandin E2 each stimulate intracellular cAMP accumulation in both mutant and control clones; however, the activation of Cl- channels in response to elevated cAMP is blocked in mutant clones, indicating direct involvement of the cAMP-dependent protein kinase. In contrast, Ca2+ ionophores retain their ability to activate the Cl- channel in T84 cells expressing the mutant regulatory subunit, suggesting that activation of the channel by means of Ca2+ does not require the participation of cAMP-dependent protein kinase activity. These clones will be useful for further studies of the interactions between the cAMP- and Ca2(+)-dependent regulatory pathways in salt-secreting epithelial cells. They can also be used to identify the mediators of Ca2(+)-dependent Cl- channel activation in isolation from interactions with the cAMP second-messenger pathway. Images PMID:2174170

  19. Role of protein synthesis in the repair of sublethal x-ray damage in a mutant Chinese hamster ovary cell line

    SciTech Connect

    Yezzi, M.J.

    1985-04-01

    A temperature-sensitive mutant for protein synthesis, CHO-TSH1, has been compared to the wild-type cell, CHO-sC1, in single- and split-radiation-dose schemes. When the exponentially growing TS mutant and the wild-type cells were treated at 40/sub 0/C for up to 2 hrs prior to graded doses of x rays, the survival curves were identical and were the same as those obtained without heat treatment. If the cultures were incubated at 40/sup 0/C for 2 hrs before a first dose and maintained at 40/sup 0/C during a 2 hr dose fractionation interval, repair of radiation damage was reduced in the mutant compared to the wild type. These observations implied that a pool of proteins was involved in the repair of sublethal x-ray damage. However, if repair was measured by the alkaline-unwinding technique under the same time and temperature schemes, no difference in the kientics of DNA strand rejoining was observed. Misrepair processes may permit restoration of DNA strand integrity but not allow functional repair. The effect of diminished repair under conditions of inhibition of protein synthesis was found to be cell-cycle dependent in survival studies with synchronized mutant cell populations. Repair was found to be almost completely eliminated if the temperature sequence described above was applied in the middle of the DNA synthetic phase. Treatment of cell populations in the middle of G/sub 1/-phase yielded repair inhibition comparable to that observed with the asynchronous cells. Splitdose experiments were done using pre-incubation with cycloheximide to chemically inhibit protein synthesis. WT cells and TS cells were treated with cycloheximide at 35/sup 0/C for 2 hrs before a first dose and during a 2 hr dose fractionation interval. 23 figs., 7 tabs.

  20. A Mutant Form of the Neisseria gonorrhoeae Pilus Secretin Protein PilQ Allows Increased Entry of Heme and Antimicrobial Compounds†

    PubMed Central

    Chen, Ching-ju; Tobiason, Deborah M.; Thomas, Christopher E.; Shafer, William M.; Seifert, H. Steven; Sparling, P. Frederick

    2004-01-01

    A spontaneous point mutation in pilQ (pilQ1) resulted in phenotypic suppression of a hemoglobin (Hb) receptor mutant (hpuAB mutant), allowing gonococci to grow on Hb as the sole source of iron. PilQ, formerly designated OMP-MC, is a member of the secretin family of proteins located in the outer membrane and is required for pilus biogenesis. The pilQ1 mutant also showed decreased piliation and transformation efficiency. Insertional inactivation of pilQ1 resulted in the loss of the Hb utilization phenotype and decreased entry of free heme. Despite the ability of the pilQ1 mutant to use Hb for iron acquisition and porphyrin, there was no demonstrable binding of Hb to the cell surface. The pilQ1 mutant was more sensitive to the toxic effect of free heme in growth medium and hypersensitive to the detergent Triton X-100 and multiple antibiotics. Double mutation in pilQ1 and tonB had no effect on these phenotypes, but a double pilQ1 pilT mutant showed a reduction in Hb-dependent growth and decreased sensitivity to heme and various antimicrobial agents. Insertional inactivation of wild-type pilQ also resulted in reduced entry of heme, Triton X-100, and some antibiotics. These results show that PilQ forms a channel that allows entry of heme and certain antimicrobial compounds and that a gain-of function point mutation in pilQ results in TonB-independent, PilT-dependent increase of entry. PMID:14729699

  1. Disrupting autophagy restores peroxisome function to an Arabidopsis lon2 mutant and reveals a role for the LON2 protease in peroxisomal matrix protein degradation.

    PubMed

    Farmer, Lisa M; Rinaldi, Mauro A; Young, Pierce G; Danan, Charles H; Burkhart, Sarah E; Bartel, Bonnie

    2013-10-01

    Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.

  2. Xyloketal-derived small molecules show protective effect by decreasing mutant Huntingtin protein aggregates in Caenorhabditis elegans model of Huntington’s disease

    PubMed Central

    Zeng, Yixuan; Guo, Wenyuan; Xu, Guangqing; Wang, Qinmei; Feng, Luyang; Long, Simei; Liang, Fengyin; Huang, Yi; Lu, Xilin; Li, Shichang; Zhou, Jiebin; Burgunder, Jean-Marc; Pang, Jiyan; Pei, Zhong

    2016-01-01

    Huntington’s disease is an autosomal-dominant neurodegenerative disorder, with chorea as the most prominent manifestation. The disease is caused by abnormal expansion of CAG codon repeats in the IT15 gene, which leads to the expression of a glutamine-rich protein named mutant Huntingtin (Htt). Because of its devastating disease burden and lack of valid treatment, development of more effective therapeutics for Huntington’s disease is urgently required. Xyloketal B, a natural product from mangrove fungus, has shown protective effects against toxicity in other neurodegenerative disease models such as Parkinson’s and Alzheimer’s diseases. To identify potential neuroprotective molecules for Huntington’s disease, six derivatives of xyloketal B were screened in a Caenorhabditis elegans Huntington’s disease model; all six compounds showed a protective effect. Molecular docking studies indicated that compound 1 could bind to residues GLN369 and GLN393 of the mutant Htt protein, forming a stable trimeric complex that can prevent the formation of mutant Htt aggregates. Taken together, we conclude that xyloketal derivatives could be novel drug candidates for treating Huntington’s disease. Molecular target analysis is a good method to simulate the interaction between proteins and drug compounds. Further, protective candidate drugs could be designed in future using the guidance of molecular docking results. PMID:27110099

  3. Trafficking defect of mutant kidney anion exchanger 1 (kAE1) proteins associated with distal renal tubular acidosis and Southeast Asian ovalocytosis.

    PubMed

    Sawasdee, Nunghathai; Udomchaiprasertkul, Wandee; Noisakran, Sansanee; Rungroj, Nanyawan; Akkarapatumwong, Varaporn; Yenchitsomanus, Pa-thai

    2006-11-24

    Compound heterozygous anion exchanger 1 (AE1) SAO/G701D mutations result in distal renal tubular acidosis with Southeast Asian ovalocytosis. Interaction, trafficking and localization of wild-type and mutant (SAO and G701D) kAE1 proteins fused with hemagglutinin, six-histidine, Myc, or green fluorescence protein (GFP) were examined in human embryonic kidney (HEK) 293 cells. When individually expressed, wild-type kAE1 was localized at cell surface while mutant kAE1 SAO and G701D were intracellularly retained. When co-expressed, wild-type kAE1 could form heterodimer with kAE1 SAO or kAE1 G701D and could rescue mutant kAE1 proteins to express on the cell surface. Co-expression of kAE1 SAO and kAE1 G701D also resulted in heterodimer formation but intracellular retention without cell surface expression, suggesting their trafficking defect and failure to rescue each other to the plasma membrane, most likely the molecular mechanism of the disease in the compound heterozygous condition. PMID:17027918

  4. Charcot-Marie-Tooth type 2B disease-causing RAB7A mutant proteins show altered interaction with the neuronal intermediate filament peripherin.

    PubMed

    Cogli, Laura; Progida, Cinzia; Thomas, Claire L; Spencer-Dene, Bradley; Donno, Claudia; Schiavo, Giampietro; Bucci, Cecilia

    2013-02-01

    Charcot-Marie-Tooth type 2B (CMT2B) is a peripheral ulcero-mutilating neuropathy caused by four missense mutations in the rab7a gene. CMT2B is clinically characterized by prominent sensory loss, distal muscle weakness leading to muscle atrophy, high frequency of foot ulcers and infections that often results in toe amputations. RAB7A is a ubiquitous small GTPase, which controls transport to late endocytic compartments. Although the biochemical and functional properties of disease-causing RAB7A mutant proteins have been investigated, it is not yet clear how the disease originates. To understand how mutations in a ubiquitous protein specifically affect peripheral neurons, we performed a two-hybrid screen using a dorsal root ganglia cDNA library with the purpose of identifying RAB7A interactors specific for these cells. We identified peripherin, an intermediate filament protein expressed primarily in peripheral neurons, as a putative RAB7A interacting protein. The interaction was confirmed by co-immunoprecipitation and pull-down experiments, and established that the interaction is direct using recombinant proteins. Silencing or overexpression of wild type RAB7A changed the soluble/insoluble rate of peripherin indicating that RAB7A is important for peripherin organization and function. In addition, disease-causing RAB7A mutant proteins bind more strongly to peripherin and their expression causes a significant increase in the amount of soluble peripherin. Since peripherin plays a role not only in neurite outgrowth during development but also in axonal regeneration after injury, these data suggest that the altered interaction between disease-causing RAB7A mutants and peripherin could play an important role in CMT2B neuropathy.

  5. Loss of growth polarity and mislocalization of septa in a Neurospora mutant altered in the regulatory subunit of cAMP-dependent protein kinase.

    PubMed Central

    Bruno, K S; Aramayo, R; Minke, P F; Metzenberg, R L; Plamann, M

    1996-01-01

    In filamentous fungi, growth polarity (i.e. hyphal extension) and formation of septa require polarized deposition of new cell wall material. To explore this process, we analyzed a conditional Neurospora crassa mutant, mcb, which showed a complete loss of growth polarity when incubated at the restrictive temperature. Cloning and DNA sequence analysis of the mcb gene revealed that it encodes a regulatory subunit of cAMP-dependent protein kinase (PKA). Unexpectedly, the mcb mutant still formed septa when grown at the restrictive temperature, indicating that polarized deposition of wall material during septation is a process that is, at least in part, independent of polarized deposition during hyphal tip extension. However, septa formed in the mcb mutant growing at the restrictive temperature are mislocalized. Both polarized growth and septation are actin-dependent processes, and a concentration of actin patches is observed at growing hyphal tips and sites where septa are being formed. In the mcb mutant growing at the restrictive temperature, actin patches are uniformly distributed over the cell cortex; however, actin patches are still concentrated at sites of septation. Our results suggest that the PKA pathway regulates hyphal growth polarity, possibly through organizing actin patches at the cell cortex. Images PMID:8918454

  6. The rye mutants identify a role for Ssn/Srb proteins of the RNA polymerase II holoenzyme during stationary phase entry in Saccharomyces cerevisiae.

    PubMed

    Chang, Y W; Howard, S C; Budovskaya, Y V; Rine, J; Herman, P K

    2001-01-01

    Saccharomyces cerevisiae cells enter into a distinct resting state, known as stationary phase, in response to specific types of nutrient deprivation. We have identified a collection of mutants that exhibited a defective transcriptional response to nutrient limitation and failed to enter into a normal stationary phase. These rye mutants were isolated on the basis of defects in the regulation of YGP1 expression. In wild-type cells, YGP1 levels increased during the growth arrest caused by nutrient deprivation or inactivation of the Ras signaling pathway. In contrast, the levels of YGP1 and related genes were significantly elevated in the rye mutants during log phase growth. The rye defects were not specific to this YGP1 response as these mutants also exhibited multiple defects in stationary phase properties, including an inability to survive periods of prolonged starvation. These data indicated that the RYE genes might encode important regulators of yeast cell growth. Interestingly, three of the RYE genes encoded the Ssn/Srb proteins, Srb9p, Srb10p, and Srb11p, which are associated with the RNA polymerase II holoenzyme. Thus, the RNA polymerase II holoenzyme may be a target of the signaling pathways responsible for coordinating yeast cell growth with nutrient availability.

  7. An Ash1-Like Protein MoKMT2H Null Mutant Is Delayed for Conidium Germination and Pathogenesis in Magnaporthe oryzae

    PubMed Central

    Cao, Zhaojun; Yin, Yue; Sun, Xuan; Han, Jun; Sun, Qing peng; Lu, Min

    2016-01-01

    Ash1 is a known H3K36-specific histone demethylase that is required for normal Hox gene expression and fertility in Drosophila and mammals. However, little is known about the expression and function of the fungal ortholog of Ash1 in phytopathogenic fungus Magnaporthe oryzae. Here we report that MoKMT2H, an Ash1-like protein, is required for conidium germination and virulence in rice. We obtained MoKMT2H null mutant (ΔMoKMT2H) using a target gene replacement strategy. In the ΔMoKMT2H null mutants, global histone methyltransferase modifications (H3K4me3, H3K9me3, H3K27me3, and H3K36me2/3) of the genome were unaffected. The ΔMoKMT2H mutants showed no defect in vegetative hyphal growth, conidium morphology, conidiation, or disease lesion formation on rice leaves. However, the MoKMT2H deletion mutants were delayed for conidium germination and consequently had decreased virulence. Taken together, our results indicated that MoKMT2H plays an important role in conidium germination during appressorium formation in the rice blast fungus and perhaps other pathogenic plant fungi. PMID:27747223

  8. pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

    PubMed Central

    Murugan, Elavazhagan; Venkatraman, Anandalakshmi; Lei, Zhou; Mouvet, Victoria; Rui Yi Lim, Rayne; Muruganantham, Nandhakumar; Goh, Eunice; Swee Lim Peh, Gary; Beuerman, Roger W.; Chaurasia, Shyam S.; Rajamani, Lakshminarayanan; Mehta, Jodhbir S.

    2016-01-01

    Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4th_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4th_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities. PMID:27030015

  9. The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism.

    PubMed

    Zaanan, Aziz; Okamoto, Koichi; Kawakami, Hisato; Khazaie, Khashayarsha; Huang, Shengbing; Sinicrope, Frank A

    2015-09-25

    In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser(69), confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr(705)) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response.

  10. Targeting mutant p53 protein and the tumor vasculature: an effective combination therapy for advanced breast tumors

    PubMed Central

    Liang, Yayun; Besch-Williford, Cynthia; Benakanakere, Indira; Thorpe, Philip E.

    2010-01-01

    Breast cancer progression depends upon the elaboration of a vasculature sufficient for the nourishment of the developing tumor. Breast tumor cells frequently contain a mutant form of p53 (mtp53), a protein which promotes their survival. The aim of this study was to determine whether combination therapy targeting mtp53 and anionic phospholipids (AP) on tumor blood vessels might be an effective therapeutic strategy for suppressing advanced breast cancer. We examined the therapeutic effects, singly, or in combination, of p53 reactivation and induction of massive apoptosis (PRIMA-1), which reactivates mtp53 and induces tumor cell apoptosis, and 2aG4, a monoclonal antibody that disrupts tumor vasculature by targeting AP on the surface of tumor endothelial cells and causes antibody-dependent destruction of tumor blood vessels, leading to ischemia and tumor cell death. Xenografts from two tumor cell lines containing mtp53, BT-474 and HCC-1428, were grown in nude mice to provide models of advanced breast tumors. After treatment with PRIMA-1 and/or 2aG4, regressing tumors were analyzed for vascular endothelial growth factor (VEGF) expression, blood vessel loss, and apoptotic markers. Individual drug treatment led to partial suppression of breast cancer progression. In contrast, combined treatment with PRIMA-1 and 2aG4 was extremely effective in suppressing tumor growth in both models and completely eradicated approximately 30% of tumors in the BT-474 model. Importantly, no toxic effects were observed in any treatment group. Mechanistic studies determined that PRIMA-1 reactivated mtp53 and also exposed AP on the surface of tumor cells as determined by enhanced 2aG4 binding. Combination treatment led to significant induction of tumor cell apoptosis, loss of VEGF expression, as well as destruction of tumor blood vessels. Furthermore, combination treatment severely disrupted tumor blood vessel perfusion in both tumor models. The observed in vitro PRIMA-1-induced exposure of

  11. Purification, Crystallization and Preliminary X-ray Diffraction Analysis of the Phage T4 Vertex Protein Gp24 and its Mutant Forms

    SciTech Connect

    Boeshans,K.; Liu, F.; Peng, G.; Idler, W.; Jang, S.; Marekov, L.; Black, L.; Ahvazi, B.

    2006-01-01

    The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6 {angstrom} resolution compared to wild-type gp24 at 3.80 {angstrom} resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.

  12. Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm.

    PubMed

    Song, Yuyu; Nagy, Maria; Ni, Weiming; Tyagi, Navneet K; Fenton, Wayne A; López-Giráldez, Francesc; Overton, John D; Horwich, Arthur L; Brady, Scott T

    2013-04-01

    Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.

  13. Purification, crystallization and preliminary X-ray diffraction of wild-type and mutant recombinant human transforming growth factor β-induced protein (TGFBIp)

    PubMed Central

    Runager, Kasper; García-Castellanos, Raquel; Valnickova, Zuzana; Kristensen, Torsten; Nielsen, Niels Chr.; Klintworth, Gordon K.; Gomis-Rüth, F. Xavier; Enghild, Jan J.

    2009-01-01

    Transforming growth factor β-induced protein (TGFBIp) has been linked to several corneal dystrophies as certain point mutations in the protein may give rise to a progressive accumulation of insoluble protein material in the human cornea. Little is known about the biological functions of this extracellular protein, which is expressed in various tissues throughout the human body. However, it has been found to interact with a number of extracellular matrix macromolecules such as collagens and proteoglycans. Structural information about TGFBIp might prove to be a valuable tool in the elucidation of its function and its role in corneal dystrophies caused by mutations in the TGFBI gene. A simple method for the purification of wild-type and mutant forms of recombinant human TGFBIp from human cells under native conditions is presented here. Moreover, the crystallization and preliminary X-ray analysis of TGFBIp are reported. PMID:19255489

  14. Role of pH in the appearance of the fluorescent state of chromo protein asCP595 and its mutant KFP

    NASA Astrophysics Data System (ADS)

    Rusanov, Alexander L.; Zubova, Nadya; Savitsky, Alexander P.

    2007-02-01

    At the present time the phenomenon of "fluorescence kindling," which is typical of asCP595 protein and some of its mutants, is of great interest for the studies of intracellular traffic. The range of changing of intracellular pH may be rather wide, however, the effect of pH on the fluorescent state of this protein is poorly investigated. Our studies have revealed that the fluorescence intensity of asCP595 increases significantly in alkaline conditions. In addition, the observed change in the position of excitation, emission, and absorption maxima indicates the appearance of new spectral forms of the protein. These conformers are characteristics of new fluorescent states. The changes in the absorption spectrum are indicative of a new dark form of the protein at alkaline pH.

  15. A mechanism for propagated SOD1 misfolding from frustration analysis of a G85R mutant protein assembly.

    PubMed

    Healy, Eamonn F

    2016-09-30

    Application of landscape theory and the dehydron hypothesis to a crystal structure of a G85R mutant superoxide dismutase (SOD1) tetrameric complex allows for the description of a prion-like hypothesis that serves to explain propagated SOD1 misfolding. We have developed two conformational-change scenarios, one local to the ESL at the complex interface, and a second displacement at the ESL of the otherdimeric subunit. When taken together these provide for a prion-like mechanism that can serve to explain the observed conversion of wtSOD1 to a misfolded form by the G85R mutant. PMID:27591900

  16. Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster

    PubMed Central

    Rodriguez-Fernandez, Imilce A.; Dell’Angelica, Esteban C.

    2015-01-01

    The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions–which together covered most of the autosomal chromosomes–to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated

  17. Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril.

    PubMed Central

    Weerkamp, A H; Handley, P S; Baars, A; Slot, J W

    1986-01-01

    The subcellular distribution of the cell wall-associated protein antigens of Streptococcus salivarius HB, which are involved in specific adhesive properties of the cells, was studied. Mutants which had lost the adhesive properties and lacked the antigens at the cell surface were compared with the parent strain. Immunoelectron microscopy of cryosections of cells labeled with affinity-purified, specific antisera and colloidal gold-protein A complexes was used to locate the antigens. Antigen C (AgC), a glycoprotein involved in attachment to host surfaces, was mainly located in the fibrillar layer outside the cell wall. A smaller amount of label was also found throughout the cytoplasmic area in the form of small clusters of gold particles, which suggests a macromolecular association. Mutant HB-7, which lacks the wall-associated AgC, accumulated AgC reactivity intracellularly. Intracellular AgC was often found associated with isolated areas of increased electron density, but sometimes seemed to fill the entire interior of the cell. Antigen B (AgB), a protein responsible for interbacterial coaggregation, was also located in the fibrillar layer, although its distribution differed from that of the wall-associated AgC since AgB was found predominantly in the peripheral areas. A very small amount of label was also found in the cytoplasmic area as discrete gold particles. Mutant HB-V5, which lacks wall-associated AgB, was not labeled in the fibrillar coat, but showed the same weak intracellular label as the parent strain. Immunolabeling with serum against AgD, another wall-associated protein but of unknown function, demonstrated its presence in the fibrillar layer of strain HB. Negatively stained preparations of whole cells of wild-type S. salivarius and mutants that had lost wall-associated AgB or AgC revealed that two classes of short fibrils are carried on the cell surface at the same time. AgB and AgC are probably located on separate classes of short, protease

  18. Mutants at the 2-Fold Interface of Adeno-associated Virus Type 2 (AAV2) Structural Proteins Suggest a Role in Viral Transcription for AAV Capsids

    PubMed Central

    Aydemir, Fikret; Salganik, Maxim; Resztak, Justyna; Singh, Jasbir; Bennett, Antonette; Agbandje-McKenna, Mavis

    2016-01-01

    ABSTRACT We previously reported that an amino acid substitution, Y704A, near the 2-fold interface of adeno-associated virus (AAV) was defective for transcription of the packaged genome (M. Salganik, F. Aydemir, H. J. Nam, R. McKenna, M. Agbandje-McKenna, and N. Muzyczka, J Virol 88:1071–1079, 2013, doi: http://dx.doi.org/10.1128/JVI.02093-13). In this report, we have characterized the defect in 6 additional capsid mutants located in a region ∼30 Å in diameter on the surface of the AAV type 2 (AAV2) capsid near the 2-fold interface. These mutants, which are highly conserved among primate serotypes, displayed a severe defect (3 to 6 logs) in infectivity. All of the mutants accumulated significant levels of uncoated DNA in the nucleus, but none of the mutants were able to accumulate significant amounts of genomic mRNA postinfection. In addition, wild-type (wt) capsids that were bound to the conformational antibody A20, which is known to bind the capsid surface in the region of the mutants, were also defective for transcription. In all cases, the mutant virus particles, as well as the antibody-bound wild-type capsids, were able to enter the cell, travel to the nucleus, uncoat, and synthesize a second strand but were unable to transcribe their genomes. Taken together, the phenotype of these mutants provides compelling evidence that the AAV capsid plays a role in the transcription of its genome, and the mutants map this functional region on the surface of the capsid near the 2-fold interface. This appears to be the first example of a viral structural protein that is also involved in the transcription of the viral genome that it delivers to the nucleus. IMPORTANCE Many viruses package enzymes within their capsids that assist in expressing their genomes postinfection, e.g., retroviruses. A number of nonenveloped viruses, including AAV, carry proteases that are needed for capsid maturation or for capsid modification during infection. We describe here what appears to

  19. Increased adenosine levels in mice expressing mutant glial fibrillary acidic protein in astrocytes result in failure of induction of LTP reversal (depotentiation) in hippocampal CA1 neurons.

    PubMed

    Fujii, Satoshi; Tanaka, Kenji F; Ikenaka, Kazuhiro; Yamazaki, Yoshihiko

    2014-08-26

    Astrocytes regulate the activity of neighboring neurons by releasing chemical transmitters, including ATP. Adenosine levels in the cerebrospinal fluid of mice that express a mutant human glial fibrillary acidic protein in astrocytes are slightly elevated compared to those in wild type mice and this might result from the observed increased release by mutant astrocytes of ATP, which can be used to produce adenosine. Using hippocampal slices from these mutant mice, we examined whether the increased endogenous adenosine levels in the hippocampus modulate the reversal of long-term potentiation (LTP), i.e. depotentiation (DP), in CA1 neurons. In hippocampal slices from wild type mice, a stable LTP was induced by tetanic stimulation consisting of 100 pulses at 100 Hz, and this was reversed by a train of low frequency stimulation (LFS) of 500 pulses at 1 Hz applied 30 min later. This induction of DP was inhibited by application of either 100 nM adenosine or 0.5 nM N(6)-cyclopentyladenosine, an adenosine A1 receptor agonist, during LFS, indicating that the increase in extracellular adenosine levels attenuated DP induction by acting on adenosine A1 receptors. In contrast, although a stable LTP was also induced in hippocampal slices from mutant mice, induction of DP was inhibited, but DP could be induced by application, during LFS, of 50 nM 8-cyclopentyltheophylline, an adenosine A1 receptor antagonist. These results suggest that a small increase in extracellular adenosine levels resulting from increased ATP release by astrocytes results in attenuation of DP in hippocampal CA1 neurons in the mutant mice.

  20. TARGETING THE ONCOGENIC MUC1-C PROTEIN INHIBITS MUTANT EGFR-MEDIATED SIGNALING AND SURVIVAL IN NON-SMALL CELL LUNG CANCER CELLS

    PubMed Central

    Kharbanda, Akriti; Rajabi, Hasan; Jin, Caining; Tchaicha, Jeremy; Kikuchi, Eiki; Wong, Kwok-Kin; Kufe, Donald

    2014-01-01

    Purpose Non-small cell lung cancers (NSCLC) that express the EGF receptor (EGFR) with activating mutations frequently develop resistance to EGFR kinase inhibitors. The mucin 1 (MUC1) heterodimeric protein is aberrantly overexpressed in NSCLC cells and confers a poor prognosis; however, the functional involvement of MUC1 in mutant EGFR signaling is not known. Experimental Design Targeting the oncogenic MUC1 C-terminal subunit (MUC1-C) in NSCLC cells harboring mutant EGFR was studied for effects on signaling, growth, clonogenic survival and tumorigenicity. Results Stable silencing of MUC1-C in H1975/EGFR(L858R/T790M) cells resulted in downregulation of AKT signaling and inhibition of growth, colony formation and tumorigenicity. Similar findings were obtained when MUC1-C was silenced in gefitinib-resistant PC9GR cells expressing EGFR(delE746_A750/T790M). The results further show that expression of a MUC1-C(CQC→AQA) mutant, which blocks MUC1-C homodimerization, suppresses EGFR(T790M), AKT and MEK→ERK activation, colony formation and tumorigenicity. In concert with these results, treatment of H1975 and PC9GR cells with GO-203, a cell-penetrating peptide that blocks MUC1-C homodimerization, resulted in inhibition of EGFR, AKT and MEK→ERK signaling and in loss of survival. Combination studies of GO-203 and afatinib, an irreversible inhibitor of EGFR, further demonstrate that these agents are synergistic in inhibiting growth of NSCLC cells harboring the activating EGFR(T790M) or EGFR(delE746-A750) mutants. Conclusions These findings indicate that targeting MUC1-C inhibits mutant EGFR signaling and survival, and thus represents a potential approach alone and in combination for the treatment of NSCLCs resistant to EGFR kinase inhibitors. PMID:25189483

  1. Increased adenosine levels in mice expressing mutant glial fibrillary acidic protein in astrocytes result in failure of induction of LTP reversal (depotentiation) in hippocampal CA1 neurons.

    PubMed

    Fujii, Satoshi; Tanaka, Kenji F; Ikenaka, Kazuhiro; Yamazaki, Yoshihiko

    2014-08-26

    Astrocytes regulate the activity of neighboring neurons by releasing chemical transmitters, including ATP. Adenosine levels in the cerebrospinal fluid of mice that express a mutant human glial fibrillary acidic protein in astrocytes are slightly elevated compared to those in wild type mice and this might result from the observed increased release by mutant astrocytes of ATP, which can be used to produce adenosine. Using hippocampal slices from these mutant mice, we examined whether the increased endogenous adenosine levels in the hippocampus modulate the reversal of long-term potentiation (LTP), i.e. depotentiation (DP), in CA1 neurons. In hippocampal slices from wild type mice, a stable LTP was induced by tetanic stimulation consisting of 100 pulses at 100 Hz, and this was reversed by a train of low frequency stimulation (LFS) of 500 pulses at 1 Hz applied 30 min later. This induction of DP was inhibited by application of either 100 nM adenosine or 0.5 nM N(6)-cyclopentyladenosine, an adenosine A1 receptor agonist, during LFS, indicating that the increase in extracellular adenosine levels attenuated DP induction by acting on adenosine A1 receptors. In contrast, although a stable LTP was also induced in hippocampal slices from mutant mice, induction of DP was inhibited, but DP could be induced by application, during LFS, of 50 nM 8-cyclopentyltheophylline, an adenosine A1 receptor antagonist. These results suggest that a small increase in extracellular adenosine levels resulting from increased ATP release by astrocytes results in attenuation of DP in hippocampal CA1 neurons in the mutant mice. PMID:25017946

  2. Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

    PubMed Central

    Saitoh, Aya; Takase, Tomoyuki; Kitaki, Hiroyuki; Miyazaki, Yuji; Kiyosue, Tomohiro

    2015-01-01

    Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation. PMID:26237185

  3. The effect of NaCl on stomatal opening in Arabidopsis wild type and agb1 heterotrimeric G-protein mutant plants.

    PubMed

    Yu, Yunqing; Assmann, Sarah M

    2016-01-01

    Salinity is a major agricultural problem that affects crop yield. Na(+) is transported to the shoot through the transpiration stream. The mutant of the sole Arabidopsis heterotrimeric G protein β subunit, agb1, is hypersensitive to salinity in part due to a higher transpiration rate. Here, we investigated the direct effect of Na(+) on stomatal opening using detached epidermal peels of wild type and agb1 plants. In both genotypes, NaCl is equally as effective as KCl in mediating stomatal opening at the concentrations tested. In both genotypes, ABA is less effective in inhibiting Na(+) mediated stomatal opening than K(+) mediated stomatal opening. The agb1 mutant is hyposensitive to ABA inhibition of K(+)-mediated but not Na(+)-mediated stomatal opening. These results suggest that the greater transpiration observed in agb1 plants grown in saline conditions is likely not mediated by differential genotypic direct effects of Na(+) on stomatal apertures. PMID:26431457

  4. Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice

    PubMed Central

    Pinto, Rute Maria; Caporale, Marco; Piras, Ilaria M.; Taggart, Aislynn; Seehusen, Frauke; Hahn, Kerstin; Janowicz, Anna; de Souza, William Marciel; Baumgärtner, Wolfgang; Shi, Xiaohong

    2016-01-01

    ABSTRACT Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro. Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. IMPORTANCE The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that

  5. DNMT3A R882 mutants interact with polycomb proteins to block haematopoietic stem and leukaemic cell differentiation

    PubMed Central

    Koya, Junji; Kataoka, Keisuke; Sato, Tomohiko; Bando, Masashige; Kato, Yuki; Tsuruta-Kishino, Takako; Kobayashi, Hiroshi; Narukawa, Kensuke; Miyoshi, Hiroyuki; Shirahige, Katsuhiko; Kurokawa, Mineo

    2016-01-01

    Despite the clinical impact of DNMT3A mutation on acute myeloid leukaemia, the molecular mechanisms regarding how this mutation causes leukaemogenesis in vivo are largely unknown. Here we show that, in murine transplantation experiments, recipients transplanted with DNMT3A mutant-transduced cells exhibit aberrant haematopoietic stem cell (HSC) accumulation. Differentiation-associated genes are downregulated without accompanying changes in methylation status of their promoter-associated CpG islands in DNMT3A mutant-transduced stem/progenitor cells, representing a DNA methylation-independent role of mutated DNMT3A. DNMT3A R882H also promotes monoblastic transformation in vitro in combination with HOXA9. Molecularly, the DNMT3A mutant interacts with polycomb repressive complex 1 (PRC1), causing transcriptional silencing, revealing a DNA methylation-independent role of DNMT3A mutation. Suppression of PRC1 impairs aberrant HSC accumulation and monoblastic transformation. From our data, it is shown that DNMT3A mutants can block the differentiation of HSCs and leukaemic cells via PRC1. This interaction could be targetable in DNMT3A-mutated leukaemias. PMID:27010239

  6. Physicochemical origin of high correlation between thermal stability of a protein and its packing efficiency: a theoretical study for staphylococcal nuclease mutants

    PubMed Central

    Oda, Koji; Kinoshita, Masahiro

    2015-01-01

    There is an empirical rule that the thermal stability of a protein is related to the packing efficiency or core volume of the folded state and the protein tends to exhibit higher stability as the backbone and side chains are more closely packed. Previously, the wild type and its nine mutants of staphylococcal nuclease were compared by examining their folded structures. The results obtained were as follows: The stability was not correlated with the number of intramolecular hydrogen bonds, intramolecular electrostatic interaction energy, or degree of burial of the hydrophobic surface; though the empirical rule mentioned above held, it was not the proximate cause of higher stability; and the number of van der Waals contacts NvdW, or equivalently, the intramolecular van der Waals interaction energy was an important factor governing the stability. Here we revisit the wild type and its nine mutants of staphylococcal nuclease using our statistical-mechanical theory for hydration of a protein. A molecular model is employed for water. We show that the pivotal factor is the magnitude of the water-entropy gain upon folding. The gain originates from an increase in the total volume available to the translational displacement of water molecules coexisting with the protein in the system. The magnitude is highly correlated with the denaturation temperature Tm. Moreover, the apparent correlation between NvdW and Tm as well as the empirical rule is interpretable (i.e., their physicochemical meanings can be clarified) on the basis of the water-entropy effect. PMID:27493849

  7. Identification and characterization of lysine-rich proteins and starch biosynthesis genes in the opaque2 mutant by transcriptional and proteomic analysis

    PubMed Central

    2013-01-01

    Background The opaque2 mutant is valuable for producing maize varieties with enhanced nutritional value. However, the exact mechanisms by which it improves protein quality and creates a soft endosperm texture are unclear. Given the importance of improving nutritional quality in grain crops, a better understanding of the physiological basis for these traits is necessary. Results In this study, we combined transcript profiling and proteomic analysis to better understand which genes and proteins are altered by opaque2 in the W64A inbred line. These analyses showed that the accumulation of some lysine-rich proteins, such as sorbitol dehydrogenase and glyceraldehyde3-phosphate dehydrogenase, was increased in mature kernels and may contribute substantially to the lysine content of opaque2 endosperm. Some defense proteins such as beta-glucosidase aggregating factor were strongly down regulated and may be regulated directly by opaque2. The mutant also had altered expression of a number of starch biosynthesis genes and this was associated with a more highly crystalline starch. Conclusions The results of these studies revealed specific target genes that can be investigated to further improve nutritional quality and agronomic performance of high lysine maize lines, particularly those based on the presence of the opaque2 mutation. Alteration of amylopectin branching patterns in opaque2 starch could contribute to generation of the soft, starchy endosperm. PMID:23586588

  8. Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression.

    PubMed

    Valenti, Fabio; Ganci, Federica; Fontemaggi, Giulia; Sacconi, Andrea; Strano, Sabrina; Blandino, Giovanni; Di Agostino, Silvia

    2015-03-20

    Genomic instability (IN) is a common feature of many human cancers. The TP53 tumour suppressor gene is mutated in approximately half of human cancers. Here, we show that BRCA1 and RAD17 genes, whose derived proteins play a pivotal role in DNA damage repair, are transcriptional targets of gain-of-function mutant p53 proteins. Indeed, high levels of mutp53 protein facilitate DNA damage accumulation and severely impair BRCA1 and RAD17 expression in proliferating cancer cells. The recruitment of mutp53/E2F4 complex onto specific regions of BRCA1 and RAD17 promoters leads to the inhibition of their expression. BRCA1 and RAD17 mRNA expression is reduced in HNSCC patients carrying TP53 mutations when compared to those bearing wt-p53 gene. Furthermore, the analysis of gene expression databases for breast cancer patients reveals that low expression of DNA repair genes correlates significantly with reduced relapse free survival of patients carrying TP53 gene mutations. Collectively, these findings highlight the direct involvement of transcriptionally active gain of function mutant p53 proteins in genomic instability through the impairment of DNA repair mechanisms.

  9. THI1, a protein involved in the biosynthesis of thiamin in Arabidopsis thaliana: structural analysis of THI1(A140V) mutant.

    PubMed

    Garcia, Assuero F; Dyszy, Fabio; Munte, Claudia E; Demarco, Ricardo; Beltramini, Leila M; Oliva, Glaucius; Costa-Filho, Antonio J; Araujo, Ana P U

    2014-06-01

    In eukaryotes, there are still steps of the vitamin B1 biosynthetic pathway not completely understood. In Arabidopsis thaliana, THI1 protein has been associated with the synthesis of the thiazole ring, a finding supported by the identification of a thiamine pyrophosphate (TPP)-like compound in its structure. Here, we investigated THI1 and its mutant THI1(A140V), responsible for the thiamin auxotrophy in a A. thaliana mutant line, aiming to clarify the impact of this mutation in the stability and activity of THI1. Recently, the THI1 orthologue (THI4) was revealed to be responsible for the donation of the sulfur atom from a cysteine residue to the thiazole ring in the thiamine intermediate. In this context, we carried out a cysteine quantification in THI1 and THI1(A140V) using electron spin resonance (ESR). These data showed that THI1(A140V) contains more sulfur-containing cysteines than THI1, indicating that the function as a sulfur donor is conserved, but the rate of donation reaction is somehow affected. Also, the bound compounds were isolated from both proteins and are present in different amounts in each protein. Unfolding studies presented differences in melting temperatures and also in the concentration of guanidine at which half of the protein unfolds, thus showing that THI1(A140V) has its conformational stability affected by the mutation. Hence, despite keeping its function in the early steps during the synthesis of TPP precursor, our studies have shown a decrease in the THI1(A140V) stability, which might be slowing down the biological activity of the mutant, and thus contributing to thiamin auxotrophy.

  10. A Screen for Dominant Negative Mutants of SEC18 Reveals a Role for the AAA Protein Consensus Sequence in ATP Hydrolysis

    PubMed Central

    Steel, Gregor J.; Harley, Carol; Boyd, Alan; Morgan, Alan

    2000-01-01

    An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum–Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure–function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr394→Pro). Thr394 is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast α-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts. PMID:10749934

  11. Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium

    PubMed Central

    Desbonnet, Charlene; Tait-Kamradt, Amelia; Garcia-Solache, Monica; Dunman, Paul; Coleman, Jeffrey; Arthur, Michel

    2016-01-01

    ABSTRACT The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins”) is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP). Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA. Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system. PMID:27048803

  12. The vhs1 mutant form of herpes simplex virus virion host shutoff protein retains significant internal ribosome entry site-directed RNA cleavage activity.

    PubMed

    Lu, P; Saffran, H A; Smiley, J R

    2001-01-01

    The virion host shutoff (vhs) protein of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated turnover of host and viral mRNAs during HSV infection. As well, it induces endoribonucleolytic cleavage of RNA substrates when produced in a rabbit reticulocyte lysate (RRL) in vitro translation system. The vhs1 point mutation (Thr 214-->Ile) eliminates vhs function during virus infection and in transiently transfected mammalian cells and was therefore previously considered to abolish vhs activity. Here we demonstrate that the vhs1 mutant protein induces readily detectable endoribonuclease activity on RNA substrates bearing the internal ribosome entry site of encephalomyocarditis virus in the RRL assay system. These data document that the vhs1 mutation does not eliminate catalytic activity and raise the possibility that the vhs-dependent endoribonuclease employs more than one mode of substrate recognition.

  13. Identification of Arabidopsis rat Mutants

    PubMed Central

    Zhu, Yanmin; Nam, Jaesung; Humara, Jaime M.; Mysore, Kirankumar S.; Lee, Lan-Ying; Cao, Hongbin; Valentine, Lisa; Li, Jingling; Kaiser, Anthony D.; Kopecky, Andrea L.; Hwang, Hau-Hsuan; Bhattacharjee, Saikat; Rao, Praveen K.; Tzfira, Tzvi; Rajagopal, Jyothi; Yi, HoChul; Veena; Yadav, Badam S.; Crane, Yan M.; Lin, Kui; Larcher, Yves; Gelvin, Matthew J.K.; Knue, Marnie; Ramos, Cynthia; Zhao, Xiaowen; Davis, Susan J.; Kim, Sang-Ic; Ranjith-Kumar, C.T.; Choi, Yoo-Jin; Hallan, Vipin K.; Chattopadhyay, Sudip; Sui, Xiangzhen; Ziemienowicz, Alicja; Matthysse, Ann G.; Citovsky, Vitaly; Hohn, Barbara; Gelvin, Stanton B.

    2003-01-01

    Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants. PMID:12805582

  14. Activation of Jun kinase/stress-activated protein kinase by GTPase-deficient mutants of G alpha 12 and G alpha 13.

    PubMed

    Prasad, M V; Dermott, J M; Heasley, L E; Johnson, G L; Dhanasekaran, N

    1995-08-01

    Signal transduction pathways regulated by G12 and G13 heterotrimeric G proteins are largely unknown. Expression of activated, GTPase-deficient mutants of alpha 12 and alpha 13 alter physiological responses such as Na+/H+ exchanger activity, but the effector pathways controlling these responses have not been defined. We have found that the expression of GTPase-deficient mutants of alpha 12 (alpha 12Q229L) or alpha 13 (alpha 13Q226L) leads to robust activation of the Jun kinase/stress-activated protein kinase (JNK/SAPK) pathway. Inducible alpha 12Q229L and alpha 13Q226L expression vectors stably transfected in NIH 3T3 cells demonstrated JNK/SAPK activation but not extracellular response/mitogen-activated protein kinase activation. Transient transfection of alpha 12Q229L and alpha 13Q226L also activated the JNK/SAPK pathway in COS-1 cells. Expression of the GTPase-deficient mutant of alpha q (alpha qQ209L) but not alpha i (alpha iQ205L) or alpha s (alpha sQ227L) was also able to activate the JNK/SAPK pathway. Functional Ras signaling was required for alpha 12Q229L and alpha 13Q226L activation of the JNK/SAPK pathway; expression of competitive inhibitory N17Ras inhibited JNK/SAPK activation in response to both alpha 12Q229L and alpha 13Q226L. The results describe for the first time a Ras-dependent signal transduction pathway involving JNK/SAPK regulated by alpha 12 and alpha 13. PMID:7629196

  15. Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

    PubMed

    Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

    2014-08-01

    5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

  16. Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases.

    PubMed Central

    Paravicini, G; Horazdovsky, B F; Emr, S D

    1992-01-01

    vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole. Images PMID:1498362

  17. Spermine Binding to Parkinson’s Protein α-Synuclein and its Disease-Related A30P and A53T Mutants

    PubMed Central

    Grabenauer, Megan; Bernstein, Summer L.; Lee, Jennifer C.; Wyttenbach, Thomas; Dupuis, Nicholas F.; Gray, Harry B.; Winkler, Jay R.; Bowers, Michael T.

    2008-01-01

    The aggregation of α-synuclein (α-syn), a protein implicated in Parkinson’s disease (PD), is believed to progress through the formation of a partially folded intermediate. Using nano-electrospray ionization (ESI) mass spectrometry combined with ion mobility measurements we found evidence for a highly compact partially folded family of structures for α-syn and its disease-related A53T mutant with net charges of −6, −7 and −8. For the other early-onset PD mutant, A30P, this highly compact population was only evident when the protein had a net charge of −6. When bound to spermine near physiologic pH, all three proteins underwent a charge reduction from the favored solution charge state of −10 to a net charge of −6. This charge reduction is accompanied by a dramatic size reduction of about a factor of two (cross section of 2600Å2 (−10 charge state) down to 1430Å2 (−6 charge state)). We conclude that spermine increases the aggregation rate of α-syn by inducing a collapsed conformation, which then proceeds to form aggregates. PMID:18693700

  18. A Truncated NLR Protein, TIR-NBS2, Is Required for Activated Defense Responses in the exo70B1 Mutant

    PubMed Central

    Nishimura, Marc T.; Vogel, John P.; Liu, Na; Liu, Simu; Zhao, Yaofei; Dangl, Jeffery L.; Tang, Dingzhong

    2015-01-01

    During exocytosis, the evolutionarily conserved exocyst complex tethers Golgi-derived vesicles to the target plasma membrane, a critical function for secretory pathways. Here we show that exo70B1 loss-of-function mutants express activated defense responses upon infection and express enhanced resistance to fungal, oomycete and bacterial pathogens. In a screen for mutants that suppress exo70B1 resistance, we identified nine alleles of TIR-NBS2 (TN2), suggesting that loss-of-function of EXO70B1 leads to activation of this nucleotide binding domain and leucine-rich repeat-containing (NLR)-like disease resistance protein. This NLR-like protein is atypical because it lacks the LRR domain common in typical NLR receptors. In addition, we show that TN2 interacts with EXO70B1 in yeast and in planta. Our study thus provides a link between the exocyst complex and the function of a ‘TIR-NBS only’ immune receptor like protein. Our data are consistent with a speculative model wherein pathogen effectors could evolve to target EXO70B1 to manipulate plant secretion machinery. TN2 could monitor EXO70B1 integrity as part of an immune receptor complex. PMID:25617755

  19. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells.

    PubMed

    Peterson, S R; Kurimasa, A; Oshimura, M; Dynan, W S; Bradbury, E M; Chen, D J

    1995-04-11

    The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an approximately 350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway.

  20. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells

    SciTech Connect

    Peterson, S.R. |; Kurimasa, Akihiro; Oshimura, Mitsuo; Dynan, W.S.; Bradbury, E.M. |; Chen, D.J.

    1995-04-11

    The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an {approx}350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway. 38 refs., 3 figs.

  1. Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

    PubMed

    Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

    2016-10-01

    We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression. PMID:27309759

  2. Decreased Amounts of Cell Wall-Associated Protein A and Fibronectin-Binding Proteins in Staphylococcus aureus sarA Mutants due to Up-Regulation of Extracellular Proteases

    PubMed Central

    Karlsson, Anna; Saravia-Otten, Patricia; Tegmark, Karin; Morfeldt, Eva; Arvidson, Staffan

    2001-01-01

    Data have been presented indicating that Staphylococcus aureus cell surface protein can be degraded by extracellular proteases produced by the same bacterium. We have found that in sarA mutant cells, which produce high amounts of four major extracellular proteases (staphylococcal serine protease [V8 protease] [SspA], cysteine protease [SspB], aureolysin [metalloprotease] [Aur], and staphopain [Scp]), the levels of cell-bound fibronectin-binding proteins (FnBPs) and protein A were very low compared to those of wild-type cells, in spite of unaltered or increased transcription of the corresponding genes. Cultivation of sarA mutant cells in the presence of the global protease inhibitor α2-macroglobulin resulted in a 16-fold increase in cell-bound FnBPs, indicating that extracellular proteases were responsible for the decreased amounts of FnBPs in sarA mutant cells. The protease inhibitor E64 had no effect on the level of FnBPs, indicating that cysteine proteases were not involved. Inactivation of either ssp or aur in the prototype S. aureus strain 8325-4 resulted in a threefold increase in the amount of cell-bound FnBPs. Inactivation of the same protease genes in a sarA mutant of 8325-4 resulted in a 10- to 20-fold increase in cell-bound protein A. As the serine protease requires aureolysin to be activated, it can thus be concluded that the serine protease is the most important protease in the release of cell-bound FnBPs and protein A. PMID:11447146

  3. Acyl protein thioesterase 1 and 2 (APT-1, APT-2) inhibitors palmostatin B, ML348 and ML349 have different effects on NRAS mutant melanoma cells.

    PubMed

    Vujic, Igor; Sanlorenzo, Martina; Esteve-Puig, Rosaura; Vujic, Marin; Kwong, Andrew; Tsumura, Aaron; Murphy, Ryan; Moy, Adrian; Posch, Christian; Monshi, Babak; Rappersberger, Klemens; Ortiz-Urda, Susana

    2016-02-01

    Oncogenic NRAS mutations are frequent in melanoma and lead to increased downstream signaling and uncontrolled cell proliferation. Since the direct inhibition of NRAS is not possible yet, modulators of NRAS posttranslational modifications have become an area of interest. Specifically, interfering with NRAS posttranslational palmitoylation/depalmitoylation cycle could disturb proper NRAS localization, and therefore decrease cell proliferation and downstream signaling. Here, we investigate the expression and function of NRAS depalmitoylating acyl protein thioesterases 1 and 2 (APT-1, APT-2) in a panel of NRAS mutant melanoma cells. First, we show that all melanoma cell lines examined express APT-1 and APT-2. Next, we show that siRNA mediated APT-1 and APT-2 knock down and that the specific APT-1 and -2 inhibitors ML348 and ML349 have no biologically significant effects in NRAS mutant melanoma cells. Finally, we test the dual APT-1 and APT-2 inhibitor palmostatin B and conclude that palmostatin B has effects on NRAS downstream signaling and cell viability in NRAS mutant melanoma cells, offering an interesting starting point for future studies. PMID:26771141

  4. Excitation energy transfer and charge separation are affected in Arabidopsis thaliana mutants lacking light-harvesting chlorophyll a/b binding protein Lhcb3.

    PubMed

    Adamiec, Małgorzata; Gibasiewicz, Krzysztof; Luciński, Robert; Giera, Wojciech; Chełminiak, Przemysław; Szewczyk, Sebastian; Sipińska, Weronika; van Grondelle, Rienk; Jackowski, Grzegorz

    2015-12-01

    The composition of LHCII trimers as well as excitation energy transfer and charge separation in grana cores of Arabidopsis thaliana mutant lacking chlorophyll a/b binding protein Lhcb3 have been investigated and compared to those in wild-type plants. In grana cores of lhcb3 plants we observed increased amounts of Lhcb1 and Lhcb2 apoproteins per PSII core. The additional copies of Lhcb1 and Lhcb2 are expected to substitute for Lhcb3 in LHCII trimers M as well as in the LHCII "extra" pool, which was found to be modestly enlarged as a result of the absence of Lhcb3. Time-resolved fluorescence measurements reveal a deceleration of the fast phase of excitation dynamics in grana cores of the mutant by ~15 ps, whereas the average fluorescence lifetime is not significantly altered. Monte Carlo modeling predicts a slowing down of the mean hopping time and an increased stabilization of the primary charge separation in the mutant. Thus our data imply that absence of apoprotein Lhcb3 results in detectable differences in excitation energy transfer and charge separation.

  5. Platelet endothelial cell adhesion molecule targeted oxidant-resistant mutant thrombomodulin fusion protein with enhanced potency in vitro and in vivo.

    PubMed

    Carnemolla, Ronald; Greineder, Colin F; Chacko, Ann-Marie; Patel, Kruti Rajan; Ding, Bi-Sen; Zaitsev, Sergei; Esmon, Charles T; Muzykantov, Vladimir R

    2013-11-01

    Thrombomodulin (TM) is a glycoprotein normally present in the membrane of endothelial cells that binds thrombin and changes its substrate specificity to produce activated protein C (APC) that has antithrombotic and anti-inflammatory features. To compensate for loss of endogenous TM in pathology, we have fused recombinant TM with single chain variable fragment (scFv) of an antibody to mouse platelet endothelial cell adhesion molecule-1 (PECAM). This fusion, anti-PECAM scFv/TM, anchors on the endothelium, stimulates APC production, and provides therapeutic benefits superior to sTM in animal models of acute thrombosis and inflammation. However, in conditions of oxidative stress typical of vascular inflammation, TM is inactivated via oxidation of the methionine 388 (M388) residue. Capitalizing on the reports that M388L mutation renders TM resistant to oxidative inactivation, in this study we designed a mutant anti-PECAM scFv/TM M388L. This mutant has the same APC-producing capacity and binding to target cells, yet, in contrast to wild-type fusion, it retains APC-producing activity in an oxidizing environment in vitro and in vivo. Therefore, oxidant resistant mutant anti-PECAM scFv/TM M388L is a preferable targeted biotherapeutic to compensate for loss of antithrombotic and anti-inflammatory TM functions in the context of vascular oxidative stress. PMID:23965383

  6. The structure of apo protein-tyrosine phosphatase 1B C215S mutant: More than just an S → O change

    PubMed Central

    Scapin, Giovanna; Patel, Sangita; Patel, Vira; Kennedy, Brian; Asante-Appiah, Ernest

    2001-01-01

    Protein-tyrosine phosphatases catalyze the hydrolysis of phosphate monoesters via a two-step mechanism involving a covalent phospho-enzyme intermediate. Biochemical and site-directed mutagenesis experiments show that the invariant Cys residue present in the PTPase signature motif (H/V)CX5R(S/T) (i.e., C215 in PTP1B) is absolutely required for activity. Mutation of the invariant Cys to Ser results in a catalytically inactive enzyme, which still is capable of binding substrates and inhibitors. Although it often is assumed that substrate-trapping mutants such as the C215S retain, in solution, the structural and binding properties of wild-type PTPases, significant differences have been found in the few studies that have addressed this issue, suggesting that the mutation may lead to structural/conformational alterations in or near the PTP1B binding site. Several crystal structures of apo-WT PTP1B, and of WT- and C215S-mutant PTP1B in complex with different ligands are available, but no structure of the apo-PTP1B C215S has ever been reported. In all previously reported structures, residues of the PTPase signature motif have an identical conformation, while residues of the WPD loop (a surface loop which includes the catalytic Asp) assume a different conformation in the presence or absence of ligand. These observations led to the hypothesis that the different spectroscopic and thermodynamic properties of the mutant protein may be the result of a different conformation for the WPD loop. We report here the structure of the apo-PTP1B C215S mutant, which reveals that, while the WPD loop is in the open conformation observed in the apo WT enzyme crystal structure, the residues of the PTPases signature motif are in a dramatically different conformation. These results provide a structural basis for the differences in spectroscopic properties and thermodynamic parameters in inhibitor binding observed for the wild-type and mutant enzymes. PMID:11468356

  7. Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT

    SciTech Connect

    Liu, Fabao; You, Xiaona; Chi, Xiumei; Wang, Tao; Ye, Lihong; Niu, Junqi; Zhang, Xiaodong

    2014-02-07

    Highlights: • Relative to wild type HBx, HBX mutant HBxΔ127 strongly enhances cell proliferation. • Relative to wild type HBx, HBxΔ127 remarkably up-regulates miR-215 in hepatoma cells. • HBxΔ127-elevated miR-215 promotes cell proliferation via targeting PTPRT mRNA. - Abstract: The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.

  8. Rer1 and calnexin regulate endoplasmic reticulum retention of a peripheral myelin protein 22 mutant that causes type 1A Charcot-Marie-Tooth disease

    PubMed Central

    Hara, Taichi; Hashimoto, Yukiko; Akuzawa, Tomoko; Hirai, Rika; Kobayashi, Hisae; Sato, Ken

    2014-01-01

    Peripheral myelin protein 22 (PMP22) resides in the plasma membrane and is required for myelin formation in the peripheral nervous system. Many PMP22 mutants accumulate in excess in the endoplasmic reticulum (ER) and lead to the inherited neuropathies of Charcot-Marie-Tooth (CMT) disease. However, the mechanism through which PMP22 mutants accumulate in the ER is unknown. Here, we studied the quality control mechanisms for the PMP22 mutants L16P and G150D, which were originally identified in mice and patients with CMT. We found that the ER-localised ubiquitin ligase Hrd1/SYVN1 mediates ER-associated degradation (ERAD) of PMP22(L16P) and PMP22(G150D), and another ubiquitin ligase, gp78/AMFR, mediates ERAD of PMP22(G150D) as well. We also found that PMP22(L16P), but not PMP22(G150D), is partly released from the ER by loss of Rer1, which is a Golgi-localised sorting receptor for ER retrieval. Rer1 interacts with the wild-type and mutant forms of PMP22. Interestingly, release of PMP22(L16P) from the ER was more prominent with simultaneous knockdown of Rer1 and the ER-localised chaperone calnexin than with the knockdown of each gene. These results suggest that CMT disease-related PMP22(L16P) is trapped in the ER by calnexin-dependent ER retention and Rer1-mediated early Golgi retrieval systems and partly degraded by the Hrd1-mediated ERAD system. PMID:25385046

  9. Reduced Dosage of pos-1 Suppresses Mex Mutants and Reveals Complex Interactions Among CCCH Zinc-Finger Proteins During Caenorhabditis elegans Embryogenesis

    PubMed Central

    Tenlen, Jennifer R.; Schisa, Jennifer A.; Diede, Scott J.; Page, Barbara D.

    2006-01-01

    Cell fate specification in the early C. elegans embryo requires the activity of a family of proteins with CCCH zinc-finger motifs. Two members of the family, MEX-5 and MEX-6, are enriched in the anterior of the early embryo where they inhibit the accumulation of posterior proteins. Embryos from mex-5 single-mutant mothers are inviable due to the misexpression of SKN-1, a transcription factor that can specify mesoderm and endoderm. The aberrant expression of SKN-1 causes a loss of hypodermal and neuronal tissue and an excess of pharyngeal muscle, a Mex phenotype (muscle excess). POS-1, a third protein with CCCH motifs, is concentrated in the posterior of the embryo where it restricts the expression of at least one protein to the anterior. We discovered that reducing the dosage of pos-1(+) can suppress the Mex phenotype of mex-5(−) embryos and that POS-1 binds the 3′-UTR of mex-6. We propose that the suppression of the Mex phenotype by reducing pos-1(+) is due to decreased repression of mex-6 translation. Our detailed analyses of these protein functions reveal complex interactions among the CCCH finger proteins and suggest that their complementary expression patterns might be refined by antagonistic interactions among them. PMID:17028349

  10. Use of a Cellulase-Derepressed Mutant of Cellulomonas in the Production of a Single-Cell Protein Product from Cellulose †

    PubMed Central

    Hitchner, E. V.; Leatherwood, J. M.

    1980-01-01

    A cellulase-derepressed mutant of a Cellulomonas species was used to produce single-cell protein from crystalline cellulose. In preliminary tests, maximum yield of single-cell protein was obtained at 30°C (pH 7.0) with urea as the nitrogen source. A continuous-flow foam flotation procedure was developed for rapid and efficient separation of bacteria from the culture liquid and cellulose residue. A pH of 4.5 was optimum for foam flotation of this organism. In preliminary trials, recovery was 85% of the cells with the flotation procedure. Cellulomonas was 68% true protein and had an essential amino acid profile featuring a high lysine content (6.5% of protein). The Cellulomonas product was evaluated nutritionally with weanling rats. The net protein utilization value for the protein supplemented with methionine was 50.4% Weight gain of rats on the Cellulomonas diet was similar to that of rats fed a casein diet. PMID:16345511

  11. Characterization of three putative Lon proteases of Thermus thermophilus HB27 and use of their defective mutants as hosts for production of heterologous proteins.

    PubMed

    Maehara, Tomoko; Hoshino, Takayuki; Nakamura, Akira

    2008-03-01

    In the genome of a thermophilic bacterium, Thermus thermophilus HB27, three genes, TTC0418, TTC0746 and TTC1975, were annotated as ATP-dependent protease La (Lon). Sequence comparisons indicated that TTC0418 and TTC0746 showed significant similarities to bacterial LonA-type proteases, such as Escherichia coli Lon protease, especially in regions corresponding to domains for ATP-binding and hydrolysis, and for proteolysis, but TTC1975 exhibited a similarity only at the C-terminal proteolytic domain. The enzymatic analyses, using purified recombinant proteins produced by E. coli, revealed that TTC0418 and TTC0746 exhibited peptidase and protease activities against two synthetic peptides and casein, respectively, in an ATP-dependent manner, and at the same time, both the enzymes had significant ATPase activities in the presence of substrates. On the other hand, TTC1975 possessed a protease activity against casein, but addition of ATP did not enhance this activity. Moreover, a T. thermophilus mutant deficient in both TTC0418 and TTC0746 showed a similar growth characteristic to an E. coli lon mutant, i.e., a growth defect lag after a nutritional downshift. These results indicate that TTC0418 and TTC0746 are actually members of bacterial LonA-type proteases with different substrate specificities, whereas TTC1975 should not be classified as a Lon protease. Finally, the effects of mutations deficient in these proteases were assessed on production of several heterologous gene products from Pyrococcus horikoshii and Geobacillus stearothermophilus. It was shown that TTC0746 mutation was more effective in improving production than the other two mutations, especially for production of P. horikoshii alpha-mannosidase and G. stearothermophilus alpha-amylase, indicating that the TTC0746 mutant of T. thermophilus HB27 may be useful for production of heterologous proteins from thermophiles and hyperthermophiles. PMID:18157502

  12. A missense mutation in the VHYNP motif of a DELLA protein causes a semi-dwarf mutant phenotype in Brassica napus.

    PubMed

    Liu, Chao; Wang, Jilin; Huang, Tiandai; Wang, Fang; Yuan, Fang; Cheng, Xiaomao; Zhang, Yan; Shi, Shuwen; Wu, Jiangsheng; Liu, Kede

    2010-07-01

    Although dwarf genes have been widely used to improve lodging resistance and enhance harvest index in cereal crops, lodging is still a serious problem in rapeseed (Brassica napus) production. A semi-dwarf B. napus mutant, ds-1, was identified through EMS mutagenesis of a microspore-cultured DH line. The mutant had a significant reduction in height due to a lower first branch position and shorter internodes when compared with wild-type cultivars. This dwarfism was inherited as a single semi-dominant gene, ds-1. DS-1 locus was mapped to chromosome A6, and co-segregated with a microsatellite marker BnEMS1125 derived from the gene BnRGA. BnRGA encodes a DELLA protein that functions as a GA signaling repressor. The expression of a mutant BnRGA allele from ds-1, Bnrga-ds, caused dwarf phenotypes in Arabidopsis. Comparative sequencing of RGA open-reading frames (ORFs) of ds-1 and wild-type cultivars revealed a single proline (P)-to-leucine (L) substitution that may lead to a gain-of-function mutation in GA signaling. The expression of the Arabidopsis homolog, Atrga-ds, bearing this site-directed mutation also rendered dwarf phenotypes in Arabidopsis, which demonstrated that the P-to-L mutation in the VHYNP motif of Bnrga-ds is responsible for the dwarfism. A yeast two-hybrid assay confirmed that this mutation inhibited the interaction between Bnrga-ds/Atrga-ds and the GA receptor, AtGID1A, in the presence of GA(3), suggesting that the conserved proline residue in the VHYNP motif of DELLA protein directly participates in DELLA-GID1 interaction. Identification and characterization of the dwarf gene ds-1 will facilitate its utilization in improving lodging resistance in Brassica breeding.

  13. Genetic Complementation and Kinetic Analyses of Rhodobacter capsulatus ORF1696 Mutants Indicate that the ORF1696 Protein Enhances Assembly of the Light-Harvesting I Complex

    PubMed Central

    Young, C. S.; Reyes, R. C.; Beatty, J. T.

    1998-01-01

    Rhodobacter capsulatus ORF1696 mutant strains were created by insertion of antibiotic resistance cartridges at different sites within the ORF1696 gene in a strain that lacks the light-harvesting II (LHII) complex. Steady-state absorption spectroscopy profiles and the kinetics of the light-harvesting I (LHI) complex assembly and decay were used to evaluate the function of the ORF1696 protein in various strains. All of the mutant strains were found to be deficient in the LHI complex, including one (ΔNae) with a disruption located 13 codons before the 3′ end of the gene. A 5′-proximal disruption after the 31st codon of ORF1696 resulted in a mutant strain (ΔMun) with a novel absorption spectrum. The two strains with more 3′ disruptions (ΔStu and ΔNae) were restored nearly to the parental strain phenotype when trans complemented with a plasmid expressing the ORF1696 gene, but ΔMun was not. The absorption spectrum of ΔMun resembled that of a strain which had a polar mutation in ORF1696. We suggest that a rho-dependent transcription termination site exists between the MunI and proximal StuI sites of ORF1696. A comparison of LHI complex assembly kinetics showed that assembly occurred 2.6-fold faster in the parental strain than in strain ΔStu. In contrast, LHI complex decay occurred 1.7-fold faster in the ORF1696 parental strain than in ΔStu. These results indicate that the ORF1696 protein has a major effect on LHI complex assembly, and models of ORF1696 function are proposed. PMID:9537372

  14. Transcriptional profiling reveals differential expression of a neuropeptide-like protein and pseudogenes in aryl hydrocarbon receptor-1 mutant Caenorhabditis elegans.

    PubMed

    Aarnio, Vuokko; Heikkinen, Liisa; Peltonen, Juhani; Goldsteins, Gundars; Lakso, Merja; Wong, Garry

    2014-03-01

    The aryl hydrocarbon receptor (AHR) functions in higher organisms in development, metabolism and toxic responses. Its Caenorhabditis elegans (C. elegans) ortholog, AHR-1, facilitates neuronal development, growth and movement. We investigated the effect of AHR mutation on the transcriptional profile of L4 stage C. elegans using RNA-seq and quantitative real time PCR in order to understand better AHR-1 function at the genomic level. Illumina HiSeq 2000 sequencing yielded 51.1, 61.2 and 54.0 million reads from wild-type controls, ahr-1(ia03) and ahr-1(ju145) mutants, respectively, providing detection of over 18,000 transcripts in each sample. Fourteen transcripts were over-expressed and 125 under-expressed in both ahr-1 mutants when compared to wild-type. Under-expressed genes included soluble guanylate cyclase (gcy) family genes, some of which were previously demonstrated to be regulated by AHR-1. A neuropeptide-like protein gene, nlp-20, and a F-box domain protein gene fbxa-192 and its pseudogenes fbxa-191 and fbxa-193 were also under-expressed. Conserved xenobiotic response elements were identified in the 5' flanking regions of some but not all of the gcy, nlp-20, and fbxa genes. These results extend previous studies demonstrating control of gcy family gene expression by AHR-1, and furthermore suggest a role of AHR-1 in regulation of a neuropeptide gene as well as pseudogenes.

  15. Impaired Acid Catalysis by Mutation of a Protein Loop Hinge Residue in a YopH Mutant Revealed by Crystal Structures

    SciTech Connect

    Brandao, T.; Robinson, H; Johnson, S; Hengge, A

    2009-01-01

    Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presence of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.

  16. Examination of stability of mutant photosynthetic reaction center of Rhodobacter sphaeroides I(L177)H and determination of location of bacteriochlorophyll covalently bound to the protein.

    PubMed

    Fufina, T Y; Vasilieva, L G; Shuvalov, V A

    2010-02-01

    We demonstrated earlier that as a result of the I(L177)H mutation in the photosynthetic reaction center (RC) of the bacterium Rhodobacter sphaeroides, one of the bacteriochlorophylls (BChl) binds with the L-subunit, simultaneously raising coordination stability of the central magnesium atom of the bacteriochlorophyll associated with the protein. In this study, spectral properties of wild type RC and I(L177)H in the presence of urea and SDS as well as at 48 degrees C were examined. It is shown that the I(L177)H mutation decreases the RC stability. Under denaturing conditions, some changes indicating breakdown of oligomeric structure of the complex and loss of interaction between pigments and their protein environment are observed in I(L177)H RC spectra. In addition, pheophytinization of bacteriochlorophylls occurs in both types of RC in the presence of SDS. However, an 811-nm band is observed in the spectrum of the mutant RC under these conditions, which indicates retention of one of the BChl molecules in the protein binding site and stable coordination of its central magnesium atom. It is shown that in both types of RC, monomeric BChl B(B) can be modified by sodium borohydride treatment and then extracted by acetone-methanol mixture. Spectral properties of the BChl covalently bound with the protein in I(L177)H RC do not change. The results demonstrate that BChl P(A) is the molecule of BChl tightly bound with the L-subunit in mutant RC as it was supposed earlier. PMID:20367608

  17. A novel mutant 10Ala/Arg together with mutant 144Ser/Arg of hepatitis B virus X protein involved in hepatitis B virus-related hepatocarcinogenesis in HepG2 cell lines.

    PubMed

    Shi, Ying; Wang, Junwei; Wang, Yuhe; Wang, Anna; Guo, Hongliang; Wei, Feili; Mehta, Sanjay R; Espitia, Stephen; Smith, Davey M; Liu, Longgen; Zhang, Yulin; Chen, Dexi

    2016-02-28

    Hepatitis B virus (HBV) infection-related hepatocellular carcinoma (HCC) represents a major health problem worldwide. HBV X (HBx) protein is the most common open reading frame that may undergo mutations, resulting in the development of HCC. This study aimed to determine specific HBx mutations that differentiate the central- and para-tumor tissues, and identify their association with HCC development. HBx gene from HCC tumor and para-tumor tissues of 47 HCC patients was amplified, sequenced and statistically analyzed. A novel combination of 2 mutations at residues 10 and 144 was identified which might play a significant role in HCC development. Expression vectors carrying HBx with the specific mutations were constructed and transfected into HepG2 and p53-null HepG2 cells. Compared to wild type (WT) and single mutation of HBx at residue 10 or 144, the 10/144 double mutations strongly up-regulated p21 expression and prolonged G1/S transition in WT- and p53-null HepG2 cells. Apoptosis was also inhibited by HBx harboring 10/44 double-mutation. Binding of 10/144 double-mutant HBx to p53 was lower than WT HBx. Conclusively, the 10/144 double mutation of HBx might play a crucial role in HCC formation.

  18. Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of the current study was to evaluate the His-tag pr...

  19. A relaxed (rel) mutant of Streptomyces coelicolor A3(2) with a missing ribosomal protein lacks the ability to accumulate ppGpp, A-factor and prodigiosin.

    PubMed

    Ochi, K

    1990-12-01

    A relaxed (rel) mutant was found among 70 thiopeptin-resistant isolates of Streptomyces coelicolor A3(2) which arose spontaneously. The ability of the rel mutant to accumulate ppGpp during Casamino acid deprivation was reduced 10-fold compared to the wild-type. Analysis of the ribosomal proteins by two-dimensional PAGE revealed that the mutant lacked a ribosomal protein, tentatively designated ST-L11. It was therefore classified as a relC mutant. The mutant was defective in producing A-factor and the pigmented antibiotic prodigiosin, in both liquid and agar cultures, but produced agarase normally. Production of actinorhodin, another pigmented antibiotic, was also abnormal; it appeared suddenly in agar cultures after 10 d incubation. Although aerial mycelium still formed, its appearance was markedly delayed. Whereas liquid cultures of the parent strain accumulated ppGpp, agar cultures accumulated only trace amounts. Instead, a substance characterized only as an unidentified HPLC peak accumulated intracellularly in the late growth phase, just before aerial mycelium formation and antibiotic production. This substance did not accumulate in mutant cells. It was found in S. lividans 66 and S. parvulus, but not in seven other Streptomyces species tested. The significance of these observations, and the relationship of the mutant to earlier rel isolates of Streptomyces is discussed.

  20. Sequence-only evolutionary and predicted structural features for the prediction of stability changes in protein mutants

    PubMed Central

    2013-01-01

    Background Even a single amino acid substitution in a protein sequence may result in significant changes in protein stability, structure, and therefore in protein function as well. In the post-genomic era, computational methods for predicting stability changes from only the sequence of a protein are of importance. While evolutionary relationships of protein mutations can be extracted from large protein databases holding millions of protein sequences, relevant evolutionary features for the prediction of stability changes have not been proposed. Also, the use of predicted structural features in situations when a protein structure is not available has not been explored. Results We proposed a number of evolutionary and predicted structural features for the prediction of stability changes and analysed which of them capture the determinants of protein stability the best. We trained and evaluated our machine learning method on a non-redundant data set of experimentally measured stability changes. When only the direction of the stability change was predicted, we found that the best performance improvement can be achieved by the combination of the evolutionary features mutation likelihood and SIFTscore in conjunction with the predicted structural feature secondary structure. The same two evolutionary features in the combination with the predicted structural feature accessible surface area achieved the lowest error when the prediction of actual values of stability changes was assessed. Compared to similar studies, our method achieved improvements in prediction performance. Conclusion Although the strongest feature for the prediction of stability changes appears to be the vector of amino acid identities in the sequential neighbourhood of the mutation, the most relevant combination of evolutionary and predicted structural features further improves prediction performance. Even the predicted structural features, which did not perform well on their own, turn out to be beneficial

  1. EXAFS of Klebsiella pneumoniae nitrogenase MoFe protein from wild-type and nif V mutant strains

    SciTech Connect

    Eidsness, M.K.; Flank, A.M.; Smith, B.E.; Flood, A.C.; Garner, C.D.; Cramer. S.P.

    1986-05-14

    The enzyme nitrogenase catalyzes the biological reduction of N/sub 2/ to NH/sub 3/. In Klebsiella pneumoniae a cluster of 17 genes in seven transcriptional units has been associated with nitrogen fixation. The nitrogenase enzyme from the nif V mutants is relatively ineffective at dinitrogen reduction, is more efficient than the wild-type enzyme at HCN reduction, and has its hydrogen evolution activity inhibited up to 80% by CO. This altered substrate specificity has been shown to be associated with the iron-molybdenum cofactor, FeMo-co, of the enzyme. X-ray absorption spectroscopy has been a valuable tool for probing the molybdenum environment of wild-type nitrogenase, and the authors report here similar studies on the Nif V/sup -/ enzyme.

  2. The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins.

    PubMed Central

    Danielsen, M; Northrop, J P; Ringold, G M

    1986-01-01

    We have isolated mouse glucocorticoid receptor (GR) cDNAs which, when expressed in transfected mammalian cells, produce a fully functional GR protein. Sequence analysis reveals an open reading frame of 2349 bp which could encode a protein of approximately 86,000 daltons. We have also isolated two receptor cDNAs from mouse S49 nuclear transfer-deficient (nt-) cells which encode mutant forms of the receptor protein. One cDNA encodes a protein that is unable to bind hormone and represents the endogenous hormone binding deficient receptor recently discovered in S49 cells. The lesion in this receptor is due to a single amino acid substitution (Glu-546 to Gly). The second cDNA from nt- cells produces a receptor protein that is able to bind hormone but has reduced nuclear binding. This cDNA, therefore, encodes for the S49 nt- receptor which has been shown to have reduced affinity for DNA. The lesion maps to a single amino acid substitution (Arg-484 to His) located in a highly Cys, Lys, Arg-rich region of the protein previously implicated in DNA binding. Our studies provide unambiguous identification of receptor domains and specific amino acids critical for the hormone and DNA binding properties of this transcriptional regulatory protein. Contained within the first 106 amino acids of the mouse GR is a stretch of nine glutamines with two prolines which are related to the family of transcribed repetitive elements, opa, found in Drosophila melanogaster. A truncated receptor lacking these 106 amino acids is functionally indistinguishable from the wild-type receptor. Images Fig. 2. Fig. 5. Fig. 6. Fig. 7. PMID:3780669

  3. Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants.

    PubMed

    Farias, Davi F; Peijnenburg, Ad A C M; Grossi-de-Sá, Maria F; Carvalho, Ana F U

    2015-01-01

    Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular evolution to the cry8Ka1 gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC50 2.83 μg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods. PMID:26513483

  4. Food safety knowledge on the Bt mutant protein Cry8Ka5 employed in the development of coleopteran-resistant transgenic cotton plants.

    PubMed

    Farias, Davi F; Peijnenburg, Ad A C M; Grossi-de-Sá, Maria F; Carvalho, Ana F U

    2015-01-01

    Insecticidal Cry proteins from Bacillus thuringiensis (Bt) have been exploited in the development of genetically modified (GM) crops for pest control. However, several pests are still difficult to control such as the coleopteran boll weevil Anthonomus grandis. By applying in vitro molecular evolution to the cry8Ka1 gene sequence, variants were generated with improved activity against A. grandis. Among them, Cry8Ka5 mutant protein showed coleoptericidal activity 3-fold higher (LC50 2.83 μg/mL) than that of the original protein (Cry8Ka1). Cry8Ka5 has been used in breeding programs in order to obtain coleopteran-resistant cotton plants. Nevertheless, there is some concern in relation to the food safety of transgenic crops, especially to the heterologously expressed proteins. In this context, our research group has performed risk assessment studies on Cry8Ka5, using the tests recommended by Codex as well as tests that we proposed as alternative and/or complementary approaches. Our results on the risk analysis of Cry8Ka5 taken together with those of other Cry proteins, point out that there is a high degree of certainty on their food safety. It is reasonable to emphasize that most safety studies on Cry proteins have essentially used the Codex approach. However, other methodologies would potentially provide additional information such as studies on the effects of Cry proteins and derived peptides on the indigenous gastrointestinal microbiota and on intestinal epithelial cells of humans. Additionally, emerging technologies such as toxicogenomics potentially will offer sensitive alternatives for some current approaches or methods.

  5. Ras-mutant cancer cells display B-Raf binding to Ras that activates extracellular signal-regulated kinase and is inhibited by protein kinase A phosphorylation.

    PubMed

    Li, Yanping; Takahashi, Maho; Stork, Philip J S

    2013-09-20

    The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.

  6. Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters

    PubMed Central

    Shirogane, Yuta; Suzuki, Satoshi O.; Ikegame, Satoshi; Koga, Ritsuko

    2013-01-01

    Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein. PMID:23255801

  7. Changes of alternative oxidase activity, capacity and protein content in leaves of Cucumis sativus wild-type and MSC16 mutant grown under different light intensities.

    PubMed

    Florez-Sarasa, Igor; Ostaszewska, Monika; Galle, Alexander; Flexas, Jaume; Rychter, Anna M; Ribas-Carbo, Miquel

    2009-12-01

    In vitro studies demonstrated that alternative oxidase (AOX) is biochemically regulated by a sulfhydryl-disulfide system, interaction with alpha-ketoacids, ubiquinone pool redox state and protein content among others. However, there is still scarce information about the in vivo regulation of the AOX. Cucumis sativus wild-type (WT) and MSC16 mutant plants were grown under two different light intensities and were used to analyze the relationship between the amount of leaf AOX protein and its in vivo capacity and activity at night and day periods. WT and MSC16 plants presented lower total respiration (V(t)), cytochrome oxidase pathway (COP) activity (v(cyt)) and alternative oxidase pathway (AOP) activity (v(alt)) when grown at low light (LL), although growth light intensity did not change the amount of cytochrome oxidase (COX) nor AOX protein. Changes of v(cyt) related to growing light conditions suggested a substrate availability and energy demand control. On the other hand, the total amount of AOX protein present in the tissue does not play a role in the regulation neither of the capacity nor of the activity of the AOP in vivo. Soluble carbohydrates were directly related to the activity of the AOP. However, although differences in soluble sugar contents mostly regulate the capacity of the AOP at different growth light intensities, additional regulatory mechanisms are necessary to fully explain the observed results.

  8. Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry.

    PubMed

    Chen, Hang; Hsiao, Yung-Chin; Chiang, Sum-Fu; Wu, Chia-Chun; Lin, Yu-Tsun; Liu, Hsuan; Zhao, Hong; Chen, Jinn-Shiun; Chang, Yu-Sun; Yu, Jau-Song

    2016-08-24

    The BRAF V600E mutation is one of the most common mutations implicated in the development of several types of cancer including colorectal cancer (CRC), where it is associated with aggressive disease phenotypes and poor outcomes. The status of the BRAF V600E mutation is frequently determined by direct DNA sequencing. However, no previous study has sought to quantify the BRAF V600E protein in cancer specimens. Here, we evaluated immunoenrichment coupled with two MS-based quantitative techniques, namely multiple reaction monitoring (MRM) and single ion monitoring conjugated accurate inclusion mass screening (SIM-AIMS), to detect and precisely quantify wild-type (WT) and V600E mutant BRAF proteins in DNA sequence-confirmed CRC tissue specimens. WT and V600E BRAF proteins were immunoprecipitated from a CRC cell line (HT-29), and their representative peptides ((592)IGDFGLATVK(601) and (592)IGDFGLATEK(601), respectively) were confirmed by LC-MS/MS analysis and then quantified by MRM or SIM-AIMS with spiked stable isotope-labeled peptide standards. Both assays worked well for measuring WT BRAF from different amounts of HT-29 cell lysates, but the MRM assay was more sensitive than SIM-AIMS assay for quantifying lower levels of V600E BRAF. In protein extracts (2 mg) from 11 CRC tissue specimens, the MRM assay could measure WT BRAF in all 11 cases (0.32-1.66 ng) and the V600E BRAF in two cases (0.1-0.13 ng; mutant-to-WT ratio, 0.16-0.17). The SIM-AIMS assay could also detect WT and V600E BRAF in CRC specimens, but the measured levels of both targets were lower than those determined by MRM assay. Collectively, this study provides an effective method to precisely quantify WT and V600E BRAF proteins in complex biological samples using immunoenrichment-coupled targeted MS. Since the V600E BRAF protein has emerged as an important therapeutic target for cancer, the developed assay should facilitate future BRAF-related basic and clinical studies. PMID:27497007

  9. Zinc site redesign in T4 gene 32 protein: structure and stability of cobalt(II) complexes formed by wild-type and metal ligand substitution mutants.

    PubMed

    Guo, J; Giedroc, D P

    1997-01-28

    Phage T4 gene 32 protein (gp32) is a zinc metalloprotein which binds cooperatively and preferentially to single-stranded nucleic acids and functions as a replication and recombination accessory protein. Zn(II) coordination by gp32 employs a His-Cys3 metal ligand donor set derived from the His64-X12-Cys77-X9-Cys87-X2-Cys90 sequence in the ssDNA-binding core domain of the molecule. Crystallographic studies reveal that His64 and Cys77 are derived from two independent beta-strands within a distorted three-stranded beta-sheet and are relatively more buried from solvent than are Cys87 and Cys90, which are positioned immediately before and within, respectively, an alpha-helix. In an effort to understand the origin of the stability of the metal complex, we have employed an anaerobic optical spectroscopic, competitive metal binding assay to determine the coordination geometry and association constants (Ka) for the binding of Co(II) to wild-type gp32 and a series of zinc ligand substitution mutants. At pH 7.5, 25 degrees C, wild-type gp32 binds Co(II) with a Ka approximately 1 x 10(9) M-1. Competition experiments reveal that Ka for Zn(II) is 3.0 (+/-1.0) x 10(11) M-1. We find that all non-native metal complexes retain tetrahedral or distorted tetrahedral coordination geometry but are greatly destabilized in a manner essentially of whether a new protein-derived coordination bond is formed (e.g., in H64C gp32) or not. Co(II) binding isotherms obtained for three His64 substitution mutants, H64C, H64D, and H64N gp32s, suggest that each mutant forms a dimeric Cys4 tetrathiolate intermediate complex at limiting [Co(II)]f, each then rearranges at high [Co(II)]f to form a monomolecular site of the expected geometry and Ka approximately 1 x 10(4) M-1. Like the His64 mutants, C77A gp32 appears to form at least two types of complexes over the course of a Co(II) titration: one with octahedral coordination geometry formed at low [Co(II)]f, with a second tetrahedral or five

  10. The α-subunit of the rice heterotrimeric G protein, RGA1, regulates drought tolerance during the vegetative phase in the dwarf rice mutant d1.

    PubMed

    Ferrero-Serrano, Ángel; Assmann, Sarah M

    2016-05-01

    Essential in the Green Revolution was the development of high-yielding dwarf varieties of rice (Oryza sativa L.), but their selection was not based on responses to water limitation. We studied physiological responses to progressive drought of the dwarf rice mutant, d1, in which the RGA1 gene, which encodes the GTP-binding α-subunit of the heterotrimeric G protein, is non-functional. Wild-type (WT) plants cease net carbon fixation 11 days after water is withheld, while d1 plants maintain net photosynthesis for an additional week. During drought, d1 plants exhibit greater stomatal conductance than the WT, but both genotypes exhibit the same transpirational water loss per unit leaf area. This is explained by a smaller driving force for water loss in d1 owing to its lower leaf temperatures, consistent with its more erect architecture. As drought becomes more severe, WT plants show an accelerated decline in photosynthesis, which may be exacerbated by the higher leaf temperatures in the WT. We thus show how a rice mutant with dwarf and erect leaves has a decreased susceptibility to water stress. Accordingly, it may be useful to incorporate RGA1 mutation in breeding or biotechnological strategies for development of drought-resistant rice. PMID:27194741

  11. DnaK dependence of mutant ethanol oxidoreductases evolved for aerobic function and protective role of the chaperone against protein oxidative damage in Escherichia coli

    PubMed Central

    Echave, Pedro; Esparza-Cerón, M. Angel; Cabiscol, Elisa; Tamarit, Jordi; Ros, Joaquim; Membrillo-Hernández, Jorge; Lin, E. C. C.

    2002-01-01

    The adhE gene of Escherichia coli encodes a multifunctional ethanol oxidoreductase (AdhE) that catalyzes successive reductions of acetyl-CoA to acetaldehyde and then to ethanol reversibly at the expense of NADH. Mutant JE52, serially selected for acquired and improved ability to grow aerobically on ethanol, synthesized an AdhEA267T/E568K with two amino acid substitutions that sequentially conferred improved catalytic properties and stability. Here we show that the aerobic growth ability on ethanol depends also on protection of the mutant AdhE against metal-catalyzed oxidation by the chaperone DnaK (a member of the Hsp70 family). No DnaK protection of the enzyme is evident during anaerobic growth on glucose. Synthesis of DnaK also protected E. coli from H2O2 killing under conditions when functional AdhE is not required. Our results therefore suggest that, in addition to the known role of protecting cells against heat stress, DnaK also protects numerous kinds of proteins from oxidative damage. PMID:11917132

  12. Immune escape mutants of Highly Pathogenic Avian Influenza H5N1 selected using polyclonal sera: identification of key amino acids in the HA protein.

    PubMed

    Sitaras, Ioannis; Kalthoff, Donata; Beer, Martin; Peeters, Ben; de Jong, Mart C M

    2014-01-01

    Evolution of Avian Influenza (AI) viruses--especially of the Highly Pathogenic Avian Influenza (HPAI) H5N1 subtype--is a major issue for the poultry industry. HPAI H5N1 epidemics are associated with huge economic losses and are sometimes connected to human morbidity and mortality. Vaccination (either as a preventive measure or as a means to control outbreaks) is an approach that splits the scientific community, due to the risk of it being a potential driving force in HPAI evolution through the selection of mutants able to escape vaccination-induced immunity. It is therefore essential to study how mutations are selected due to immune pressure. To this effect, we performed an in vitro selection of mutants from HPAI A/turkey/Turkey/1/05 (H5N1), using immune pressure from homologous polyclonal sera. After 42 rounds of selection, we identified 5 amino acid substitutions in the Haemagglutinin (HA) protein, most of which were located in areas of antigenic importance and suspected to be prone to selection pressure. We report that most of the mutations took place early in the selection process. Finally, our antigenic cartography studies showed that the antigenic distance between the selected isolates and their parent strain increased with passage number.

  13. Differential Phospholipid Substrates and Directional Transport by ATP-binding Cassette Proteins ABCA1, ABCA7, and ABCA4 and Disease-causing Mutants*♦

    PubMed Central

    Quazi, Faraz; Molday, Robert S.

    2013-01-01

    ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters that share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7, and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported phosphatidylserine. In contrast, ABCA4 transported phosphatidylethanolamine in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport and ATPase activities of ABCA1 and ABCA4 were reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and the corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of apoA-1 with phospholipids for HDL formation. PMID:24097981

  14. The α-subunit of the rice heterotrimeric G protein, RGA1, regulates drought tolerance during the vegetative phase in the dwarf rice mutant d1

    PubMed Central

    Ferrero-Serrano, Ángel; Assmann, Sarah M.

    2016-01-01

    Essential in the Green Revolution was the development of high-yielding dwarf varieties of rice (Oryza sativa L.), but their selection was not based on responses to water limitation. We studied physiological responses to progressive drought of the dwarf rice mutant, d1, in which the RGA1 gene, which encodes the GTP-binding α-subunit of the heterotrimeric G protein, is non-functional. Wild-type (WT) plants cease net carbon fixation 11 days after water is withheld, while d1 plants maintain net photosynthesis for an additional week. During drought, d1 plants exhibit greater stomatal conductance than the WT, but both genotypes exhibit the same transpirational water loss per unit leaf area. This is explained by a smaller driving force for water loss in d1 owing to its lower leaf temperatures, consistent with its more erect architecture. As drought becomes more severe, WT plants show an accelerated decline in photosynthesis, which may be exacerbated by the higher leaf temperatures in the WT. We thus show how a rice mutant with dwarf and erect leaves has a decreased susceptibility to water stress. Accordingly, it may be useful to incorporate RGA1 mutation in breeding or biotechnological strategies for development of drought-resistant rice. PMID:27194741

  15. Depletion of a novel SET-domain protein enhances the sterility of mes-3 and mes-4 mutants of Caenorhabditis elegans.

    PubMed Central

    Xu, L; Strome, S

    2001-01-01

    Four maternal-effect sterile genes, mes-2, mes-3, mes-4, and mes-6, are essential for germline development in Caenorhabditis elegans. Homozygous mes progeny from heterozygous mothers are themselves fertile but produce sterile progeny with underproliferated and degenerated germlines. All four mes genes encode chromatin-associated proteins, two of which resemble known regulators of gene expression. To identify additional components in the MES pathway, we used RNA-mediated interference (RNAi) to test candidate genes for enhancement of the Mes mutant phenotype. Enhancement in this assay was induction of sterility a generation earlier, in the otherwise fertile homozygous progeny of heterozygous mothers, which previous results had suggested represent a sensitized genetic background. We tested seven genes predicted to encode regulators of chromatin organization for RNAi-induced enhancement of mes-3 sterility and identified one enhancer, called set-2 after the SET domain encoded by the gene. Depletion of SET-2 also enhances the sterile phenotype of mes-4 but not of mes-2 or mes-6. set-2 encodes two alternatively spliced transcripts, set-2(l) and set-2(s), both of which are enriched in the germline of adults. In the adult germline, SET-2(L) protein is localized in mitotic and mid-late-stage meiotic nuclei but is undetectable in early pachytene nuclei. SET-2(L) protein is localized in all nuclei of embryos. The localization of SET-2(L) does not depend on any of the four MES proteins, and none of the MES proteins depend on SET-2 for their normal localization. Our results suggest that SET-2 participates along with the MES proteins in promoting normal germline development. PMID:11729150

  16. Characterization of cDNA encoding mouse DNA repair protein O sup 6 -methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli

    SciTech Connect

    Shiota, Susumu; Tano, Keizo ); von Wronski, M.A.; Brent, T.P. ); Bigner, D.D. ); Mitra, S. )

    1992-02-25

    A mouse cDNA clone encoding O{sup 6}-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O{sup 6}-alkylguanine in DNA, was cloned from a {lambda}gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.

  17. Problem-Solving Test: Tryptophan Operon Mutants

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  18. Secretory pathway retention of mutant prion protein induces p38-MAPK activation and lethal disease in mice

    PubMed Central

    Puig, Berta; Altmeppen, Hermann C.; Ulbrich, Sarah; Linsenmeier, Luise; Krasemann, Susanne; Chakroun, Karima; Acevedo-Morantes, Claudia Y.; Wille, Holger; Tatzelt, Jörg; Glatzel, Markus

    2016-01-01

    Misfolding of proteins in the biosynthetic pathway in neurons may cause disturbed protein homeostasis and neurodegeneration. The prion protein (PrPC) is a GPI-anchored protein that resides at the plasma membrane and may be misfolded to PrPSc leading to prion diseases. We show that a deletion in the C-terminal domain of PrPC (PrPΔ214–229) leads to partial retention in the secretory pathway causing a fatal neurodegenerative disease in mice that is partially rescued by co-expression of PrPC. Transgenic (Tg(PrPΔ214–229)) mice show extensive neuronal loss in hippocampus and cerebellum and activation of p38-MAPK. In cell culture under stress conditions, PrPΔ214–229 accumulates in the Golgi apparatus possibly representing transit to the Rapid ER Stress-induced ExporT (RESET) pathway together with p38-MAPK activation. Here we describe a novel pathway linking retention of a GPI-anchored protein in the early secretory pathway to p38-MAPK activation and a neurodegenerative phenotype in transgenic mice. PMID:27117504

  19. Structure of solvation water around the active and inactive regions of a type III antifreeze protein and its mutants of lowered activity

    NASA Astrophysics Data System (ADS)

    Grabowska, Joanna; Kuffel, Anna; Zielkiewicz, Jan

    2016-08-01

    Water molecules from the solvation shell of the ice-binding surface are considered important for the antifreeze proteins to perform their function properly. Herein, we discuss the problem whether the extent of changes of the mean properties of solvation water can be connected with the antifreeze activity of the protein. To this aim, the structure of solvation water of a type III antifreeze protein from Macrozoarces americanus (eel pout) is investigated. A wild type of the protein is used, along with its three mutants, with antifreeze activities equal to 54% or 10% of the activity of the native form. The solvation water of the ice-binding surface and the rest of the protein are analyzed separately. To characterize the structure of solvation shell, parameters describing radial and angular characteristics of the mutual arrangement of the molecules were employed. They take into account short-distance (first hydration shell) or long-distance (two solvation shells) effects. The obtained results and the comparison with the results obtained previously for a hyperactive antifreeze protein from Choristoneura fumiferana lead to the conclusion that the structure and amino acid composition of the active region of the protein evolved to achieve two goals. The first one is the modification of the properties of the solvation water. The second one is the geometrical adjustment of the protein surface to the specific crystallographic plane of ice. Both of these goals have to be achieved simultaneously in order for the protein to perform its function properly. However, they seem to be independent from one another in a sense that very small antifreeze activity does not imply that properties of water become different from the ones observed for the wild type. The proteins with significantly lower activity still modify the mean properties of solvation water in a right direction, in spite of the fact that the accuracy of the geometrical match with the ice lattice is lost because of the

  20. Structure of solvation water around the active and inactive regions of a type III antifreeze protein and its mutants of lowered activity.

    PubMed

    Grabowska, Joanna; Kuffel, Anna; Zielkiewicz, Jan

    2016-08-21

    Water molecules from the solvation shell of the ice-binding surface are considered important for the antifreeze proteins to perform their function properly. Herein, we discuss the problem whether the extent of changes of the mean properties of solvation water can be connected with the antifreeze activity of the protein. To this aim, the structure of solvation water of a type III antifreeze protein from Macrozoarces americanus (eel pout) is investigated. A wild type of the protein is used, along with its three mutants, with antifreeze activities equal to 54% or 10% of the activity of the native form. The solvation water of the ice-binding surface and the rest of the protein are analyzed separately. To characterize the structure of solvation shell, parameters describing radial and angular characteristics of the mutual arrangement of the molecules were employed. They take into account short-distance (first hydration shell) or long-distance (two solvation shells) effects. The obtained results and the comparison with the results obtained previously for a hyperactive antifreeze protein from Choristoneura fumiferana lead to the conclusion that the structure and amino acid composition of the active region of the protein evolved to achieve two goals. The first one is the modification of the properties of the solvation water. The second one is the geometrical adjustment of the protein surface to the specific crystallographic plane of ice. Both of these goals have to be achieved simultaneously in order for the protein to perform its function properly. However, they seem to be independent from one another in a sense that very small antifreeze activity does not imply that properties of water become different from the ones observed for the wild type. The proteins with significantly lower activity still modify the mean properties of solvation water in a right direction, in spite of the fact that the accuracy of the geometrical match with the ice lattice is lost because of the

  1. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    SciTech Connect

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong; Wei, Shi-Cheng; Liang, Yu-He Su, Xiao-Dong

    2007-09-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.

  2. A temperature sensitive mutant of heat shock protein 70 reveals an essential role during the early steps of tombusvirus replication.

    PubMed

    Wang, Robert Yung-Liang; Stork, Jozsef; Pogany, Judit; Nagy, Peter D

    2009-11-10

    By co-opting host proteins for their replication, plus-stranded RNA viruses can support robust replication and suppress host anti-viral responses. Tomato bushy stunt virus (TBSV) recruit the cellular heat shock protein 70 (Hsp70), an abundant cytosolic chaperone, into the replicase complex. By taking advantage of yeast model host, we demonstrate that the four-member SSA subfamily of HSP70 genes is essential for TBSV replication. The constitutively expressed SSA1 and SSA2, which are resident proteins in the viral replicase, can be complemented by the heat-inducible SSA3 and/or SSA4 for TBSV replication. Using a yeast strain carrying a temperature sensitive ssa1(ts), but lacking functional SSA2/3/4, we show that inactivation of Ssa1p(ts) led to a defect in membrane localization of the viral replication proteins, resulting in cytosolic distribution of the viral proteins and lack of replicase activity. An in vitro replicase assembly assay with Ssa1p(ts) revealed that functional Ssa1p is required during the replicase assembly process, but not during minus- or plus-strand synthesis. Temperature shift experiments from nonpermissive to permissive in ssa1(ts) yeast revealed that the re-activated Ssa1p(ts) could promote efficient TBSV replication in the absence of other SSA genes. We also demonstrate that the purified recombinant Ssa3p can facilitate the in vitro assembly of the TBSV replicase on yeast membranes, demonstrating that Ssa3p can fully complement the function of Ssa1p. Taken together, the cytosolic SSA subfamily of Hsp70 proteins play essential and multiple roles in TBSV replication. PMID:19748649

  3. Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize.

    PubMed

    Wu, Xiaolin; Gong, Fangping; Yang, Le; Hu, Xiuli; Tai, Fuju; Wang, Wei

    2014-01-01

    ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5), deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs), late embryogenesis abundant (LEA) proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation. PMID:25653661

  4. Exploring the potential of complex formation between a mutant DNA and the wild type protein counterpart: a MM and MD simulation approach.

    PubMed

    Roy, Sujata; Sen, Srikanta

    2006-09-01

    We have demonstrated that the methods of molecular modeling and molecular dynamics simulation might be used to assess whether a specific mutation in the DNA would destabilize a known DNA-protein complex. The approach is based on probing the changes in the interaction that would be induced into the complex if within the already formed wild type complex the mutation could be introduced. We have used Hoxc8-DNA complex as a test system where it is known that the Hoxc8 binding affinity of the DNA is completely lost upon mutation of the DNA by replacing TAAT stretch to GCCG. Mutation was obtained by changing the relevant base pairs into the DNA of the model of the corresponding wild type complex developed by homology modeling and MD simulation in water for 2.0 ns. Comparison of the structure, dynamics and interactions between the hypothetical mutant model with those of the similarly refined wild type model shows that the loss of affinity of the mutant DNA to Hoxc8 has two different origins: (i) loss of several strong H-bonds as the direct consequences of mutation and (ii) reduced H-bonds in the common parts due to a net loss or inferior H-bonding geometry induced by the mutation as indirect effects. The net change in the interaction energy between the DNA and the protein in the best possible configuration indicated the experimentally observed destabilization effects. No significant change in the groove width was observed and no correlation was found between the water-bridges and the loss of affinity. PMID:16406716

  5. Constitutive expression of a COOH-terminal leucine mutant of lysosome-associated membrane protein-1 causes its exclusive localization in low density intracellular vesicles.

    PubMed

    Akasaki, Kenji; Shiotsu, Keiko; Michihara, Akihiro; Ide, Norie; Wada, Ikuo

    2014-07-01

    Lysosome-associated membrane protein-1 (LAMP-1) is a type I transmembrane protein with a short cytoplasmic tail that possesses a lysosome-targeting signal of GYQTI(382)-COOH. Wild-type (WT)-LAMP-1 was exclusively localized in high density lysosomes, and efficiency of LAMP-1's transport to lysosomes depends on its COOH-terminal amino acid residue. Among many different COOH-terminal amino acid substitution mutants of LAMP-1, a leucine-substituted mutant (I382L) displays the most efficient targeting to late endosomes and lysosomes [Akasaki et al. (2010) J. Biochem. 148: , 669-679]. In this study, we generated two human hepatoma cell lines (HepG2 cell lines) that stably express WT-LAMP-1 and I382L, and compared their intracellular distributions. The subcellular fractionation study using Percoll density gradient centrifugation revealed that WT-LAMP-1 had preferential localization in the high density secondary lysosomes where endogenous human LAMP-1 was enriched. In contrast, a major portion of I382L was located in a low density fraction. The low density fraction also contained approximately 80% of endogenous human LAMP-1 and significant amounts of endogenous β-glucuronidase and LAMP-2, which probably represents occurrence of low density lysosomes in the I382L-expressing cells. Double immunofluorescence microscopic analyses distinguished I382L-containing intracellular vesicles from endogenous LAMP-1-containing lysosomes and early endosomes. Altogether, constitutive expression of I382L causes its aberrant intracellular localization and generation of low density lysosomes, indicating that the COOH-terminal isoleucine is critical for normal localization of LAMP-1 in the dense lysosomes.

  6. COOH-terminal collagen Q (COLQ) mutants causing human deficiency of endplate acetylcholinesterase impair the interaction of ColQ with proteins of the basal lamina.

    PubMed

    Arredondo, Juan; Lara, Marian; Ng, Fiona; Gochez, Danielle A; Lee, Diana C; Logia, Stephanie P; Nguyen, Joanna; Maselli, Ricardo A

    2014-05-01

    Collagen Q (ColQ) is a key multidomain functional protein of the neuromuscular junction (NMJ), crucial for anchoring acetylcholinesterase (AChE) to the basal lamina (BL) and accumulating AChE at the NMJ. The attachment of AChE to the BL is primarily accomplished by the binding of the ColQ collagen domain to the heparan sulfate proteoglycan perlecan and the COOH-terminus to the muscle-specific receptor tyrosine kinase (MuSK), which in turn plays a fundamental role in the development and maintenance of the NMJ. Yet, the precise mechanism by which ColQ anchors AChE at the NMJ remains unknown. We identified five novel mutations at the COOH-terminus of ColQ in seven patients from five families affected with endplate (EP) AChE deficiency. We found that the mutations do not affect the assembly of ColQ with AChE to form asymmetric forms of AChE or impair the interaction of ColQ with perlecan. By contrast, all mutations impair in varied degree the interaction of ColQ with MuSK as well as basement membrane extract (BME) that have no detectable MuSK. Our data confirm that the interaction of ColQ to perlecan and MuSK is crucial for anchoring AChE to the NMJ. In addition, the identified COOH-terminal mutants not only reduce the interaction of ColQ with MuSK, but also diminish the interaction of ColQ with BME. These findings suggest that the impaired attachment of COOH-terminal mutants causing EP AChE deficiency is in part independent of MuSK, and that the COOH-terminus of ColQ may interact with other proteins at the BL.

  7. Assembly defects of desmin disease mutants carrying deletions in the alpha-helical rod domain are rescued by wild type protein.

    PubMed

    Bär, Harald; Mücke, Norbert; Katus, Hugo A; Aebi, Ueli; Herrmann, Harald

    2007-04-01

    Most mutations of desmin that cause severe autosomal dominant forms of myofibrillar myopathy are point mutations and locate in the central alpha-helical coiled-coil rod domain. Recently, two in-frame deletions of one and three amino acids, respectively, in the alpha-helix have been described and discussed to drastically interfere with the architecture of the desmin dimer and possibly also the formation of tetramers and higher order complexes [Kaminska, A., Strelkov, S.V., Goudeau, B., Olive, M., Dagvadorj, A., Fidzianska, A., Simon-Casteras, M., Shatunov, A., Dalakas, M.C., Ferrer, I., Kwiecinski, H., Vicart, P., Goldfarb, L.G., 2004. Small deletions disturb desmin architecture leading to breakdown of muscle cells and development of skeletal or cardioskeletal myopathy. Hum. Genet. 114, 306-313.]. Therefore, it was proposed that they may poison intermediate filament (IF) assembly. We have now recombinantly synthesized both mutant proteins and subjected them to comprehensive in vitro assembly experiments. While exhibiting assembly defects when analyzed on their own, both one-to-one mixtures of the respective mutant protein with wild type desmin facilitated proper filament formation. Transient transfection studies complemented this fundamental finding by demonstrating that wild type desmin is also rescuing these assembly defects in vivo. In summary, our findings strongly question the previous hypothesis that it is assembly incompetence due to molecular rearrangements caused by the mutations, which triggers the development of disease. As an alternative, we propose that these mutations cause subtle age-dependent structural alterations of desmin IFs that eventually lead to disease. PMID:17188893

  8. Thermostability and Photostability of Photosystem II in Leaves of the Chlorina-f2 Barley Mutant Deficient in Light-Harvesting Chlorophyll a/b Protein Complexes.

    PubMed Central

    Havaux, M.; Tardy, F.

    1997-01-01

    The chlorophyll-b-less chlorina-f2 barley mutant is deficient in the major as well as some minor light-harvesting chlorophyll-protein complexes of photosystem II (LHCII). Although the LHCII deficiency had relatively minor repercussions on the leaf photosynthetic performances, the responses of photosystem II (PSII) to elevated temperatures and to bright light were markedly modified. The chlorina-f2 mutation noticeably reduced the thermostability of PSII, with thermal denaturation of PSII starting at about 35[deg]C and 38.5[deg]C in chlorina-f2 and in the wild type, respectively. The increased susceptibility of PSII to heat stress in chlorina-f2 leaves was due to the weakness of its electron donor side, with moderate heat stress causing detachment of the 33-kD extrinsic PSII protein from the oxygen-evolving complex. Prolonged dark adaptation of chlorina-f2 leaves was also observed to inhibit the PSII donor side. However, weak illumination slowly reversed the dark-induced inhibition of PSII in chlorina-f2 and cancelled the difference in PSII thermostability observed between chlorina-f2 and wild-type leaves. The mutant was more sensitive to photoinhibition than the wild type, with strong light stress impairing the PSII donor side in chlorina-f2 but not in the wild type. This difference was not observed in anaerobiosis or in the presence of 3-(3,4-dichlorophenyl)- 1,1-dimethylurea, diuron. The acceptor side of PSII was only slightly affected by the mutation and/or the aforementioned stress conditions. Taken together, our results indicate that LHCII stabilize the PSII complexes and maintain the water-oxidizing system in a functional state under varying environmental conditions. PMID:12223653

  9. The seirena B Class Floral Homeotic Mutant of California Poppy (Eschscholzia californica) Reveals a Function of the Enigmatic PI Motif in the Formation of Specific Multimeric MADS Domain Protein Complexes[C][W][OA

    PubMed Central

    Lange, Matthias; Orashakova, Svetlana; Lange, Sabrina; Melzer, Rainer; Theißen, Günter; Smyth, David R.; Becker, Annette

    2013-01-01

    The products of B class floral homeotic genes specify petal and stamen identity, and loss of B function results in homeotic conversions of petals into sepals and stamens into carpels. Here, we describe the molecular characterization of seirena-1 (sei-1), a mutant from the basal eudicot California poppy (Eschscholzia californica) that shows homeotic changes characteristic of floral homeotic B class mutants. SEI has been previously described as EScaGLO, one of four B class–related MADS box genes in California poppy. The C terminus of SEI, including the highly conserved PI motif, is truncated in sei-1 proteins. Nevertheless, like the wild-type SEI protein, the sei-1 mutant protein is able to bind CArG-boxes and can form homodimers, heterodimers, and several higher order complexes with other MADS domain proteins. However, unlike the wild type, the mutant protein is not able to mediate higher order complexes consisting of specific B, C, and putative E class related proteins likely involved in specifying stamen identity. Within the PI motif, five highly conserved N-terminal amino acids are specifically required for this interaction. Several families lack this short conserved sequence, including the Brassicaceae, and we propose an evolutionary scenario to explain these functional differences. PMID:23444328

  10. Selection of hepatitis B surface "escape" mutants during passive immune prophylaxis following liver transplantation: potential impact of genetic changes on polymerase protein function

    PubMed Central

    Shields, P; Owsianka, A; Carman, W; Boxall, E; Hubscher, S; Shaw, J; O'Donnell, K; Elias, E; Mutimer, D

    1999-01-01

    CASE REPORT—A patient is described who developed hepatitis B virus (HBV) reinfection five months following liver transplantation. Failure of hepatitis B immunoglobulin prophylaxis was associated with the emergence of mutations. HBV gene sequencing identified nucleotide substitutions associated with amino acid changes, one within the major hydrophilic region (MHR) of the HBV surface antigen at amino acid position 144 and one outside the MHR. Because of the overlapping reading frames of surface and polymerase genes, the latter surface antigen change was associated with an amino acid change in the polymerase protein. The patient developed significant allograft hepatitis and was treated with lamivudine (3TC) 100 mg daily. Rapid decline of serum HBV DNA was observed with loss of HBV e antigen and HBV surface antigen from serum. There was normalisation of liver biochemistry, and liver immunohistochemistry showed a reduction in HBV core and disappearance of HBs antigen staining.
CONCLUSION—Surface antigen encoding gene mutations associated with HBIg escape may be associated with alteration of the polymerase protein. The polymerase changes may affect sensitivity to antiviral treatment. Selection pressure on one HBV reading frame (for example, HBIg pressure on HBsAg, or nucleoside analogue pressure on polymerase protein) may alter the gene product of the overlapping frame. Such interactions are relevant to strategies employing passive immune prophylaxis and antiviral treatment.


Keywords: liver transplantation; prophylaxis; escape mutants; lamivudine PMID:10403747

  11. Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein

    PubMed Central

    Tan, Wei-Hung; Cheng, Shu-Chun; Liu, Yu-Tung; Wu, Cheng-Guo; Lin, Min-Han; Chen, Chiao-Che; Lin, Chao-Hsiung; Chou, Chi-Yuan

    2016-01-01

    Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We’ve also identified four historical mutations that are able to produce a “GST-like” S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution. PMID:27499004

  12. Differential interaction of Apolipoprotein-E isoforms with insulin receptors modulates brain insulin signaling in mutant human amyloid precursor protein transgenic mice.

    PubMed

    Chan, Elizabeth S; Chen, Christopher; Cole, Gregory M; Wong, Boon-Seng

    2015-09-08

    It is unclear how human apolipoprotein E4 (ApoE4) increases the risk for Alzheimer's disease (AD). Although Aβ levels can lead to insulin signaling impairment, these experiments were done in the absence of human ApoE. To examine ApoE role, we crossed the human ApoE-targeted replacement mice with mutant human amyloid precursor protein (APP) mice. In 26 week old mice with lower Aβ levels, the expression and phosphorylation of insulin signaling proteins remained comparable among APP, ApoE3xAPP and ApoE4xAPP mouse brains. When the mice aged to 78 weeks, these proteins were markedly reduced in APP and ApoE4xAPP mouse brains. While Aβ can bind to insulin receptor, how ApoE isoforms modulate this interaction remains unknown. Here, we showed that ApoE3 had greater association with insulin receptor as compared to ApoE4, regardless of Aβ42 concentration. In contrast, ApoE4 bound more Aβ42 with increasing peptide levels. Using primary hippocampal neurons, we showed that ApoE3 and ApoE4 neurons are equally sensitive to physiological levels of insulin. However, in the presence of Aβ42, insulin failed to elicit a downstream response only in ApoE4 hippocampal neurons. Taken together, our data show that ApoE genotypes can modulate this Aβ-mediated insulin signaling impairment.

  13. Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein.

    PubMed

    Tan, Wei-Hung; Cheng, Shu-Chun; Liu, Yu-Tung; Wu, Cheng-Guo; Lin, Min-Han; Chen, Chiao-Che; Lin, Chao-Hsiung; Chou, Chi-Yuan

    2016-01-01

    Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We've also identified four historical mutations that are able to produce a "GST-like" S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution. PMID:27499004

  14. Biological safety assessment of mutant variant of Allium sativum leaf agglutinin (mASAL), a novel antifungal protein for future transgenic application.

    PubMed

    Ghosh, Prithwi; Roy, Amit; Chakraborty, Joydeep; Das, Sampa

    2013-12-01

    Genetic engineering has established itself to be an important tool for crop improvement. Despite the success, there is always a risk of food allergy induced by alien gene products. The present study assessed the biosafety of mutant Allium sativum leaf agglutinin (mASAL), a potent antifungal protein generated by site directed mutagenesis of Allium sativum leaf agglutinin (ASAL). mASAL was cloned in pET28a+ and expressed in E. coli, and the safety assessment was carried out according to the FAO/WHO guideline (2001). Bioinformatics analysis, pepsin digestion, and thermal stability assay showed the protein to be nonallergenic. Targeted sera screening revealed no significant IgE affinity of mASAL. Furthermore, mASAL sensitized Balb/c mice showed normal histopathology of lung and gut tissue. All results indicated the least possibility of mASAL being an allergen. Thus, mASAL appears to be a promising antifungal candidate protein suitable for agronomical biotechnology.

  15. Mutation of a Cuticle Protein Gene, BmCPG10, Is Responsible for Silkworm Non-Moulting in the 2nd Instar Mutant.

    PubMed

    Wu, Fan; Wang, Pingyang; Zhao, Qiaoling; Kang, Lequn; Xia, Dingguo; Qiu, Zhiyong; Tang, Shunming; Li, Muwang; Shen, Xingjia; Zhang, Guozheng

    2016-01-01

    In the silkworm, metamorphosis and moulting are regulated by ecdysone hormone and juvenile hormone. The subject in the present study is a silkworm mutant that does not moult in the 2nd instar (nm2). Genetic analysis indicated that the nm2 mutation is controlled by a recessive gene and is homozygous lethal. Based on positional cloning, nm2 was located in a region approximately 275 kb on the 5th linkage group by eleven SSR polymorphism markers. In this specific range, according to the transcriptional expression of thirteen genes and cloning, the relative expression level of the BmCPG10 gene that encodes a cuticle protein was lower than the expression level of the wild-type gene. Moreover, this gene's structure differs from that of the wild-type gene: there is a deletion of 217 bp in its open reading frame, which resulted in a change in the protein it encoded. The BmCPG10 mRNA was detectable throughout silkworm development from the egg to the moth. This mRNA was low in the pre-moulting and moulting stages of each instar but was high in the gluttonous stage and in newly exuviated larvae. The BmCPG10 mRNA showed high expression levels in the epidermis, head and trachea, while the expression levels were low in the midgut, Malpighian tubule, prothoracic gland, haemolymph and ventral nerve cord. The ecdysone titre was determined by ELISA, and the results demonstrated that the ecdysone titre of nm2 larvae was lower than that of the wild-type larvae. The nm2 mutant could be rescued by feeding 20-hydroxyecdysone, cholesterol and 7-dehydrocholesterol (7dC), but the rescued nm2 only developed to the 4th instar and subsequently died. The moulting time of silkworms could be delayed by BmCPG10 RNAi. Thus, we speculated that the mutation of BmCPG10 was responsible for the silkworm mutant that did not moult in the 2nd instar. PMID:27096617

  16. Mutation of a Cuticle Protein Gene, BmCPG10, Is Responsible for Silkworm Non-Moulting in the 2nd Instar Mutant

    PubMed Central

    Zhao, Qiaoling; Kang, Lequn; Xia, Dingguo; Qiu, Zhiyong; Tang, Shunming; Li, Muwang; Shen, Xingjia; Zhang, Guozheng

    2016-01-01

    In the silkworm, metamorphosis and moulting are regulated by ecdysone hormone and juvenile hormone. The subject in the present study is a silkworm mutant that does not moult in the 2nd instar (nm2). Genetic analysis indicated that the nm2 mutation is controlled by a recessive gene and is homozygous lethal. Based on positional cloning, nm2 was located in a region approximately 275 kb on the 5th linkage group by eleven SSR polymorphism markers. In this specific range, according to the transcriptional expression of thirteen genes and cloning, the relative expression level of the BmCPG10 gene that encodes a cuticle protein was lower than the expression level of the wild-type gene. Moreover, this gene’s structure differs from that of the wild-type gene: there is a deletion of 217 bp in its open reading frame, which resulted in a change in the protein it encoded. The BmCPG10 mRNA was detectable throughout silkworm development from the egg to the moth. This mRNA was low in the pre-moulting and moulting stages of each instar but was high in the gluttonous stage and in newly exuviated larvae. The BmCPG10 mRNA showed high expression levels in the epidermis, head and trachea, while the expression levels were low in the midgut, Malpighian tubule, prothoracic gland, haemolymph and ventral nerve cord. The ecdysone titre was determined by ELISA, and the results demonstrated that the ecdysone titre of nm2 larvae was lower than that of the wild-type larvae. The nm2 mutant could be rescued by feeding 20-hydroxyecdysone, cholesterol and 7—dehydrocholesterol (7dC), but the rescued nm2 only developed to the 4th instar and subsequently died. The moulting time of silkworms could be delayed by BmCPG10 RNAi. Thus, we speculated that the mutation of BmCPG10 was responsible for the silkworm mutant that did not moult in the 2nd instar. PMID:27096617

  17. Activation of mutant protein kinase C{gamma} leads to aberrant sequestration and impairment of its cellular function

    SciTech Connect

    Doran, Graeme; Davies, Kay E.; Talbot, Kevin

    2008-08-01

    Mutations in protein kinase C{gamma} (PKC{gamma}) cause the neurodegenerative disease spinocerebellar ataxia type 14 (SCA14). In this study, expression of an extensive panel of known SCA14-associated PKC{gamma} mutations as fusion proteins in cell culture led to the consistent formation of cytoplasmic aggregates in response to purinoceptor stimulation. Aggregates co-stained with antibodies to phosphorylated PKC{gamma} and the early endosome marker EEA1 but failed to redistribute to the cell membrane under conditions of oxidative stress. These studies suggest that Purkinje cell damage in SCA14 may result from a reduction of PKC{gamma} activity due its aberrant sequestration in the early endosome compartment.

  18. Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype

    PubMed Central

    Plog, Stephanie; Klymiuk, Nikolai; Binder, Stefanie; Van Hook, Matthew J.; Thoreson, Wallace B.; Gruber, Achim D.; Mundhenk, Lars

    2015-01-01

    The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype. PMID:26474299

  19. Defective Dissociation of a “Slow” RecA Mutant Protein Imparts an Escherichia coli Growth Defect*

    PubMed Central

    Cox, Julia M.; Li, Hao; Wood, Elizabeth A.; Chitteni-Pattu, Sindhu; Inman, Ross B.; Cox, Michael M.

    2008-01-01

    The RecA and some related proteins possess a simple motif, called (KR)X(KR), that (in RecA) consists of two lysine residues at positions 248 and 250 at the subunit-subunit interface. This study and previous work implicate this RecA motif in the following: (a) catalyzing ATP hydrolysis in trans,(b) coordinating the ATP hydrolytic cycles of adjacent subunits, (c) governing the rate of ATP hydrolysis, and (d) coupling the ATP hydrolysis to work (in this case DNA strand exchange). The conservative K250R mutation leaves RecA nucleoprotein filament formation largely intact. However, ATP hydrolysis is slowed to less than 15% of the wild-type rate. DNA strand exchange is also slowed commensurate with the rate of ATP hydrolysis. The results reinforce the idea of a tight coupling between ATP hydrolysis and DNA strand exchange. When a plasmid-borne RecA K250R protein is expressed in a cell otherwise lacking RecA protein, the growth of the cells is severely curtailed. The slow growth defect is alleviated in cells lacking RecFOR function, suggesting that the defect reflects loading of RecA at stalled replication forks. Suppressors occur as recA gene alterations, and their properties indicate that limited dissociation by RecA K250R confers the slow growth phenotype. Overall, the results suggest that recombinational DNA repair is a common occurrence in cells. RecA protein plays a sufficiently intimate role in the bacterial cell cycle that its properties can limit the growth rate of a bacterial culture. PMID:18603529

  20. Protein Disulfide Isomerase Regulates Endoplasmic Reticulum Stress and the Apoptotic Process during Prion Infection and PrP Mutant-Induced Cytotoxicity

    PubMed Central

    Wang, Shao-Bin; Shi, Qi; Xu, Yin; Xie, Wu-Ling; Zhang, Jin; Tian, Chan; Guo, Yan; Wang, Ke; Zhang, Bao-Yun; Chen, Cao; Gao, Chen; Dong, Xiao-Ping

    2012-01-01

    Background Protein disulfide isomerase (PDI), is sorted to be enzymatic chaperone for reconstructing misfolded protein in endoplasmic reticulum lumen. Recently, PDI has been identified as a link between misfolded protein and neuron apoptosis. However, the potential for PDI to be involved in the pathogenesis of prion disease remains unknown. In this study, we propose that PDI may function as a pleiotropic regulator in the cytotoxicity induced by mutated prion proteins and in the pathogenesis of prion diseases. Methodology/Principal Findings To elucidate potential alterations of PDI in prion diseases, the levels of PDI and relevant apoptotic executors in 263K infected hamsters brain tissues were evaluated with the use of Western blots. Abnormal upregulation of PDI, Grp78 and Grp58 was detected. Dynamic assays of PDI alteration identified that the upregulation of PDI started at the early stage and persistently increased till later stage. Obvious increases of PDI and Grp78 levels were also observed in cultured cells transiently expressing PrP mutants, PrP-KDEL or PrP-PG15, accompanied by significant cytotoxicities. Excessive expression of PDI partially eased ER stress and cell apoptosis caused by accumulation of PrP-KDEL, but had less effect on cytotoxicity induced by PrP-PG15. Knockdown of endogenous PDI significantly amended cytotoxicity of PrP-PG15, but had little influence on that of PrP-KDEL. A series of membrane potential assays found that apoptosis induced by misfolded PrP proteins could be regulated by PDI via mitochondrial dysfunction. Moreover, biotin-switch assays demonstrated active S-nitrosylted modifications of PDI (SNO-PDI) both in the brains of scrapie-infected rodents and in the cells with misfolded PrP proteins. Conclusion/Significance Current data in this study highlight that PDI and its relevant executors may function as a pleiotropic regulator in the processes of different misfolded PrP proteins and at different stages during prion infection. SNO

  1. Target binding to S100B reduces dynamic properties and increases Ca2+-binding affinity for wild type and EF-hand mutant proteins

    PubMed Central

    Liriano, Melissa A.; Varney, Kristen M.; Wright, Nathan T.; Hoffman, Cassandra L.; Toth, Eric A.; Ishima, Rieko; Weber, David J.

    2012-01-01

    Mutations in the second EF-hand (D61N, D63N, D65N, E72A) of S100B were used to study its Ca2+-binding and dynamic properties in the absence and presence of abound target, TRTK-12. With D63NS100B as an exception (D63NKD = 50 ± 9 µM), Ca2+-binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (D61NKD = 412 ± 67 µM; D65NKD = 968 ± 171 µM; E72AKD = 471 ± 133 µM), when compared to wild-type protein (WTKD = 56 ± 9 µM). For the TRTK-12 complexes, the Ca2+-binding affinity to wild type (WT+TRTKKD = 12 ± 10 µM) and the EF2 mutants were increased by 5- to 19-fold versus in the absence of target (D61N+TRTKKD = 29 ± 1.2 µM; D63N+TRTKKD = 10 ± 2.2 µM; D65N+TRTKKD = 73 ± 4.4 µM; E72A+TRTKKD = 18 ± 3.7 µM). In addition, Rex, as measured using relaxation dispersion for side chain 15N resonances of Asn63 (D63NS100B) was reduced upon TRTK-12 binding when measured by nuclear magnetic resonance (NMR). Likewise, backbone motions on multiple time scales (ps-ms) throughout wild type, D61NS100B D63NS100B, and D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca2+-bound (2.0 Å) and TRTK-bound (1.2 Å) D63NS100B showed no change in Ca2+ coordination, so these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca2+-binding affinity in solution. Thus, a model for how S100B-TRTK12 complex formation increases Ca2+ binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12. PMID:22824086

  2. Protein kinase C inhibitor sotrastaurin selectively inhibits the growth of CD79 mutant diffuse large B-cell lymphomas.

    PubMed

    Naylor, Tara L; Tang, Huaping; Ratsch, Boris A; Enns, Andreas; Loo, Alice; Chen, Liqing; Lenz, Peter; Waters, Nigel J; Schuler, Walter; Dörken, Bernd; Yao, Yung-Mae; Warmuth, Markus; Lenz, Georg; Stegmeier, Frank

    2011-04-01

    The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) correlates with poor prognosis. The ABC subtype of DLBCL is associated with constitutive activation of the NF-κB pathway, and oncogenic lesions have been identified in its regulators, including CARD11/CARMA1 (caspase recruitment domain-containing protein 11), A20/TNFAIP3, and CD79A/B. In this study, we offer evidence of therapeutic potential for the selective PKC (protein kinase C) inhibitor sotrastaurin (STN) in preclinical models of DLBCL. A significant fraction of ABC DLBCL cell lines exhibited strong sensitivity to STN, and we found that the molecular nature of NF-κB pathway lesions predicted responsiveness. CD79A/B mutations correlated with STN sensitivity, whereas CARD11 mutations rendered ABC DLBCL cell lines insensitive. Growth inhibitory effects of PKC inhibition correlated with NF-κB pathway inhibition and were mediated by induction of G₁-phase cell-cycle arrest and/or cell death. We found that STN produced significant antitumor effects in a mouse xenograft model of CD79A/B-mutated DLBCL. Collectively, our findings offer a strong rationale for the clinical evaluation of STN in ABC DLBCL patients who harbor CD79 mutations also illustrating the necessity to stratify DLBCL patients according to their genetic abnormalities.

  3. Molecular analysis of lungs from pigs immunized with a mutant transferrin binding protein B-based vaccine and challenged with Haemophilus parasuis.

    PubMed

    Martínez-Martínez, S; Rodríguez-Ferri, E F; Frandoloso, R; Garrido-Pavón, J J; Zaldívar-López, S; Barreiro, C; Gutiérrez-Martín, C B

    2016-10-01

    The molecular analysis of pigs vaccinated with a mutant transferrin-binding protein B (Y167A) from Haemophilus parasuis was compared with that performed for unvaccinated challenged (UNCH) and unvaccinated unchallenged (UNUN) pigs. Microarray analysis revealed that UNCH group showed the most distinct expression profile for immune response genes, mainly for those genes involved in inflammation or immune cell trafficking. This fact was confirmed by real-time PCR, in which the greatest level of differential expression from this group were CD14, CD163, IL-8 and IL-12. In Y167A group, overexpressed genes included MAP3K8, CD14, IL-12 and CD163. Proteomics revealed that collagen α-1 and peroxiredoxins 2 and 6 were overexpressed in Y167A pigs. Our study reveals new data on genes and proteins involved in H. parasuis infection and several candidates of resistance to infection that are induced by Y167A vaccine. The expression of proinflammatory molecules from Y176A pigs is similar to their expression in UNUN pigs. PMID:27638122

  4. Kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab) coat protein and its mutants with ssDNA

    SciTech Connect

    Priyadarshini, C.G. Poornima; Savithri, H.S.

    2009-04-10

    Gemini viral assembly and transport of viral DNA into nucleus for replication, essentially involve DNA-coat protein interactions. The kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali recombinant coat protein (rCP) with DNA was studied by electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR). The rCP interacted with ssDNA with a K{sub A}, of 2.6 +- 0.29 x 10{sup 8} M{sup -1} in a sequence non-specific manner. The CP has a conserved C2H2 type zinc finger motif composed of residues C68, C72, H81 and H85. Mutation of these residues to alanine resulted in reduced binding to DNA probes. The H85A mutant rCP showed the least binding with approximately 756 fold loss in the association rate and a three order magnitude decrease in the binding affinity as compared to rCP. The CP-DNA interactions via the zinc finger motif could play a crucial role in virus assembly and in nuclear transport.

  5. Transient free radicals in iron/oxygen reconstitution of mutant protein R2 Y122F. Possible participants in electron transfer chains in ribonucleotide reductase.

    PubMed

    Sahlin, M; Lassmann, G; Pötsch, S; Sjöberg, B M; Gräslund, A

    1995-05-26

    Ferrous iron/oxygen reconstitution of the mutant R2 apoprotein Y122F leads to formation of a diferric center similar to that of the wild-type R2 protein of Escherichia coli ribonucleotide reductase. This reconstitution reaction requires two extra electrons, supplied or transferred by the protein matrix of R2. We observed several transient free radical species using stopped flow and freeze quench EPR and stopped flow UV-visible spectroscopy. Three of the radicals occur in the time window 0.1-2 s, i.e. concomitant with formation of the diferric site. They include a strongly iron-coupled radical (singlet EPR signal) observed only at < or = 77 K, a singlet EPR signal observed only at room temperature, and a radical at Tyr-356 (light absorption at 410 nm), an invariant residue proposed to be part of an electron transfer chain in catalysis. Three additional transient radicals species are observed in the time window 6 s to 20 min. Two of these are conclusively identified, by specific deuteration, as tryptophan radicals. Comparing side chain geometry and distance to the iron center with EPR characteristics of the radicals, we propose certain Trp residues in R2 as likely to harbor these transient radicals.

  6. Fusion Protein of Mutant B7-DC and Fc Enhances the Antitumor Immune Effect of GM-CSF-secreting Whole-cell Vaccine

    PubMed Central

    Kojima, Masatsugu; Murata, Satoshi; Mekata, Eiji; Takebayashi, Katsushi; Jaffee, Elizabeth M.; Tani, Tohru

    2015-01-01

    Summary B7-DC [also known as programmed death ligand 2 (PD-L2)] is a costimulatory molecule expressed predominantly on dendritic cells (DCs) and macrophages. In addition to its coinhibitory receptor, programmed death receptor 1 (PD-1), evidence suggests that B7-DC interacts with an unidentified costimulatory receptor on T cells. B7-DC mutants with selective binding capacity for the costimulatory receptor may be effective in stimulating antitumor immune responses, while avoiding the inhibitory effects of PD-1. In this study, we concomitantly administered a GM-CSF-secreting whole cell vaccine together with a fusion protein of mutant B7-DC and Fc portion (mB7-DC-Fc), which binds selectively to the costimulatory receptor. This lead to an increased number of tumor antigen-specific cytotoxic T lymphocytes both in the spleen and at the tumor site and complete elimination of established tumors in vivo. In addition, mB7-DC-Fc increased IFN-γ and IL-2 production and decreased IL-4 and IL-10 production in vitro, indicating that mB7-DC-Fc tips the Th1/Th2 balance toward Th1 dominance, which is more favorable for antitumor immunity. Furthermore, mB7-DC-Fc decreased the PD-1 + proportion of CD8 + T cells in vitro and tumor-infiltrating CD8 + T cells in vivo, suggesting that mB7-DC-Fc may maintain tumor-infiltrating CD8 + T cells in a nonexhausted state. In conclusion, mB7-DC-Fc administration during the T-cell priming phase enhances antitumor effects of vaccine by generating more tumor antigen-specific cytotoxic T lymphocytes and leading to their accumulation at the tumor site. We suggest that this combination approach may be a promising strategy for antitumor immunotherapy. PMID:24598447

  7. Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules.

    PubMed

    Nunbhakdi-Craig, Viyada; Schuechner, Stefan; Sontag, Jean-Marie; Montgomery, Lisa; Pallas, David C; Juno, Claudia; Mudrak, Ingrid; Ogris, Egon; Sontag, Estelle

    2007-05-01

    Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the B alpha regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation- (L309 Delta) and phosphorylation- (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B', and B''-type regulatory subunits in NIH 3T3 fibroblasts and neuro-2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309 Delta, and T304D mutants, which are impaired in their ability to associate with the B alpha subunit, induces their loss. Silencing of B alpha subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal B alpha-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.

  8. Core domain mutant Y220C of p53 protein has a key role in copper homeostasis in case of free fatty acids overload.

    PubMed

    Arciello, Mario; Longo, Alessia; Viscomi, Carmela; Capo, Concetta; Angeloni, Antonio; Rossi, Luisa; Balsano, Clara

    2015-12-01

    Nonalcoholic fatty liver disease (NAFLD) is a pathology that includes a wide variety of clinical conditions ranging from simple steatosis to end-stage liver diseases. Despite the huge amount of researches, the molecular basis of NAFLD are still not fully understood. Recently, it was suggested a role for p53 in NAFLD pathogenesis. Among its targets there is Synthesis of Cytochrome c Oxidase 2 (SCO2), a copper chaperone, involved in both aerobic respiration and metal cellular excretion. Copper seems to play a role in NAFLD. It was demonstrated a low hepatic copper content in NAFLD patients, which correlates with metabolic syndrome parameters. Copper homeostasis deregulation, in fact, seems to be related to lipid metabolism alteration and insulin resistance. Here we provide evidence on the role of p53 in the modulation of copper homeostasis, in an experimental model of NAFLD. We used two different hepatoma cell lines, HepG2 and Huh 7.5.1, characterized by the presence of wt p53 and its Y220C mutant, respectively, treated with a free fatty acids (FFAs) solution. Interestingly, p53 activation correlated with the intracellular copper level maintenance. We demonstrated that, in hepatoma cell lines, core domain mutant Y220C of p53 affects the modulation of SCO2 and Copper transporter 1 (CTR1), influencing, in this way, intracellular copper homeostasis in presence of FFAs accumulation, and that the 220 residue of the protein is crucial for such control. The role of p53 we highlighted may have deep implications in clinical conditions where copper homeostasis is deregulated.

  9. Streptavidin mutants

    DOEpatents

    Sano, Takeshi; Cantor, Charles R.; Vajda, Sandor; Reznik, Gabriel O.; Smith, Cassandra L.; Pandori, Mark W.

    2000-01-01

    The present invention relates to streptavidin proteins and peptides having a altered physical properties such as an increased stability or increased or decreased affinity for binding biotin. The invention also relates to methods for the detection, identification, separation and isolation of targets using streptavidin proteins or peptides. Streptavidin with increased or reduced affinity allows for the use of the streptavidin-biotin coupling systems for detection and isolation systems wherein it is necessary to remove of one or the other of the binding partners. Such systems are useful for the purification of functional proteins and viable cells. The invention also relates to nucleic acids which encode these streptavidin proteins and peptides and to recombinant cells such as bacteria, yeast and mammalian cells which contain these nucleic acids.

  10. Protein Kinase Cι Drives a NOTCH3-dependent Stem-like Phenotype in Mutant KRAS Lung Adenocarcinoma.

    PubMed

    Ali, Syed A; Justilien, Verline; Jamieson, Lee; Murray, Nicole R; Fields, Alan P

    2016-03-14

    We report that the protein kinase Cι (PKCι) oncogene controls expression of NOTCH3, a key driver of stemness, in KRAS-mediated lung adenocarcinoma (LADC). PKCι activates NOTCH3 expression by phosphorylating the ELF3 transcription factor and driving ELF3 occupancy on the NOTCH3 promoter. PKCι-ELF3-NOTCH3 signaling controls the tumor-initiating cell phenotype by regulating asymmetric cell division, a process necessary for tumor initiation and maintenance. Primary LADC tumors exhibit PKCι-ELF3-NOTCH3 signaling, and combined pharmacologic blockade of PKCι and NOTCH synergistically inhibits tumorigenic behavior in vitro and LADC growth in vivo demonstrating the therapeutic potential of PKCι-ELF3-NOTCH3 signal inhibition to more effectively treat KRAS LADC. PMID:26977885

  11. Generation of protein kinase Ck1α mutants which discriminate between canonical and non-canonical substrates

    PubMed Central

    Bustos, Victor H.; Marin, Oriano; Meggio, Flavio; Cesaro, Luca; Allende, Catherine C.; Allende, Jorge E.; Pinna, Lorenzo A.

    2005-01-01

    Protein kinase CK1 denotes a family of pleiotropic serine/threonine protein kinases implicated in a variety of cellular functions. Typically, CK1 acts as a ‘phosphate-directed’ kinase whose targeting is primed by a single phosphorylated side chain at position n−3 or n−4 relative to serine/threonine, but increasing evidence is accumulating that CK1 can also engage some of its substrates at sites that do not conform to this canonical consensus. In the present paper, we show that CK1α phosphorylates with the same efficiency phosphopeptides primed by a phosphoserine residue at either n−3 [pS(−3)] or n−4 [pS(−4)] positions. The phosphorylation efficiency of the pS(−4) peptide, and to a lesser extent that of the pS(−3) peptide, is impaired by the triple mutation of the lysine residues in the K229KQK232 stretch to alanine residues, promoting 40-fold and 6-fold increases of Km respectively. In both cases, the individual mutation of Lys232 is as detrimental as the triple mutation. A kinetic alanine-scan analysis with a series of substituted peptide substrates in which the priming phosphoserine residue was effectively replaced by a cluster of four aspartate residues was also consistent with a crucial role of Lys232 in the recognition of the acidic determinant at position n−4. In sharp contrast, the phosphorylation of β-catenin and of a peptide including the non-canonical β-catenin site (Ser45) lacking acidic/phosphorylated determinants upstream is not significantly affected by mutations in the KKQK stretch. These data provide a molecular insight into the structural features that underlie the site specificity of CK1α and disclose the possibility of developing strategies for the preferential targeting of subsets of CK1 substrates. PMID:15975091

  12. Structure of wild type and mutant F508del CFTR: A small-angle X-ray scattering study of the protein-detergent complexes.

    PubMed

    Pollock, Naomi L; Satriano, Letizia; Zegarra-Moran, Olga; Ford, Robert C; Moran, Oscar

    2016-04-01

    CFTR is an anionic channel expressed in epithelia whose mutations cause cystic fibrosis. Wild (WT) and mutated (F508del) types were over-expressed in yeast, solubilised in the detergent LPG-14 and purified. The detergent-CFTR complexes were studied by SAXS techniques using a solvent of variable density. The final result of the study is the numerical value of a set of parameters: molecular mass, volume and radius of gyration, average electron density and second moment of the electron density fluctuations inside the particles. It is also shown that in the complex the centres of gravity of CFTR and of the detergent are displaced relative to each other. The analysis of these parameters led to the determination of the size and shape of the volumes occupied by protein and by detergent in the complex. WT-CFTR to be an elongated molecule (maximum diameter ∼12.4nm) which spans a flat detergent micelle. The distance distribution function, P(r) confirms that the WT-CFTR is elongated and with an inhomogeneous electronic density. The F508del-CFTR molecule is also elongated (maximum diameter ∼13.2nm), but the associated detergent micelle hides a larger surface, plausibly related to an increased exposure of hydrophobic portions of the mutated protein. The corresponding P(r) is consistent with the presence of well defined domains, probably linked by flexible regions. These differences suggest that the full-length mutant F508del-CFTR has a detectably different conformation, in contrast to the minor differences observed for the isolated F508-containing domain. We interpret the data in terms of an incomplete post-translational assembly of the protein domains.

  13. Functional deficiency of NBN, the Nijmegen breakage syndrome protein, in a p.R215W mutant breast cancer cell line

    PubMed Central

    2014-01-01

    Background Mutations in NBN, the gene for Nijmegen Breakage Syndrome (NBS), are thought to predispose women to developing breast cancer, but a breast cancer cell line containing mutations in NBN has not yet been described. The p.R215W missense mutation occurs at sub-polymorphic frequencies in several populations. We aimed to investigate its functional impact in breast cancer cells from a carrier of this NBN mutation. Methods Breast cancer cell lines were screened by immunoblotting for NBN protein levels, and the NBN coding region was sequenced for mutation analysis. Radiosensitivity assays and functional studies were performed through immunocytochemistry and immunoblotting, and flow cytometry was employed to assess cell cycle progression. Impedance measurements were used to study the consequences of PARP1 inhibition. Statistical comparisons between cell lines were performed using t-tests. Results HCC1395 breast cancer cells exhibited reduced NBN protein levels. Direct sequencing identified the NBN p.R215W mutation in the hemizygous state, in addition to a truncation in BRCA1. Mutations in both genes were already present in the heterozygous state in the patient’s germline. HCC1395 cells were highly radiosensitive, susceptible to apoptosis and were deficient in the formation of NBN foci. There was also evidence for some impairment in the formation of γH2AX, MDC1, and 53BP1 foci after irradiation; these foci appeared smaller and irregular compared with repair foci in wild-type cells, although ATM signalling was largely unaffected. In line with their deficiency in NBN and BRCA1, HCC1395 cells were particularly sensitive to PARP1 inhibition. Conclusion Our results indicate that the p.R215W mutation in the HCC1395 breast cancer cell line impairs NBN function, making this cell line a potentially useful cellular model for studying defective NBN protein within a mutant BRCA1 background. PMID:24928521

  14. Trimethylangelicin promotes the functional rescue of mutant F508del CFTR protein in cystic fibrosis airway cells.

    PubMed

    Favia, Maria; Mancini, Maria T; Bezzerri, Valentino; Guerra, Lorenzo; Laselva, Onofrio; Abbattiscianni, Anna C; Debellis, Lucantonio; Reshkin, Stephan J; Gambari, Roberto; Cabrini, Giulio; Casavola, Valeria

    2014-07-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a protein kinase A (PKA)-activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however, the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4'-trimethylangelicin (TMA), besides inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with Pseudomonas aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild-type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 h after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities, also displays corrector properties.

  15. Molecular dynamics study of chemically engineered green fluorescent protein mutants: comparison of intramolecular fluorescence resonance energy transfer rate.

    PubMed

    Mitchell, Felicity L; Frank, Filipp; Marks, Gabriel E; Suzuki, Miho; Douglas, Kenneth T; Bryce, Richard A

    2009-04-01

    Because of its unusual spectroscopic properties, green fluorescent protein (GFP) has become a useful tool in molecular genetics, biochemistry and cell biology. Here, we computationally characterize the behavior of two GFP constructs, designed as bioprobes for enzymatic triggering using intramolecular fluorescence resonance energy transfer (FRET). These constructs differ in the location of an intramolecular FRET partner, an attached chemical chromophore (either near an N-terminal or C-terminal site). We apply the temperature replica exchange molecular dynamics method to the two flexible constructs in conjunction with a generalized Born implicit solvent model. The calculated rate of FRET was derived from the interchromophore distance, R, and orientational factor, kappa(2). In agreement with experiment, the construct with the C-terminally attached dye was predicted to have higher energy transfer rate than observed for the N-terminal construct. The molecular basis for this observation is discussed. In addition, we find that the orientational factor, kappa(2), deviates from the commonly assumed value, the implications of which are also considered.

  16. Isolation and Characterization of Noncytopathic Pestivirus Mutants Reveals a Role for Nonstructural Protein NS4B in Viral Cytopathogenicity

    PubMed Central

    Qu, Lin; McMullan, Laura K.; Rice, Charles M.

    2001-01-01

    Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viruses by the production of viral nonstructural protein NS3. However, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics and smaller plaques than NADL but remained cp. A number of independent ncp variants were isolated by puromycin selection. Remarkably, these ncp variants produced NS3 and viral RNA at levels comparable to those of the cp parent. Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasmic reticulum membrane. Other engineered substitutions for Y2441 also affected viral cytopathogenicity and viability, with Y2441V being cp, Y2441A being ncp, and Y2441D rendering the virus unable to replicate. The ncp substitutions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-linked in NADL-infected cells, indicating that they are associated as components of a multiprotein complex. Although the mechanism remains to be elucidated, these results demonstrate that mutations in NS4B can attenuate BVDV cytopathogenicity despite NS3 production. PMID:11602707

  17. Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation

    SciTech Connect

    Ansong, Charles; Yoon, Hyunjin; Porwollik, Steffen; Mottaz-Brewer, Heather; Petritis, Brianne O.; Jaitly, Navdeep; Adkins, Joshua N.; Mcclelland, Michael; Heffron, Fred; Smith, Richard D.

    2009-03-11

    In recent years the profound importance of sRNA-mediated translational/post-transcriptional regulation has been increasingly appreciated. However, the global role played by translational regulation in control of gene expression has never been elucidated in any organism for the simple reason that global proteomics methods required to accurately characterize post-transcriptional processes and the knowledge of translational control mechanisms have only become available within the last few years. The proteins Hfq and SmpB are essential for the biological activity of a range of regulatory sRNAs and thus provide a means to identify potential targets of sRNA regulation. We performed a sample-matched global proteomics and transcriptional analysis to examine the role of Hfq and SmpB in global protein translation and virulence using the Salmonella typhimurium model system. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all Salmonella proteins, respectively, with limited correlation between transcription and protein expression. This is the first report suggesting that SmpB could be a general translational regulator. The broad spectrum of proteins modulated by Hfq was also surprising including central metabolism, LPS biosynthesis, two-component regulatory systems, quorum sensing, SP1-TTSS, oxidative stress, fatty acid metabolism, nucleoside and nucleotide metabolism, envelope stress, aminoacyl-tRNA synthetases, amino acid biosynthesis, peptide transport, and motility.. The extent of global regulation of translation by Hfq is unexpected, with profound effects in all stages of Salmonella’s life cycle. Our results represent the first global systems-level analysis of translational regulation; the elucidated potential targets of sRNA regulation from our analysis will

  18. Mutant Forkhead L2 (FOXL2) proteins associated with premature ovarian failure (POF) dimerize with wild-type FOXL2, leading to altered regulation of genes associated with granulosa cell differentiation.

    PubMed

    Kuo, Fang-Ting; Bentsi-Barnes, Ikuko K; Barlow, Gillian M; Pisarska, Margareta D

    2011-10-01

    Premature ovarian failure in the autosomal dominant disorder blepharophimosis-ptosis-epicanthus inversus is due to mutations in the gene encoding Forkhead L2 (FOXL2), producing putative truncated proteins. We previously demonstrated that FOXL2 is a transcriptional repressor of the steroidogenic acute regulatory (StAR), P450SCC (CYP11A), P450aromatase (CYP19), and cyclin D2 (CCND2) genes, markers of ovarian follicle proliferation and differentiation. Furthermore, we found that mutations of FOXL2 may regulate wild-type FOXL2, leading to loss of transcriptional repression of CYP19, similar to StAR. However, the regulatory mechanisms underlying these premature ovarian failure-associated mutations remain largely unknown. Therefore, we examined the effects of a FOXL2 mutant protein on the transcriptional repression of the CYP19 promoter by the full-length protein. We found that mutant FOXL2 exerts a dominant-negative effect on the repression of CYP19 by wild-type FOXL2. Both wild-type and mutant FOXL2 and can form homo- and heterodimers. We identified a minimal -57-bp human CYP19 promoter containing two potential FOXL2-binding regions and found that both wild-type and mutant FOXL2 can bind to either of these regions. Mutational analysis revealed that either site is sufficient for transcriptional repression by wild-type FOXL2, and the dominant-negative effect of mutant FOXL2, but these are eliminated when both sites are mutated. These findings confirm that mutant FOXL2 exerts a dominant-negative effect on wild-type FOXL2's activity as a transcriptional repressor of key genes in ovarian follicle differentiation and suggest that this is likely due to heterodimer formation and possibly also competition for DNA binding.

  19. Mutant Forkhead L2 (FOXL2) Proteins Associated with Premature Ovarian Failure (POF) Dimerize with Wild-Type FOXL2, Leading to Altered Regulation of Genes Associated with Granulosa Cell Differentiation

    PubMed Central

    Kuo, Fang-Ting; Bentsi-Barnes, Ikuko K.; Barlow, Gillian M.

    2011-01-01

    Premature ovarian failure in the autosomal dominant disorder blepharophimosis-ptosis-epicanthus inversus is due to mutations in the gene encoding Forkhead L2 (FOXL2), producing putative truncated proteins. We previously demonstrated that FOXL2 is a transcriptional repressor of the steroidogenic acute regulatory (StAR), P450SCC (CYP11A), P450aromatase (CYP19), and cyclin D2 (CCND2) genes, markers of ovarian follicle proliferation and differentiation. Furthermore, we found that mutations of FOXL2 may regulate wild-type FOXL2, leading to loss of transcriptional repression of CYP19, similar to StAR. However, the regulatory mechanisms underlying these premature ovarian failure-associated mutations remain largely unknown. Therefore, we examined the effects of a FOXL2 mutant protein on the transcriptional repression of the CYP19 promoter by the full-length protein. We found that mutant FOXL2 exerts a dominant-negative effect on the repression of CYP19 by wild-type FOXL2. Both wild-type and mutant FOXL2 and can form homo- and heterodimers. We identified a minimal −57-bp human CYP19 promoter containing two potential FOXL2-binding regions and found that both wild-type and mutant FOXL2 can bind to either of these regions. Mutational analysis revealed that either site is sufficient for transcriptional repression by wild-type FOXL2, and the dominant-negative effect of mutant FOXL2, but these are eliminated when both sites are mutated. These findings confirm that mutant FOXL2 exerts a dominant-negative effect on wild-type FOXL2's activity as a transcriptional repressor of key genes in ovarian follicle differentiation and suggest that this is likely due to heterodimer formation and possibly also competition for DNA binding. PMID:21862621

  20. Manipulation of cellular GSH biosynthetic capacity via TAT-mediated protein transduction of wild-type or a dominant-negative mutant of glutamate cysteine ligase alters cell sensitivity to oxidant-induced cytotoxicity

    SciTech Connect

    Backos, Donald S.; Brocker, Chad N.; Franklin, Christopher C.

    2010-02-15

    The glutathione (GSH) antioxidant defense system plays a central role in protecting mammalian cells against oxidative injury. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH biosynthesis and is a heterodimeric holoenzyme composed of catalytic (GCLC) and modifier (GCLM) subunits. As a means of assessing the cytoprotective effects of enhanced GSH biosynthetic capacity, we have developed a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. Bacterial expression vectors encoding TAT fusion proteins of both GCL subunits were generated and recombinant fusion proteins were synthesized and purified to near homogeneity. The TAT-GCL fusion proteins were capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins resulted in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and GSH levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL. Transduction of cells with a catalytically inactive GCLC(E103A)-TAT mutant decreased cellular GCL activity in a dose-dependent manner. TAT-mediated manipulation of cellular GCL activity was also functionally relevant as transduction with wild-type GCLC(WT)-TAT or mutant GCLC(E103A)-TAT conferred protection or enhanced sensitivity to H{sub 2}O{sub 2}-induced cell death, respectively. These findings demonstrate that TAT-mediated transduction of wild-type or dominant-inhibitory mutants of the GCL subunits is a viable means of manipulating cellular GCL activity to assess the effects of altered GSH biosynthetic capacity.

  1. Environments of the four tryptophans in the extracellular domain of human tissue factor: comparison of results from absorption and fluorescence difference spectra of tryptophan replacement mutants with the crystal structure of the wild-type protein.

    PubMed Central

    Hasselbacher, C A; Rusinova, E; Waxman, E; Rusinova, R; Kohanski, R A; Lam, W; Guha, A; Du, J; Lin, T C; Polikarpov, I

    1995-01-01

    The local environments of the four tryptophan residues of the extracellular domain of human tissue factor (sTF) were assessed from difference absorption and fluorescence spectra. The difference spectra were derived by subtracting spectra from single Trp-to-Phe or Trp-to-Tyr replacement mutants from the corresponding spectrum of the wild-type protein. Each of the mutants was capable of enhancing the proteolytic activity of factor VIIa showing that the mutations did not introduce major structural changes, although the mutants were more susceptible to denaturation by guanidinium chloride. The difference spectra indicate that the Trp residues are buried to different extents within the protein matrix. This evaluation was compared with the x-ray crystal structure of sTF. There is excellent agreement between predictions from the difference spectra and the environments of the Trp residues observed in the x-ray crystal structure, demonstrating that difference absorption and particularly fluorescence spectra derived from functional single-Trp replacement mutants can be used to obtain information about the local environments of individual Trp residues in multi-tryptophan proteins. Images FIGURE 7 FIGURE 8 PMID:7669897

  2. Mutational analysis of human T-cell leukemia virus type I Tax: regions necessary for function determined with 47 mutant proteins.

    PubMed Central

    Semmes, O J; Jeang, K T

    1992-01-01

    We have made 47 mutations that span the length of the human T-cell leukemia virus type I (HTLV-I) Tax open reading frame. Of the 47 mutations, 38 were substitutions of single amino acids, 5 were missense changes in two or more amino acids, and 4 were deletions. A subset of these mutations includes individual changes of all 26 naturally occurring serines to alanines. By assaying each mutant protein separately on the HTLV-I long terminal repeat (LTR) and the human immunodeficiency virus type 1 (HIV-1) LTR in parallel, we were able to identify regions of Tax selectively necessary for each promoter. A small region in the carboxyl terminus, amino acids 315 to 325, was found to be selectively important for activation of the HTLV-I LTR. Three changes at serine 113, serine 160, and serine 258 were found to specifically affect function on the HIV-1 LTR. Surprisingly, we found that the great preponderance of missense changes (32 of 42) in Tax did not affect function. Images PMID:1433511

  3. Gene Mapping of a Mutant Mungbean (Vigna radiata L.) Using New Molecular Markers Suggests a Gene Encoding a YUC4-like Protein Regulates the Chasmogamous Flower Trait

    PubMed Central

    Chen, Jingbin; Somta, Prakit; Chen, Xin; Cui, Xiaoyan; Yuan, Xingxing; Srinives, Peerasak

    2016-01-01

    Mungbean (Vigna radiata L.) is a cleistogamous plant in which flowers are pollinated before they open, which prevents yield improvements through heterosis. We previously generated a chasmogamous mutant (CM) mungbean in which open flowers are pollinated. In this study, we developed insertion/deletion (indel) markers based on the transcriptome differences between CM and Sulu-1 (i.e., normal flowering) plants. An F2 population derived from a cross between CM and Sulu-1 was used for gene mapping. Segregation analyses revealed that a single recessive gene regulates the production of chasmogamous flowers. Using newly developed indel and simple sequence repeat markers, the cha gene responsible for the chasmogamous flower trait was mapped to a 277.1-kb segment on chromosome 6. Twelve candidate genes were detected in this segment, including Vradi06g12650, which encodes a YUCCA family protein associated with floral development. A single base pair deletion producing a frame-shift mutation and a premature stop codon in Vradi06g12650 was detected only in CM plants. This suggested that Vradi06g12650 is a cha candidate gene. Our results provide important information for the molecular breeding of chasmogamous mungbean lines, which may serve as new genetic resources for hybrid cultivar development. PMID:27375671

  4. An Adenovirus Type 5 Mutant with the Preterminal Protein Gene Deleted Efficiently Provides Helper Functions for the Production of Recombinant Adeno-Associated Virus

    PubMed Central

    Maxwell, Ian H.; Maxwell, Francoise; Schaack, Jerome

    1998-01-01

    Production of recombinant adeno-associated virus (rAAV) requires helper functions that have routinely been provided by infection of the producer cells with adenovirus. Complete removal and/or inactivation of progeny adenovirus, present in such rAAV preparations, presents significant difficulty. Here, we report that an adenovirus type 5 (Ad5) mutant with the preterminal protein (pTP) gene deleted can provide helper function for the growth of rAAV. At high multiplicity, Ad5dl308ΔpTP was as efficient as the phenotypically wild-type Ad5dl309 in permitting growth of rAAV. Use of Ad5dl308ΔpTP, which is incapable of replication in the absence of complementation for pTP, as a helper avoids the need to remove contaminating adenovirus infectious activity by heat inactivation or by purification. Comparison of the transducing ability of rAAV generated with either Ad5dl308ΔpTP or Ad5dl309 as a helper demonstrated that the heat inactivation protocol generally used does not remove all of the helper Ad5dl309 function. PMID:9733887

  5. A novel mutation in the SLC25A15 gene in a Turkish patient with HHH syndrome: functional analysis of the mutant protein.

    PubMed

    Ersoy Tunalı, Nagehan; Marobbio, Carlo M T; Tiryakioğlu, N Ozan; Punzi, Giuseppe; Saygılı, Seha K; Onal, Hasan; Palmieri, Ferdinando

    2014-05-01

    The hyperornithinemia-hyperammonemia-homocitrullinuria syndrome is a rare autosomal recessive disorder caused by the functional deficiency of the mitochondrial ornithine transporter 1 (ORC1). ORC1 is encoded by the SLC25A15 gene and catalyzes the transport of cytosolic ornithine into mitochondria in exchange for citrulline. Although the age of onset and the severity of the symptoms vary widely, the disease usually manifests in early infancy. The typical clinical features include protein intolerance, lethargy, episodic confusion, cerebellar ataxia, seizures and mental retardation. In this study, we identified a novel p.Ala15Val (c.44C>T) mutation by genomic DNA sequencing in a Turkish child presenting severe tantrum, confusion, gait disturbances and loss of speech abilities in addition to hyperornithinemia, hyperammonemia and homocitrullinuria. One hundred Turkish control chromosomes did not possess this variant. The functional effect of the novel mutation was assessed by both complementation of the yeast ORT1 null mutant and transport assays. Our study demonstrates that the A15V mutation dramatically interferes with the transport properties of ORC1 since it was shown to inhibit ornithine transport nearly completely. PMID:24721342

  6. Small Molecule DFPM Derivative-Activated Plant Resistance Protein Signaling in Roots Is Unaffected by EDS1 Subcellular Targeting Signal and Chemical Genetic Isolation of victr R-Protein Mutants.

    PubMed

    Kunz, Hans-Henning; Park, Jiyoung; Mevers, Emily; García, Ana V; Highhouse, Samantha; Gerwick, William H; Parker, Jane E; Schroeder, Julian I

    2016-01-01

    The small molecule DFPM ([5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione) was recently shown to trigger signal transduction via early effector-triggered immunity signaling genes including EDS1 and PAD4 in Arabidopsis thaliana accession Col-0. Chemical genetic analyses of A. thaliana natural variants identified the plant Resistance protein-like Toll/Interleukin1 Receptor (TIR)-Nucleotide Binding (NB)-Leucine-Rich Repeat (LRR) protein VICTR as required for DFPM-mediated root growth arrest. Here a chemical genetic screen for mutants which disrupt DFPM-mediated root growth arrest in the Col-0 accession identified new mutant alleles of the TIR-NB-LRR gene VICTR. One allele, victr-6, carries a Gly216-to-Asp mutation in the Walker A domain supporting an important function of the VICTR nucleotide binding domain in DFPM responses consistent with VICTR acting as a canonical Resistance protein. The essential nucleo-cytoplasmic regulator of TIR-NB-LRR-mediated effector-triggered immunity, EDS1, was reported to have both nuclear and cytoplasmic actions in pathogen resistance. DFPM was used to investigate the requirements for subcellular EDS1 localization in DFPM-mediated root growth arrest. EDS1-YFP fusions engineered to localize mainly in the cytoplasm or the nucleus by tagging with a nuclear export signal (NES) or a nuclear localization signal (NLS), respectively, were tested. We found that wild-type EDS1-YFP and both the NES and NLS-tagged EDS1 variants were induced by DFPM treatments and fully complemented eds1 mutant plants in root responses to DFPM, suggesting that enrichment of EDS1 in either compartment could confer DFPM-mediated root growth arrest. We further found that a light and O2-dependent modification of DFPM is necessary to mediate DFPM signaling in roots. Chemical analyses including Liquid Chromatography-Mass Spectrometry and High-Resolution Atmospheric Pressure Chemical Ionization Mass Spectrometry identified a DFPM modification product that is

  7. Small Molecule DFPM Derivative-Activated Plant Resistance Protein Signaling in Roots Is Unaffected by EDS1 Subcellular Targeting Signal and Chemical Genetic Isolation of victr R-Protein Mutants

    PubMed Central

    Mevers, Emily; García, Ana V.; Highhouse, Samantha; Gerwick, William H.; Parker, Jane E.; Schroeder, Julian I.

    2016-01-01

    The small molecule DFPM ([5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione) was recently shown to trigger signal transduction via early effector-triggered immunity signaling genes including EDS1 and PAD4 in Arabidopsis thaliana accession Col-0. Chemical genetic analyses of A. thaliana natural variants identified the plant Resistance protein-like Toll/Interleukin1 Receptor (TIR)-Nucleotide Binding (NB)-Leucine-Rich Repeat (LRR) protein VICTR as required for DFPM-mediated root growth arrest. Here a chemical genetic screen for mutants which disrupt DFPM-mediated root growth arrest in the Col-0 accession identified new mutant alleles of the TIR-NB-LRR gene VICTR. One allele, victr-6, carries a Gly216-to-Asp mutation in the Walker A domain supporting an important function of the VICTR nucleotide binding domain in DFPM responses consistent with VICTR acting as a canonical Resistance protein. The essential nucleo-cytoplasmic regulator of TIR-NB-LRR-mediated effector-triggered immunity, EDS1, was reported to have both nuclear and cytoplasmic actions in pathogen resistance. DFPM was used to investigate the requirements for subcellular EDS1 localization in DFPM-mediated root growth arrest. EDS1-YFP fusions engineered to localize mainly in the cytoplasm or the nucleus by tagging with a nuclear export signal (NES) or a nuclear localization signal (NLS), respectively, were tested. We found that wild-type EDS1-YFP and both the NES and NLS-tagged EDS1 variants were induced by DFPM treatments and fully complemented eds1 mutant plants in root responses to DFPM, suggesting that enrichment of EDS1 in either compartment could confer DFPM-mediated root growth arrest. We further found that a light and O2-dependent modification of DFPM is necessary to mediate DFPM signaling in roots. Chemical analyses including Liquid Chromatography-Mass Spectrometry and High-Resolution Atmospheric Pressure Chemical Ionization Mass Spectrometry identified a DFPM modification product that is

  8. Small Molecule DFPM Derivative-Activated Plant Resistance Protein Signaling in Roots Is Unaffected by EDS1 Subcellular Targeting Signal and Chemical Genetic Isolation of victr R-Protein Mutants.

    PubMed

    Kunz, Hans-Henning; Park, Jiyoung; Mevers, Emily; García, Ana V; Highhouse, Samantha; Gerwick, William H; Parker, Jane E; Schroeder, Julian I

    2016-01-01

    The small molecule DFPM ([5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione) was recently shown to trigger signal transduction via early effector-triggered immunity signaling genes including EDS1 and PAD4 in Arabidopsis thaliana accession Col-0. Chemical genetic analyses of A. thaliana natural variants identified the plant Resistance protein-like Toll/Interleukin1 Receptor (TIR)-Nucleotide Binding (NB)-Leucine-Rich Repeat (LRR) protein VICTR as required for DFPM-mediated root growth arrest. Here a chemical genetic screen for mutants which disrupt DFPM-mediated root growth arrest in the Col-0 accession identified new mutant alleles of the TIR-NB-LRR gene VICTR. One allele, victr-6, carries a Gly216-to-Asp mutation in the Walker A domain supporting an important function of the VICTR nucleotide binding domain in DFPM responses consistent with VICTR acting as a canonical Resistance protein. The essential nucleo-cytoplasmic regulator of TIR-NB-LRR-mediated effector-triggered immunity, EDS1, was reported to have both nuclear and cytoplasmic actions in pathogen resistance. DFPM was used to investigate the requirements for subcellular EDS1 localization in DFPM-mediated root growth arrest. EDS1-YFP fusions engineered to localize mainly in the cytoplasm or the nucleus by tagging with a nuclear export signal (NES) or a nuclear localization signal (NLS), respectively, were tested. We found that wild-type EDS1-YFP and both the NES and NLS-tagged EDS1 variants were induced by DFPM treatments and fully complemented eds1 mutant plants in root responses to DFPM, suggesting that enrichment of EDS1 in either compartment could confer DFPM-mediated root growth arrest. We further found that a light and O2-dependent modification of DFPM is necessary to mediate DFPM signaling in roots. Chemical analyses including Liquid Chromatography-Mass Spectrometry and High-Resolution Atmospheric Pressure Chemical Ionization Mass Spectrometry identified a DFPM modification product that is

  9. Members of a Novel Family of Mammalian Protein Kinases Complement the DNA-Negative Phenotype of a Vaccinia Virus ts Mutant Defective in the B1 Kinase

    PubMed Central

    Boyle, Kathleen A.; Traktman, Paula

    2004-01-01

    Temperature-sensitive (ts) mutants of vaccinia virus defective in the B1 kinase demonstrate a conditionally lethal defect in DNA synthesis. B1 is the prototypic member of a new family of protein kinases (vaccinia virus-related kinases, or VRK) that possess distinctive B1-like sequence features within their catalytic motifs (R. J. Nichols and P. Traktman, J. Biol. Chem., in press). Given the striking sequence similarity between B1 and the VRK enzymes, we proposed that they might share overlapping substrate specificity. We therefore sought to determine whether the human and mouse VRK1 enzymes (hVRK1 and mVRK1, respectively) could complement a B1 deficiency in vivo. Recombinant ts2 viruses expressing hVRK1, mVRK1, or wild-type B1 were able to synthesize viral DNA at high temperature, but those expressing the more distantly related human casein kinase 1α2 could not. Complementation required the enzymatic activity of hVRK1, since a catalytically inactive allele of hVRK1 was unable to confer a temperature-insensitive phenotype. Interestingly, rescue of viral DNA synthesis was not coupled to the ability to phosphorylate H5, the only virus-encoded protein shown to be a B1 substrate in vivo. Expression of hVRK1 during nonpermissive ts2 infections restored virus production and plaque formation, whereas expression of mVRK1 resulted in an intermediate level of rescue. Taken together, these observations indicate that enzymatically active cellular VRK1 kinases can perform the function(s) of B1 required for genome replication, most likely due to overlapping specificity for cellular and/or viral substrates. PMID:14747564

  10. Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants

    PubMed Central

    Lee, Sunmin; Tsutsumi, Shinji; Yim, Kendrick; Rivas, Candy; Alarcon, Sylvia; Schwartz, Harvey; Khamit-Kush, Kofi; Scroggins, Bradley T.; Beebe, Kristin; Trepel, Jane B.; Neckers, Len

    2015-01-01

    The two cytosolic/nuclear isoforms of the molecular chaperone HSP90, stress-inducible HSP90α and constitutively expressed HSP90β, fold, assemble and maintain the three-dimensional structure of numerous client proteins. Because many HSP90 clients are important in cancer, several HSP90 inhibitors have been evaluated in the clinic. However, little is known concerning possible unique isoform or conformational preferences of either individual HSP90 clients or inhibitors. In this report, we compare the relative interaction strength of both HSP90α and HSP90β with the transcription factors HSF1 and HIF1α, the kinases ERBB2 and MET, the E3-ubiquitin ligases KEAP1 and RHOBTB2, and the HSP90 inhibitors geldanamycin and ganetespib. We observed unexpected differences in relative client and drug preferences for the two HSP90 isoforms, with HSP90α binding each client protein with greater apparent affinity compared to HSP90β, while HSP90β bound each inhibitor with greater relative interaction strength compared to HSP90α. Stable HSP90 interaction was associated with reduced client activity. Using a defined set of HSP90 conformational mutants, we found that some clients interact strongly with a single, ATP-stabilized HSP90 conformation, only transiently populated during the dynamic HSP90 chaperone cycle, while other clients interact equally with multiple HSP90 conformations. These data suggest different functional requirements among HSP90 clientele that, for some clients, are likely to be ATP-independent. Lastly, the two inhibitors examined, although sharing the same binding site, were differentially able to access distinct HSP90 conformational states. PMID:26517842

  11. O2 activation by non-heme diiron proteins: identification of a symmetric mu-1,2-peroxide in a mutant of ribonucleotide reductase.

    PubMed

    Moënne-Loccoz, P; Baldwin, J; Ley, B A; Loehr, T M; Bollinger, J M

    1998-10-20

    Non-heme diiron clusters occur in a number of enzymes (e.g., ribonucleotide reductase, methane monooxygenase, and Delta9-stearoyl-ACP desaturase) that activate O2 for chemically difficult oxidation reactions. In each case, a kinetically labile peroxo intermediate is believed to form when O2 reacts with the diferrous enzyme, followed by O-O bond cleavage and the formation of high-valent iron intermediates [formally Fe(IV)] that are thought to be the reactive oxidants. Greater kinetic stability of a peroxodiiron(III) intermediate in protein R2 of ribonucleotide reductase was achieved by the iron-ligand mutation Asp84 --> Glu and the surface mutation Trp48 --> Phe. Here, we present the first definitive evidence for a bridging, symmetrical peroxo adduct from vibrational spectroscopic studies of the freeze-trapped intermediate of this mutant R2. Isotope-sensitive bands are observed at 870, 499, and 458 cm-1 that are assigned to the intraligand peroxo stretching frequency and the asymmetric and symmetric Fe-O2-Fe stretching frequencies, respectively. Similar results have been obtained in the resonance Raman spectroscopic study of a peroxodiferric species of Delta9-stearoyl-ACP desaturase [Broadwater, J. A., Ai, J., Loehr, T. M., Sanders-Loehr, J., and Fox, B. G. (1998) Biochemistry 37, 14664-14671]. Similarities among these adducts and transient species detected during O2 activation by methane monooxygenase hydroxylase, ferritin, and wild-type protein R2 suggest the symmetrical peroxo adduct as a common intermediate in the diverse oxidation reactions mediated by members of this class.

  12. Abscisic Acid–Responsive Guard Cell Metabolomes of Arabidopsis Wild-Type and gpa1 G-Protein Mutants[C][W

    PubMed Central

    Jin, Xiaofen; Wang, Rui-Sheng; Zhu, Mengmeng; Jeon, Byeong Wook; Albert, Reka; Chen, Sixue; Assmann, Sarah M.

    2013-01-01

    Individual metabolites have been implicated in abscisic acid (ABA) signaling in guard cells, but a metabolite profile of this specialized cell type is lacking. We used liquid chromatography–multiple reaction monitoring mass spectrometry for targeted analysis of 85 signaling-related metabolites in Arabidopsis thaliana guard cell protoplasts over a time course of ABA treatment. The analysis utilized ∼350 million guard cell protoplasts from ∼30,000 plants of the Arabidopsis Columbia accession (Col) wild type and the heterotrimeric G-protein α subunit mutant, gpa1, which has ABA-hyposensitive stomata. These metabolomes revealed coordinated regulation of signaling metabolites in unrelated biochemical pathways. Metabolites clustered into different temporal modules in Col versus gpa1, with fewer metabolites showing ABA-altered profiles in gpa1. Ca2+-mobilizing agents sphingosine-1-phosphate and cyclic adenosine diphosphate ribose exhibited weaker ABA-stimulated increases in gpa1. Hormone metabolites were responsive to ABA, with generally greater responsiveness in Col than in gpa1. Most hormones also showed different ABA responses in guard cell versus mesophyll cell metabolomes. These findings suggest that ABA functions upstream to regulate other hormones, and are also consistent with G proteins modulating multiple hormonal signaling pathways. In particular, indole-3-acetic acid levels declined after ABA treatment in Col but not gpa1 guard cells. Consistent with this observation, the auxin antagonist α-(phenyl ethyl-2-one)-indole-3-acetic acid enhanced ABA-regulated stomatal movement and restored partial ABA sensitivity to gpa1. PMID:24368793

  13. The single N-glycan deletion mutant of soluble ErbB3 protein attenuates heregulin β1-induced tumor progression by blocking of the HIF-1 and Nrf2 pathway

    SciTech Connect

    Takamiya, Rina Takahashi, Motoko; Uehara, Yasuaki; Ariki, Shigeru; Hashimoto, Jiro; Hasegawa, Yoshihiro; Kuroki, Yoshio

    2014-11-21

    Highlights: • The sErbB3 N418Q mutant blocks heregulin β1 induced nuclear accumulation of HIF-1α. • The sErbB3 N418Q mutant attenuates cancer cell migration induced by heregulin β1. • The sErbB3 N418Q mutant blocks heregulin β1 induced nuclear accumulation of Nrf2. • The sErbB3 N418Q mutant may be a potential therapeutic application for tumor. - Abstract: It has been well documented that activation of the ErbB3–PI3K–Akt pathway is implicated in tumor survival and progression. We previously demonstrated that the single N-glycan deletion mutant of soluble ErbB3 protein (sErbB3 N418Q) attenuates heregulin β1-induced ErbB3 signaling. The active PI3K–Akt pathway augments the nuclear accumulation of hypoxia inducible factor (HIF)-1α, which activates the transcription of many target genes and drives cancer progression. In this study, we focused on the effects of sErbB3 N418Q mutant on nuclear accumulation of HIF-1α. Pretreatment with the sErbB3 N418Q mutant suppressed heregulin β1-induced HIF-1α activation in MCF7 cells. Similar results were also obtained in other breast cancer cell lines, T47D and BT474. Interestingly, these suppressive effects were not observed with the sErbB3 wild type. In addition, pretreatment with the sErbB3 N418Q mutant suppressed the cell migration of MCF7 cells induced by heregulin β1. Furthermore, incubation with heregulin β1 also induced the nuclear accumulation of Nrf2, and this effect was also reduced by the sErbB3 N418Q mutant, but not the sErbB3 wild type. These findings indicated that the sErbB3 N418Q mutant suppressed malignant formation of cancer cells by blocking of the HIF-1α and Nrf2 pathways.

  14. Proteomic analysis of proteins related to rice grain chalkiness using iTRAQ and a novel comparison system based on a notched-belly mutant with white-belly

    PubMed Central

    2014-01-01

    Background Grain chalkiness is a complex trait adversely affecting appearance and milling quality, and therefore has been one of principal targets for rice improvement. Eliminating chalkiness from rice has been a daunting task due to the complex interaction between genotype and environment and the lack of molecular markers. In addition, the molecular mechanisms underlying grain chalkiness formation are still imperfectly understood. Results We identified a notched-belly mutant (DY1102) with high percentage of white-belly, which only occurs in the bottom part proximal to the embryo. Using this mutant, a novel comparison system that can minimize the effect of genetic background and growing environment was developed. An iTRAQ-based comparative display of the proteins between the bottom chalky part and the upper translucent part of grains of DY1102 was performed. A total of 113 proteins responsible for chalkiness formation was identified. Among them, 70 proteins are up-regulated and 43 down-regulated. Approximately half of these differentially expressed proteins involved in central metabolic or regulatory pathways including carbohydrate metabolism (especially cell wall synthesis) and protein synthesis, folding and degradation, providing proteomic confirmation of the notion that chalkiness formation involves diverse but delicately regulated pathways. Protein metabolism was the most abundant category, accounting for 27.4% of the total differentially expressed proteins. In addition, down regulation of PDIL 2–3 and BiP was detected in the chalky tissue, indicating the important role of protein metabolism in grain chalkiness formation. Conclusions Using this novel comparison system, our comprehensive survey of endosperm proteomics in the notched-belly mutant provides a valuable proteomic resource for the characterization of pathways contributing to chalkiness formation at molecular and biochemical levels. PMID:24924297

  15. Construction of mouse adenovirus type 1 mutants.

    PubMed

    Cauthen, Angela N; Welton, Amanda R; Spindler, Katherine R

    2007-01-01

    Mouse adenovirus provides a model for studying adenovirus pathogenesis in the natural host. The ability to make viral mutants allows the investigation of specific mouse adenoviral gene contributions to virus-host interactions. Methods for propagation and titration of wild-type mouse adenovirus, production of viral DNA and viral DNA-protein complex, and transfection of mouse cells to obtain mouse adenovirus mutants are described in this chapter. Plaque purification, propagation, and titration of the mutant viruses are also presented.

  16. cAMP-specific phosphodiesterase HSPDE4D3 mutants which mimic activation and changes in rolipram inhibition triggered by protein kinase A phosphorylation of Ser-54: generation of a molecular model.

    PubMed Central

    Hoffmann, R; Wilkinson, I R; McCallum, J F; Engels, P; Houslay, M D

    1998-01-01

    Ser-13 and Ser-54 were shown to provide the sole sites for the protein kinase A (PKA)-mediated phosphorylation of the human cAMP-specific phosphodiesterase isoform HSPDE4D3. The ability of PKA to phosphorylate and activate HSPDE4D3 was mimicked by replacing Ser-54 with either of the negatively charged amino acids, aspartate or glutamate, within the consensus motif of RRES54. The PDE4 selective inhibitor rolipram ¿4-[3-(cyclopentoxy)-4-methoxyphenyl]-2-pyrrolidone¿ inhibited both PKA-phosphorylated HSPDE4D3 and the Ser-54-->Asp mutant, with an IC50 value that was approximately 8-fold lower than that seen for the non-PKA-phosphorylated enzyme. Lower IC50 values for inhibition by rolipram were seen for a wide range of non-activated residue 54 mutants, except for those which had side-chains able to serve as hydrogen-bond donors, namely the Ser-54-->Thr, Ser-54-->Tyr and Ser-54-->Cys mutants. The Glu-53-->Ala mutant exhibited an activity comparable with that of the PKA phosphorylated native enzyme and the Ser-54-->Asp mutant but, in contrast to the native enzyme, was insensitive to activation by PKA, despite being more rapidly phosphorylated by this protein kinase. The activated Glu-53-->Ala mutant exhibited a sensitivity to inhibition by rolipram which was unchanged from that of the native enzyme. The double mutant, Arg-51-->Ala/Arg-52-->Ala, showed no change in either enzyme activity or rolipram inhibition from the native enzyme and was incapable of providing a substrate for PKA phosphorylation at Ser-54. No difference in inhibition by dipyridamole was seen for the native enzyme and the Ser-54-->Asp and Ser-54-->Ala mutants. A model is proposed which envisages that phosphorylation by PKA triggers at least two distinct conformational changes in HSPDE4D3; one of these gives rise to enzyme activation and another enhances sensitivity to inhibition by rolipram. Activation of HSPDE4D3 by PKA-mediated phosphorylation is suggested to involve disruption of an ion

  17. A vaccine based on a mutant transferrin binding protein B of Haemophilus parasuis induces a strong T-helper 2 response and bacterial clearance after experimental infection.

    PubMed

    Martínez-Martínez, Sonia; Frandoloso, Rafael; Rodríguez-Ferri, Elías-Fernando; García-Iglesias, María-José; Pérez-Martínez, Claudia; Álvarez-Estrada, Álvaro; Gutiérrez-Martín, César-Bernardo

    2016-10-15

    This study aimed to characterize the type of immune response induced by an experimental vaccine based on a mutant Haemophilus parasuis transferrin binding protein (Tbp) B (Y167A) defective in its ability to bind porcine transferrin. Clinical and pathological signs, bacterial clearance, antibody response and the cytokine profile in alveolar macrophages and spleen after the vaccination and challenge of twenty-two colostrum-deprived pigs with 10(8) CFU of H. parasuis were analysed. Pigs vaccinated with Y167A were compared to those vaccinated with native TbpB (nTbpB), those treated with a commercial bacterin (CB) against Glässer's disease, those unvaccinated challenged (CH) and those unvaccinated unchallenged (UNCH) pigs. The rectal temperatures of Y167A pigs resembled those of UNCH pigs and were significantly lower than those of the nTbpB, CB and CH animals. A major reduction in pathological changes of the challenged pigs was observed in the Y167A group. H. parasuis was cleared from 88.9% of the samples from Y167A pigs versus 60.0% and 55.6% from those of the CB and nTbpB groups, respectively. The antibody response elicited by Y167A by ELISA was notably higher than that observed for nTbpB and CB pigs and was capable of preventing the expression and secretion of IL-8. The expression of IL-4 and IL-5, which were associated with the specific antibody levels, suggests that the main mechanism of protection conferred by Y167A vaccine is based on a strong T-helper 2 response. PMID:27590421

  18. A mouse model of Townes-Brocks syndrome expressing a truncated mutant Sall1 protein is protected from acute kidney injury.

    PubMed

    Hirsch, Sara; El-Achkar, Tarek; Robbins, Lynn; Basta, Jeannine; Heitmeier, Monique; Nishinakamura, Ryuichi; Rauchman, Michael

    2015-11-15

    It has been postulated that developmental pathways are reutilized during repair and regeneration after injury, but functional analysis of many genes required for kidney formation has not been performed in the adult organ. Mutations in SALL1 cause Townes-Brocks syndrome (TBS) and nonsyndromic congenital anomalies of the kidney and urinary tract, both of which lead to childhood kidney failure. Sall1 is a transcriptional regulator that is expressed in renal progenitor cells and developing nephrons in the embryo. However, its role in the adult kidney has not been investigated. Using a mouse model of TBS (Sall1TBS), we investigated the role of Sall1 in response to acute kidney injury. Our studies revealed that Sall1 is expressed in terminally differentiated renal epithelia, including the S3 segment of the proximal tubule, in the mature kidney. Sall1TBS mice exhibited significant protection from ischemia-reperfusion injury and aristolochic acid-induced nephrotoxicity. This protection from acute injury is seen despite the presence of slowly progressive chronic kidney disease in Sall1TBS mice. Mice containing null alleles of Sall1 are not protected from acute kidney injury, indicating that expression of a truncated mutant protein from the Sall1TBS allele, while causative of congenital anomalies, protects the adult kidney from injury. Our studies further revealed that basal levels of the preconditioning factor heme oxygenase-1 are elevated in Sall1TBS kidneys, suggesting a mechanism for the relative resistance to injury in this model. Together, these studies establish a functional role for Sall1 in the response of the adult kidney to acute injury. PMID:26311113

  19. Motor neuron cell death in wobbler mutant mice follows overexpression of the G-protein-coupled, protease-activated receptor for thrombin.

    PubMed Central

    Festoff, B. W.; D'Andrea, M. R.; Citron, B. A.; Salcedo, R. M.; Smirnova, I. V.; Andrade-Gordon, P.

    2000-01-01

    BACKGROUND: Mechanisms underlying neurodegeneration are actively sought for new therapeutic strategies. Transgenic, knockout and genetic mouse models greatly aid our understanding of the mechanisms for neuronal cell death. A naturally occurring, autosomal recessive mutant, known as wobbler, and mice transgenic for familial amyotrophic lateral sclerosis (FALS) superoxide dismutase (SOD)1 mutations are available, but the molecular mechanisms remain equally unknown. Both phenotypes are detectable after birth. Wobbler is detectable in the third week of life, when homozygotes (wr/wr) exhibit prominent gliosis and significant motor neuron loss in the cervical, but not in lumbar, spinal cord segments. To address molecular mechanisms, we evaluated "death signals" associated with the multifunctional serine protease, thrombin, which leads to apoptotic motor neuronal cell death in culture by cleavage of a G-protein coupled, protease-activated receptor 1 (PAR-1). MATERIALS AND METHODS: Thrombin activities were determined with chromogenic substrate assays, Western immunoblots and immunohistochemistry were performed with anti-PAR-1 to observe localizations of the receptor and anti-GFAP staining was used to monitor astrocytosis. PAR-1 mRNA levels and locations were determined by reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridizations. Cell death was monitored with in situ DNA fragmentation assays. RESULTS: In preliminary studies we found a 5-fold increase in PAR-1 mRNA in cervical spinal cords from wr/wr, compared with wild-type (wt) littermates. Our current studies suggested that reactive astrocytosis and motor neuron cell death were causally linked with alterations in thrombin signaling. PAR-1 protein expression was increased, as demonstrated by immunocytochemistry and confirmed with in situ hybridization, in phenotypic wr/wr motor neurons, compared with wt, but not in astrocytes. This increase was much greater in cervical, compared with lumbar

  20. Identification of a site in the phosphocarrier protein, HPr, which influences its interactions with sugar permeases of the bacterial phosphotransferase system: kinetic analyses employing site-specific mutants.

    PubMed Central

    Koch, S; Sutrina, S L; Wu, L F; Reizer, J; Schnetz, K; Rak, B; Saier, M H

    1996-01-01

    The permeases of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS), the sugar-specific enzymes II, are energized by sequential phosphoryl transfer from phosphoenolpyruvate to (i) enzyme I, (ii) the phosphocarrier protein HPr, (iii) the enzyme IIA domains of the permeases, and (iv) the enzyme IIBC domains of the permeases which transport and phosphorylate their sugar substrates. A number of site-specific mutants of HPr were examined by using kinetic approaches. Most of the mutations exerted minimal effects on the kinetic parameters characterizing reactions involving phosphoryl transfer from phospho-HPr to various sugars. However, when the well-conserved aspartyl 69 residue in HPr was changed to a glutamyl residue, the affinities for phospho-HPr of the enzymes II specific for mannitol, N-acetylglucosamine, and beta-glucosides decreased markedly without changing the maximal reaction rates. The same mutation reduced the spontaneous rate of phosphohistidyl HPr hydrolysis but did not appear to alter the rate of phosphoryl transfer from phospho-enzyme I to HPr. When the adjacent glutamyl residue 70 in HPr was changed to a lysyl residue, the Vmax values of the reactions catalyzed by the enzymes II were reduced, but the Km values remained unaltered. Changing this residue to alanine exerted little effect. Site-specific alterations in the C terminus of the beta-glucoside enzyme II which reduced the maximal reaction rate of phosphoryl transfer about 20-fold did not alter the relative kinetic parameters because of the aforementioned mutations in HPr. Published three-dimensional structural analyses of HPr and the complex of HPr with the glucose-specific enzyme IIA (IIAGlc) (homologous to the beta-glucoside and N-acetylglucosamine enzyme IIA domains) have revealed that residues 69 and 70 in HPr are distant from the active phosphorylation site and the IIAGlc binding interface in HPr. The results reported therefore suggest that residues D-69 and E-70 in

  1. Dynamic docking and electron-transfer between cytochrome b5 and a suite of myoglobin surface-charge mutants. Introduction of a functional-docking algorithm for protein-protein complexes.

    PubMed

    Liang, Zhao-Xun; Kurnikov, Igor V; Nocek, Judith M; Mauk, A Grant; Beratan, David N; Hoffman, Brian M

    2004-03-10

    Horse myoglobin (Mb) provides a convenient "workbench" for probing the effects of electrostatics on binding and reactivity in the dynamic [Mb, cytochrome b(5)] electron-transfer (ET) complex. We have combined mutagenesis and heme neutralization to prepare a suite of six Mb surface-charge variants: the [S92D]Mb and [V67R]Mb mutants introduce additional charges on the "front" face, and incorporation of the heme di-ester into each of these neutralizes the charge on the heme propionates which further increases the positive charge on the "front" face. For this set of mutants, the nominal charge of Mb changes by -1 to +3 units relative to that for native Mb. For each member of this set, we have measured the bimolecular quenching rate constant (k(2)) for the photoinitiated (3)ZnDMb --> Fe(3+)b(5) ET reaction as a function of ionic strength. We find: (i) a dramatic decoupling of binding and reactivity, in which k(2) varies approximately 10(3)-fold within the suite of Mbs without a significant change in binding affinity; (ii) the ET reaction occurs within the "thermodynamic" or "rapid exchange" limit of the "Dynamic Docking" model, in which a large ensemble of weakly bound protein-protein configurations contribute to binding, but only a few are reactive, as shown by the fact that the zero-ionic-strength bimolecular rate constant varies exponentially with the net charge on Mb; (iii) Brownian dynamic docking profiles allow us to visualize the microscopic basis of dynamic docking. To describe these results we present a new theoretical approach which mathematically combines PATHWAY donor/acceptor coupling calculations with Poisson-Boltzmann-based electrostatics estimates of the docking energetics in a Monte Carlo (MC) sampling framework that is thus specially tailored to the intermolecular ET problem. This procedure is extremely efficient because it targets only the functionally active complex geometries by introducing a "reactivity filter" into the computations themselves

  2. A Porcine Circovirus Type 2 (PCV2) Mutant with 234 Amino Acids in Capsid Protein Showed More Virulence In Vivo, Compared with Classical PCV2a/b Strain

    PubMed Central

    Guo, Longjun; Fu, Yujie; Wang, Yiping; Lu, Yuehua; Wei, Yanwu; Tang, Qinghai; Fan, Peihu; Liu, Jianbo; Zhang, Long; Zhang, Feiyan; Huang, Liping; Liu, Dan; Li, Shengbin; Wu, Hongli; Liu, Changming

    2012-01-01

    Background Porcine circovirus type 2 (PCV2) is considered to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which has become a serious economic problem for the swine industry worldwide. The major genotypes, PCV2a and PCV2b, are highly prevalent in the pig population and are present worldwide. However, another newly emerging PCV2b genotype mutant, which has a mutation in its ORF2-encoded capsid protein, has been sporadically present in China, as well as in other countries. It is therefore important to determine the relative virulence of the newly emerging PCV2b genotype mutant, compared with the existing PCV2a and PCV2b genotypes, and to investigate whether the newly emerging mutant virus induces more severe illness. Methodology/Principal Findings Twenty healthy, 30-day-old, commercial piglets served as controls or were challenged with PCV2a, PCV2b and the newly emerging mutant virus. A series of indexes representing different parameters were adopted to evaluate virulence, including clinical signs, serological detection, viral load and distribution, changes in immune cell subsets in the peripheral blood, and evaluation of pathological lesions. The newly emerging PCV2 mutant demonstrated more severe signs compatible with PMWS, characterized by wasting, coughing, dyspnea, diarrhea, rough hair-coat and depression. Moreover, the pathological lesions and viremia, as well as the viral loads in lymph nodes, tonsils and spleen, were significantly more severe (P<0.05) for piglets challenged with the newly emerging mutant compared with those in the groups challenged with PCV2a and PCV2b. In addition, a significantly lower average daily weight gain (P<0.05) was recorded in the group challenged with the newly emerging PCV2 mutant than in the groups challenged with the prevailing PCV2a and PCV2b. Conclusions This is believed to be the first report to confirm the enhanced virulence of the newly emerging PCV2 mutant in vivo. PMID:22829951

  3. Membrane-Associated Proteins of a Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis Activate the Inflammatory Response through Toll-Like Receptor 2

    PubMed Central

    Ingalls, Robin R.; Lien, Egil; Golenbock, Douglas T.

    2001-01-01

    The recent isolation of a lipopolysaccharide (LPS)-deficient mutant of Neisseria meningitidis has allowed us to explore the roles of other gram-negative cell wall components in the host response to infection. The experiments in this study were designed to examine the ability of this mutant strain to activate cells. Although it was clearly less potent than the parental strain, we found the LPS-deficient mutant to be a capable inducer of the inflammatory response in monocytic cells, inducing a response similar to that seen with Staphylococcus aureus. Cellular activation by the LPS mutant was related to expression of CD14, a high-affinity receptor for LPS and other microbial products, as well as Toll-like receptor 2, a member of the Toll family of receptors recently implicated in host responses to gram-positive bacteria. In contrast to the parental strain, the synthetic LPS antagonist E5564 did not inhibit the LPS-deficient mutant. We conclude that even in the absence of LPS, the gram-negative cell wall remains a potent inflammatory stimulant, utilizing signaling pathways independent of those involved in LPS signaling. PMID:11254578

  4. Mutant p53: One, No One, and One Hundred Thousand

    PubMed Central

    Walerych, Dawid; Lisek, Kamil; Del Sal, Giannino

    2015-01-01

    Encoded by the mutated variants of the TP53 tumor suppressor gene, mutant p53 proteins are getting an increased experimental support as active oncoproteins promoting tumor growth and metastasis. p53 missense mutant proteins are losing their wild-type tumor suppressor activity and acquire oncogenic potential, possessing diverse transforming abilities in cell and mouse models. Whether various mutant p53s differ in their oncogenic potential has been a matter of debate. Recent discoveries are starting to uncover the existence of mutant p53 downstream programs that are common to different mutant p53 variants. In this review, we discuss a number of studies on mutant p53, underlining the advantages and disadvantages of alternative experimental approaches that have been used to describe the numerous mutant p53 gain-of-function activities. Therapeutic possibilities are also discussed, taking into account targeting either individual or multiple mutant p53 proteins in human cancer. PMID:26734571

  5. ECB deacylase mutants

    DOEpatents

    Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

    2002-01-01

    A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

  6. Early Events in the Amyloid Formation of the A546T Mutant of Transforming Growth Factor β-Induced Protein in Corneal Dystrophies Compared to the Nonfibrillating R555W and R555Q Mutants.

    PubMed

    Koldsø, Heidi; Andersen, Ole Juul; Nikolajsen, Camilla Lund; Scavenius, Carsten; Sørensen, Charlotte S; Underhaug, Jarl; Runager, Kasper; Nielsen, Niels Chr; Enghild, Jan J; Schiøtt, Birgit

    2015-09-15

    The human transforming growth factor β-induced protein (TGFBIp) is involved in several types of corneal dystrophies where protein aggregation and amyloid fibril formation severely impair vision. Most disease-causing mutations are located in the last of four homologous fasciclin-1 (FAS1) domains of the protein, and it has been shown that when isolated, the fourth FAS1 domain (FAS1-4) mimics the behavior of full-length TGFBIp. In this study, we use molecular dynamics simulations and principal component analysis to study the wild-type FAS1-4 domain along with three disease-causing mutations (R555W, R555Q, and A546T) to decipher any internal difference in dynamical properties of the domains that may explain their varied stabilities and aggregation properties. In addition, we use a protein-protein docking method in combination with chemical cross-linking experiments and mass spectrometry of the cross-linked species to obtain information about interaction faces between identical FAS1-4 domains. The results show that the pathogenic mutations A546T and R555W affect the packing in the hydrophobic core of FAS1-4 in different directions. We further show that the FAS1-4 monomers associate using their β-rich regions, consistent with peptides observed to be part of the amyloid fibril core in lattice corneal dystrophy patients.

  7. Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

    PubMed

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Park, Bo-Kyoung; Hahn, Tae-Wook

    2016-06-01

    Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus. PMID:27057678

  8. Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

    PubMed

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Park, Bo-Kyoung; Hahn, Tae-Wook

    2016-06-01

    Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus.

  9. Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant1[OPEN

    PubMed Central

    Mary, Viviane; Schnell Ramos, Magali; Gillet, Cynthia; Socha, Amanda L.; Giraudat, Jérôme; Agorio, Astrid; Merlot, Sylvain; Clairet, Colin; Kim, Sun A.; Punshon, Tracy; Guerinot, Mary Lou; Thomine, Sébastien

    2015-01-01

    To improve seed iron (Fe) content and bioavailability, it is crucial to decipher the mechanisms that control Fe storage during seed development. In Arabidopsis (Arabidopsis thaliana) seeds, most Fe is concentrated in insoluble precipitates, with phytate in the vacuoles of cells surrounding the vasculature of the embryo. NATURAL RESISTANCE ASSOCIATED-MACROPHAGE PROTEIN3 (AtNRAMP3) and AtNRAMP4 function redundantly in Fe retrieval from vacuoles during germination. When germinated under Fe-deficient conditions, development of the nramp3nramp4 double mutant is arrested as a consequence of impaired Fe mobilization. To identify novel genes involved in seed Fe homeostasis, we screened an ethyl methanesulfonate-mutagenized population of nramp3nramp4 seedlings for mutations suppressing their phenotypes on low Fe. Here, we report that, among the suppressors, two independent mutations in the VACUOLAR IRON TRANSPORTER1 (AtVIT1) gene caused the suppressor phenotype. The AtVIT1 transporter is involved in Fe influx into vacuoles of endodermal and bundle sheath cells. This result establishes a functional link between Fe loading in vacuoles by AtVIT1 and its remobilization by AtNRAMP3 and AtNRAMP4. Moreover, analysis of subcellular Fe localization indicates that simultaneous disruption of AtVIT1, AtNRAMP3, and AtNRAMP4 limits Fe accumulation in vacuolar globoids. PMID:26232490

  10. Absence of the major light harvesting antenna proteins alters the redox properties of photosystem II reaction centres in the chlorine F2 mutant of barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although the chlorina F2 mutant of barley specifically exhibits reduced levels of the major light harvesting polypeptides (Lhcb) associated with photosystem II, thermoluminescence measurements of photosystem reaction centre photochemistry revealed that S2/S3QB- charge recombinations were shifted to ...

  11. Analyzing the 3D Structure of Human Carbonic Anhydrase II and Its Mutants Using Deep View and the Protein Data Bank

    ERIC Educational Resources Information Center

    Ship, Noam J.; Zamble, Deborah B.

    2005-01-01

    The self directed study of a 3D image of a biomolecule stresses the complex nature of the intra- and intermolecular interactions that come together to define its structure. This is made up of a series of in vitro experiments with a wild-type and mutants forms of human carbonic anhydrase II (hCAII) that examine the structure function relationship…

  12. Suppression of Arbuscule Degeneration in Medicago truncatula phosphate transporter4 Mutants is Dependent on the Ammonium Transporter 2 Family Protein AMT2;3.

    PubMed

    Breuillin-Sessoms, Florence; Floss, Daniela S; Gomez, S Karen; Pumplin, Nathan; Ding, Yi; Levesque-Tremblay, Veronique; Noar, Roslyn D; Daniels, Dierdra A; Bravo, Armando; Eaglesham, James B; Benedito, Vagner A; Udvardi, Michael K; Harrison, Maria J

    2015-04-01

    During arbuscular mycorrhizal (AM) symbiosis, the plant gains access to phosphate (Pi) and nitrogen delivered by its fungal symbiont. Transfer of mineral nutrients occurs at the interface between branched hyphae called arbuscules and root cortical cells. In Medicago truncatula, a Pi transporter, PT4, is required for symbiotic Pi transport, and in pt4, symbiotic Pi transport fails, arbuscules degenerate prematurely, and the symbiosis is not maintained. Premature arbuscule degeneration (PAD) is suppressed when pt4 mutants are nitrogen-deprived, possibly the result of compensation by PT8, a second AM-induced Pi transporter. However, PAD is also suppressed in nitrogen-starved pt4 pt8 double mutants, negating this hypothesis and furthermore indicating that in this condition, neither of these symbiotic Pi transporters is required for symbiosis. In M. truncatula, three AMT2 family ammonium transporters are induced during AM symbiosis. To test the hypothesis that suppression of PAD involves AMT2 transporters, we analyzed double and triple Pi and ammonium transporter mutants. ATM2;3 but not AMT2;4 was required for suppression of PAD in pt4, while AMT2;4, but not AMT2;3, complemented growth of a yeast ammonium transporter mutant. In summary, arbuscule life span is influenced by PT4 and ATM2;3, and their relative importance varies with the nitrogen status of the plant.

  13. Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

    SciTech Connect

    Choudhury, Kamalika Roy; Bhattacharyya, Nitai P.

    2015-01-02

    Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

  14. A study of the K(+)-site mutant of ascorbate peroxidase: mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side.

    PubMed

    Cheek, J; Mandelman, D; Poulos, T L; Dawson, J H

    1999-02-01

    A series of ferric and ferrous derivatives of wild-type ascorbate peroxidase (APX) and of an engineered K(+)-site mutant of APX that has had its potassium cation binding site removed have been examined by electronic absorption and magnetic circular dichroism (MCD) spectroscopy at 4 degrees C. Wild-type ferric APX has spectroscopic properties that are very similar to those of ferric cytochrome c peroxidase (CCP) and likely exists primarily as a five-coordinate high-spin heme ligated on the proximal side by a histidine at pH 7. There is also evidence for minority contributions from six-coordinate high- and low-spin species (histidine-water, histidine-hydroxide, and bis-histidine). The K(+)-site mutant of APX varies considerably in the electronic absorption and MCD spectra in both the ferric and ferrous states when compared with spectra of the wild-type APX. The electronic absorption and MCD spectra of the engineered K(+)-site APX mutant are essentially identical to those of cytochrome b5, a known bis-imidazole (histidine) ligated heme system. It therefore appears that the K(+)-site mutant of APX has undergone a conformational change to yield a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH. This conformational change is the result of mutagenesis of the protein to remove the K(+)-binding site which is located approximately 8 A from the peroxide binding pocket. Thus, mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side. PMID:10499104

  15. Mutation-Induced Changes in the Protein Environment and Site Energies in the (M)L214G Mutant of the Rhodobacter sphaeroides Bacterial Reaction Center.

    PubMed

    Jankowiak, Ryszard; Rancova, Olga; Chen, Jinhai; Kell, Adam; Saer, Rafael G; Beatty, J Thomas; Abramavicius, Darius

    2016-08-18

    This work focuses on the low-temperature (5 K) photochemical (transient) hole-burned (HB) spectra within the P870 absorption band, and their theoretical analysis, for the (M)L214G mutant of the photosynthetic Rhodobacter sphaeroides bacterial reaction center (bRC). To provide insight into system-bath interactions of the bacteriochlorophyll a (BChl a) special pair, i.e., P870, in the mutated bRC, the optical line shape function for the P870 band is calculated numerically. On the basis of the modeling studies, we demonstrate that (M)L214G mutation leads to a heterogeneous population of bRCs with modified (increased) total electron-phonon coupling strength of the special pair BChl a and larger inhomogeneous broadening. Specifically, we show that after mutation in the (M)L214G bRC a large fraction (∼50%) of the bacteriopheophytin (HA) chromophores shifts red and the 800 nm absorption band broadens, while the remaining fraction of HA cofactors retains nearly the same site energy as HA in the wild-type bRC. Modeling using these two subpopulations allowed for fits of the absorption and nonresonant (transient) HB spectra of the mutant bRC in the charge neutral, oxidized, and charge-separated states using the Frenkel exciton Hamiltonian, providing new insight into the mutant's complex electronic structure. Although the average (M)L214G mutant quantum efficiency of P(+)QA(-) state formation seems to be altered in comparison with the wild-type bRC, the average electron transfer time (measured via resonant transient HB spectra within the P870 band) was not affected. Thus, mutation in the vicinity of the electron acceptor (HA) does not tune the charge separation dynamics. Finally, quenching of the (M)L214G mutant excited states by P(+) is addressed by persistent HB spectra burned within the B band in chemically oxidized samples.

  16. Activation of mitogen-activated protein kinase is necessary but not sufficient for proliferation of human thyroid epithelial cells induced by mutant Ras.

    PubMed

    Gire, V; Marshall, C J; Wynford-Thomas, D

    1999-08-26

    Given the high frequency of ras oncogene activation in several common human cancers, its signal pathways are an important target for novel therapy. For practical reasons, however, these have been studied mainly in the context of transformation of established fibroblast cell lines, whereas ras acts at an earlier stage in human tumorigenesis and predominantly on epithelial cells. Here we have developed a more directly relevant model - human primary thyroid epithelial cells - which are a major target of naturally-occurring Ras mutation, and in which expression of mutant Ras in culture induces clonal expansion without morphological transformation, closely reproducing the phenotype of the corresponding tumour in vivo. Transient or stable expression of mutant H-ras (by scrapeloading or retroviral infection) at levels which stimulated proliferation induced sustained activation and translocation of MAP kinase (MAPK) in these cells. Inhibition of the MAPK pathway at the level of MAPKK, by expression of a dominant-negative mutant or by the pharmacological inhibitor PD98059, efficiently blocked the proliferative response. Conversely, selective activation of MAPK by a constitutively-active MAPKK1 mutant failed to mimic the action of Ras and, although this was achievable with activated Raf, micro-injection of anti-ras antibodies showed that this still required endogenous wild-type Ras function. In contrast to recent results obtained with a rodent thyroid cell line (WRT), therefore, activation of the MAPK pathway is necessary, but not sufficient, for the proliferogenic action of mutant Ras on primary human thyroid cells. These data emphasize the unreliability of extrapolation from cell lines and establish the feasibility of using a more representative human epithelial model for Ras signalling studies.

  17. Identification and genetic definition of a bovine papillomavirus type 1 E7 protein and absence of a low-copy-number phenotype exhibited by E5, E6, or E7 viral mutants.

    PubMed Central

    Jareborg, N; Alderborn, A; Burnett, S

    1992-01-01

    The bovine papillomavirus type 1 (BPV-1) genome replicates as a multiple-copy plasmid in murine C127 cells transformed to neoplasia by virus infection or by transfection with BPV-1 DNA. It was reported previously that BPV-1 genomes harboring frameshift mutations in the E6 or E7 open reading frame (ORF) replicated in C127 cells transformed by these mutants at a low copy number. Furthermore, the characterization of a BPV-1 mRNA in which the E6 and E7 ORFs were spliced together in frame has led to the assumption that an E6/7 fusion protein is expressed in virus-transformed C127 cells. To define the number and nature of the E6 and E7 gene products expressed in BPV-1-transformed cells, we performed immunoprecipitation experiments with antisera raised to bacterially expressed BPV-1 E6 and E7 fusion proteins. By employing cell culture conditions which induce BPV-1 E2 transactivator expression and viral early region transcription in virus-transformed C127 cell lines, we detected a single immunoprecipitated E6 protein species with an apparent molecular mass of 17 kDa and a single E7 protein species with an apparent molecular mass of 15 kDa. To characterize further these E6 and E7 proteins, C127 cells were transformed by transfection with BPV-1 genomes containing mutations predicted to prevent expression of specific E6 or E7 gene products, and the transformed cells were subjected to immunoprecipitation analysis with the E6 or E7 antiserum. The results of these experiments confirmed that the E6 and E7 ORFs encode distinct proteins and failed to establish the existence of an E6/7 fusion protein. We did not find a significant difference in the viral genome copy number between clonal C127 cell lines transformed by wild-type BPV-1 or by mutant viral genomes unable to express the E6 or the E7 protein. Furthermore, in contrast to two previous reports suggesting that expression of the BPV-1 E5 gene was required for the establishment or maintenance of a high viral plasmid copy number

  18. The multiple roles of light-harvesting chlorophyll a/b-protein complexes define structure and optimize function of Arabidopsis chloroplasts: a study using two chlorophyll b-less mutants.

    PubMed

    Kim, Eun-Ha; Li, Xiao-Ping; Razeghifard, Reza; Anderson, Jan M; Niyogi, Krishna K; Pogson, Barry J; Chow, Wah Soon

    2009-08-01

    The multiple roles of light-harvesting chlorophyll a/b-protein complexes in the structure and function of Arabidopsis chloroplasts were investigated using two chlorophyll b-less mutants grown under metal halide lamps with a significant far-red component. In ch1-3, all six light-harvesting proteins of photosystem (PS) II were greatly decreased; in ch1-3lhcb5, Lhcb5 was completely absent while the other five proteins were further decreased. The thylakoids of ch1-3 were less negatively-charged than the wild type, and those of ch1-3lhcb5 were even less so. Despite the expected weaker electrostatic repulsion, however, thylakoids in leaves of the mutants were not well stacked, an effect we attribute to lower van der Waals attraction, lower electrostatic attraction between opposite charges, and the absence or instability of PSII supercomplexes and peripheral light-harvesting trimers. The quantum yield of oxygen evolution in leaves decreased from 0.109 (wild type) to 0.087 (ch1-3) and 0.081 (ch1-3lhcb5) O(2) (photon absorbed)(-1); we attribute this decrease to an excessive spillover from PSII to PSI, a limited PSII antenna, and increased light-independent thermal dissipation in PSII in the mutants. Destabilization of the donor side of PSII, indicated by slower electron donation to the redox-active tyrosine Y(Z)(*) in ch1-3, probably enhanced PSII susceptibility to photoinactivation, increased the non-functional PSII complexes in vivo, and further inactivated PSII complexes in vitro. The evolution of chlorophyll b-containing chloroplasts seems to fine-tune oxygenic photosynthesis.

  19. XRCC1 and CYP2E1 polymorphisms as susceptibility factors of plasma mutant p53 protein and anti-p53 antibody expression in vinyl chloride monomer-exposed polyvinyl chloride workers.

    PubMed

    Wong, Ruey-Hong; Du, Chung-Li; Wang, Jung-Der; Chan, Chang-Chuan; Luo, Jiin-Chyuan J; Cheng, Tsun-Jen

    2002-05-01

    Mutant p53 protein and anti-p53 antibody in circulating blood can be detectedamong individuals with mutations of the p53 tumor suppressor gene. Plasma mutant p53 protein and anti-p53 antibody have also been associated with vinyl chloride monomer (VCM) exposure, although the mechanism of VCM-related carcinogenesis remains unclear. Polymorphisms of metabolic and DNA repair genes have been implicated in chemical exposure-related carcinogenesis. The aim of this study is to explore the association between polymorphisms of metabolic and DNA repair genes with mutant p53 protein and anti-p53 antibody expression induced by VCM. Study subjects comprised 333 male workers occupationally exposed to VCM. Plasma mutant p53 protein and anti-p53 antibody detected with ELISA were grouped together as p53 overexpression. Genotypes of cytochrome P450 2E1 (CYP2E1), aldehyde dehydrogenase 2 (ALDH2), glutathione S-transferase T1 (GSTT1), and X-ray repair cross-complementing group 1 (XRCC1, exon 10) genes were identified by the PCR. High VCM exposure group had significantly higher p53 overexpression as compared with low exposure group [odds ratio (OR), 2.1; 95% confidence interval (CI), 1.1-3.8]. Individuals having experienced a high VCM exposure and displaying a XRCC1 Gln-Gln genotype had a highest risk of p53 overexpression among those having different combinations of VCM exposure and XRCC1 genotypes (OR, 6.5; 95% CI, 1.7-24.2). Interestingly, those subjects reflecting a CYP2E1 c2c2 genotype among the low VCM-exposure group demonstrated a greater risk of p53 overexpression (OR, 9.8; 95% CI, 1.2-81.6) as compared with those experiencing a low VCM exposure and CYP2E1 c1c1/c1c2 genotypes. Additional analysis revealed that individuals possessing more susceptible XRCC1 Gln-Gln, CYP2E1 c2c2, ALDH2 1-2/2-2, and non-null GSTT1 genotypes were more likely to reveal p53 overexpression. Our results suggest that susceptible XRCC1 and CYP2E1 genotypes may modulate the mutation of the p53 gene among

  20. Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: shutoff of host DNA and protein synthesis gene dosage experiments, identification of a restrictive host, and possible biological significance.

    PubMed

    Snustad, D P; Bursch, C J; Parson, K A; Hefeneider, S H

    1976-04-01

    The shutoff of host DNA synthesis is delayed until about 8 to 10 min after infection when Escherichia coli B/5 cells were infected with bacteriophage T4 mutants deficient in the ability to induce nuclear disruption (ndd mutants). The host DNA synthesized after infection with ndd mutants is stable in the absence of T4 endonucleases II and IV, but is unstable in the presence of these nucleases. Host protein synthesis, as indicated by the inducibility of beta-galactosidase and sodium dodecyl sulfate-polyacrylamide gel patterns of isoptopically labeled proteins synthesize after infection, is shut off normally in ndd-infected cells, even in the absence of host DNA degradation. The Cal Tech wild-type strain of E. coli CT447 was found to restrict growth of the ndd mutants. Since T4D+ also has a very low efficiency of plating on CT447, we have isolated a nitrosoguanidine-induced derivative of CT447 which yields a high T4D+ efficiency of plating while still restricting the ndd mutants. Using this derivative, CT447 T4 plq+ (for T4 plaque+), we have shown that hos DNA degradation and shutoff of host DNA synthesis occur after infection with either ndd98 X 5 (shutoff delayed) or T4D+ (shutoff normal) with approximately the same kinetics as in E. coli strain B/5. Nuclear disruption occurs after infection of CT447 with ndd+ phage, but not after infection with ndd- phage. The rate of DNA synthesis after infection of CT447 T4 plq+ with ndd98 X 5 is about 75% of the rate observed after infection with T4D+ while the burst size of ndd98 X 5 is only 3.5% of that of T4D+. The results of gene dosage experiments using the ndd restrictive host C5447 suggest that the ndd gene product is required in stoichiometric amounts. The observation by thin-section electron microscopy of two distinct pools of DNA, one apparently phage DNA and the other host DNA, in cells infected with nuclear disruption may be a compartmentalization mechanism which separates the pathways of host DNA degradation and

  1. Complementation of the embryo-lethal T-DNA insertion mutant of AUXIN-BINDING-PROTEIN 1 (ABP1) with abp1 point mutated versions reveals crosstalk of ABP1 and phytochromes

    PubMed Central

    Effendi, Yunus; Ferro, Noel; Labusch, Corinna; Geisler, Markus; Scherer, Günther F. E.

    2015-01-01

    The function of the extracytoplasmic AUXIN-BINDING-PROTEIN1 (ABP1) is largely enigmatic. We complemented a homozygous T-DNA insertion null mutant of ABP1 in Arabidopsis thaliana Wassilewskia with three mutated and one wild-type (wt) ABP1 cDNA, all tagged C-terminally with a strepII–FLAG tag upstream the KDEL signal. Based on in silico modelling, the abp1 mutants were predicted to have altered geometries of the auxin binding pocket and calculated auxin binding energies lower than the wt. Phenotypes linked to auxin transport were compromised in these three complemented abp1 mutants. Red light effects, such as elongation of hypocotyls in constant red (R) and far-red (FR) light, in white light supplemented by FR light simulating shade, and inhibition of gravitropism by R or FR, were all compromised in the complemented lines. Using auxin- or light-induced expression of marker genes, we showed that auxin-induced expression was delayed already after 10min, and light-induced expression within 60min, even though TIR1/AFB or phyB are thought to act as receptors relevant for gene expression regulation. The expression of marker genes in seedlings responding to both auxin and shade showed that for both stimuli regulation of marker gene expression was altered after 10–20min in the wild type and phyB mutant. The rapidity of expression responses provides a framework for the mechanics of functional interaction of ABP1 and phyB to trigger interwoven signalling pathways. PMID:25392478

  2. Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels.

    PubMed Central

    Kyöstiö, S R; Owens, R A; Weitzman, M D; Antoni, B A; Chejanovsky, N; Carter, B J

    1994-01-01

    The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in gene regulation and replication of the virus. We studied here the regulation of mRNA transcribed from the AAV p5 and p19 promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p5 transcript encodes the larger Rep proteins, Rep78 and Rep68, while the p19 transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation in the rep gene accumulated higher levels of p5 and p19 mRNA than a plasmid containing the wild-type AAV genome. Addition of increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p5 and p19 mRNA accumulation from pNTC3, indicating that the levels of both transcripts were decreased by the Rep proteins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that p5 and p19 mRNA accumulation was inhibited 5- to 10-fold by Rep78 and Rep68 and 2- to 3-fold by Rep52 and Rep40. Analysis of carboxyl-terminal truncation mutants of Rep78 showed that the ability of Rep78 to decrease p5 and p19 mRNA levels was lost when 159 or more amino acids were deleted. Rep78 and Rep68 mutants deleted for the methionine at residue 225 showed decreased abilities to down-regulate both p5 and p19 transcript levels, while mutants containing a substitution of glycine for the methionine resembled the wild-type Rep78. A Rep78 protein with a mutation in the putative nucleoside triphosphate binding site inhibited expression from p5 but not from p19, suggesting that the regulation of p5 transcript levels by Rep78 and Rep68 differs from that of p19. A deletion analysis of AAV cis sequences revealed that an intact terminal repeat was not required for negative regulation of p5 and p19 transcript levels and that the regulation of p19 mRNA levels by Rep78 did not require the presence

  3. Differential analysis in Proteome of Space Induced Rice and Soybean Mutants

    NASA Astrophysics Data System (ADS)

    Wang, W.; Lu, B.; Gu, D.; Han, S.; Gao, Y.; Sun, Y.

    To investigate the change trends of proteome induced in space environment we chose 3 Rice mutants 2 Soybean mutants and the seeds which were selected as high yields high tillering rice blast resistance soybean insect pest resistance and wider leaf shape individually after abroad Recoverable Satellite JB-1 for 15 days in 1996 and their corresponding controls Two-dimensional gel electrophoresis 2-D with Coomassie Brilliant Blue staining and PDQuest TM software analysis found that In 6 rice samples 329 pm 35 protein spots were detected in controls whereas 298 pm 37 protein spots detected in mutants representing a 9 decrease 69 pm 27 protein spots were lost in mutants while 37 pm 14 protein spots appeared additionally showing 11 protein spots were lost in mutants 58 protein spots were significantly regulated in mutants with 16 pm 7 up- and 42 pm 18 down-regulated which occupied 5 and 14 of the total average mutants spots separately In 3 soybean leaf samples 263 pm 12 protein spots were detected in controls whereas 255 pm 20 protein spots detected in mutants representing a 3 decrease 49 pm 10 protein spots were lost in mutants while 36 pm 16 protein spots appeared additionally showing 5 protein spots lost in mutants 51 protein spots were significantly regulated in mutants with 25 pm 7 up- and 26 pm 15 down-regulated which occupied 9 8 and 10 2 of the total average mutants spots separately In 3 soybean seed samples 208 pm 41 protein spots were

  4. Protein glycosylation defects in the Saccharomyces cerevisiae mnn7 mutant class. Support for the stop signal proposed for regulation of outer chain elongation.

    PubMed

    Ballou, L; Alvarado, E; Tsai, P K; Dell, A; Ballou, C E

    1989-07-15

    Total cell mannoprotein was isolated from Saccharomyces cerevisiae X2180 mutants that have defects in elongation of the outer chain attached to the N-linked core oligosaccharides (mnn7, mnn8, mnn9, and mnn10) (Ballou, L., Cohen, R. E., and Ballou, C. E. (1980) J. Biol. Chem. 255, 5986-5991). Comparison of the oligosaccharides released by endoglucosaminidase H digestion confirmed that the mnn9 mutation eliminates all but two mannoses of the outer chain, whereas the mnn8 and mnn10 strains produce outer chains of variable but similar lengths. The isolate designated mnn7 was found to be allelic with mnn8. Haploid mutants of the type mnn8 mnn9 or mnn9 mnn10 had the mnn9 phenotype, which established that the mnn9 defect is dominant and presumably acts at a processing step prior to the steps affected by mnn8 and mnn10. Analysis of the mnn1 mnn2 mnn10 oligosaccharides revealed that the heterogeneous outer chain contained 6-16 alpha 1----6-linked mannose units and each was terminated by a single alpha 1----2-linked mannose unit, whereas the core lacked one such unit that was present in the mnn9 oligosaccharide. The results are consistent with and support the hypothesis (Gopal, P. K., and Ballou, C. E. (1988) Proc. Natl. Acad. Sci. U.S.A. 84, 8824-8828) that addition of such a side-chain mannose unit is associated with termination of outer chain elongation in these mutants and may serve as a stop signal that regulates outer chain synthesis in the parent wild-type strain. PMID:2663843

  5. 'Let the phage do the work': Using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants

    SciTech Connect

    Teschke, Carolyn M.; Parent, Kristin N.

    2010-06-05

    The amino acid sequence of viral capsid proteins contains information about their folding, structure and self-assembly processes. While some viruses assemble from small preformed oligomers of coat proteins, other viruses such as phage P22 and herpesvirus assemble from monomeric proteins (Fuller and King, 1980). The subunit assembly process is strictly controlled through protein:protein interactions such that icosahedral structures are formed with specific symmetries, rather than aberrant structures. dsDNA viruses commonly assemble by first forming a precursor capsid that serves as a DNA packaging machine. DNA packaging is accompanied by a conformational transition of the small precursor procapsid into a larger capsid for isometric viruses. Here we highlight the pseudo-atomic structures of phage P22 coat protein and rationalize several decades of data about P22 coat protein folding, assembly and maturation generated from a combination of genetics and biochemistry.

  6. [Integration and intramolecular transposition of the TnBP3 Bordetella pertussis transposon in the Escherichia coli K-12 cells -- mutant for the phosphoenolpyruvate-dependent phosphotransferase system Hpr protein ].

    PubMed

    Sivov, I G; Bol'shakova, T N; Karataev, G I

    2001-07-01

    Integration of a plasmid carrying the TnBP3 transposon of Bordetella pertussis into the chromosome of Escherichia coli and transpositions of the integrated structure within a chromosome in the wild-type and mutant cells ptsH devoid of the major Hpr protein of the phosphoenolpyruvate-dependent phosphotransferase system were studied. When transposed to a new chromosome site, the integrated structure was precisely (or almost precisely) excised from the metY gene sequence, which resulted in restoration of the Met+ phenotype. The integration and transposition events were only observed in the E. coli cells carrying the ptsH+ allele. The ptsH mutations inhibited integration and intramolecular transposition, which were restored after phenotypic or genetic suppression of the ptsH mutation. The intensity of the processes studied were suggested to depend on the integrity of a chain that ensures transferring of the phosphoryl residue by proteins of the phosphotransferase system in E. coli K12. The results obtained indicate that the ptsH mutants of E. coli can serve as the optimal host for cloning of fragments carrying repeated sequences of B. pertussis, which may apply to the repeated sequences of other microorganisms.

  7. BikDDA, a mutant of Bik with longer half-life expression protein, can be a novel therapeutic gene for triple-negative breast cancer.

    PubMed

    Jiao, Shiping; Wu, Minqing; Ye, Feng; Tang, Hailin; Xie, Xinhua; Xie, Xiaoming

    2014-01-01

    Our previous studies showed that BikDD, a constitutively active mutant form of Bik, exhibited powerful antitumor effects in preclinical pancreatic, lung and breast cancer models. Howerver, the antitumor activity of BikDD in triple-negative breast cancer (TNBC) is unknown. Here we show that aberrant expression of p-ERK1/2 was a meaningful molecular phenotype in TNBC patients, and can be an obstacle for treatment because of the converse correlation with Bik. A novel mutant, BikDDA, in which Ser124 was changed to Alanine to block BikDD phosphorylation by p-ERK1/2 prevented subsequent ubiquitin-proteasome degradation. BikDDA showed a prolonged half-life and enhanced pro-apoptotic ability in TNBC cells compared with BikDD. Moreover, aberrant expression of p-ERK1/2 was associated with 5-fluorouracil resistance in breast cancer patients and BikDDA enhanced the therapeutic effects of 5-fluorouracil in vitro.

  8. BikDDA, a Mutant of Bik with Longer Half-Life Expression Protein, Can Be a Novel Therapeutic Gene for Triple-Negative Breast Cancer

    PubMed Central

    Jiao, Shiping; Wu, Minqing; Ye, Feng; Tang, Hailin; Xie, Xinhua; Xie, Xiaoming

    2014-01-01

    Our previous studies showed that BikDD, a constitutively active mutant form of Bik, exhibited powerful antitumor effects in preclinical pancreatic, lung and breast cancer models. Howerver, the antitumor activity of BikDD in triple-negative breast cancer (TNBC) is unknown. Here we show that aberrant expression of p-ERK1/2 was a meaningful molecular phenotype in TNBC patients, and can be an obstacle for treatment because of the converse correlation with Bik. A novel mutant, BikDDA, in which Ser124 was changed to Alanine to block BikDD phosphorylation by p-ERK1/2 prevented subsequent ubiquitin-proteasome degradation. BikDDA showed a prolonged half-life and enhanced pro-apoptotic ability in TNBC cells compared with BikDD. Moreover, aberrant expression of p-ERK1/2 was associated with 5-fluorouracil resistance in breast cancer patients and BikDDA enhanced the therapeutic effects of 5-fluorouracil in vitro. PMID:24637719

  9. Evolved Osmotolerant Escherichia coli Mutants Frequently Exhibit Defective N-Acetylglucosamine Catabolism and Point Mutations in Cell Shape-Regulating Protein MreB

    PubMed Central

    Winkler, James D.; Garcia, Carlos; Olson, Michelle; Callaway, Emily

    2014-01-01

    Biocatalyst robustness toward stresses imposed during fermentation is important for efficient bio-based production. Osmotic stress, imposed by high osmolyte concentrations or dense populations, can significantly impact growth and productivity. In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable of in situ DNA exchange) and asexual Escherichia coli strains under sodium chloride (NaCl) stress. All isolates had significantly improved growth under selection and could grow in up to 0.80 M (47 g/liter) NaCl, a concentration that completely inhibits the growth of the unevolved parental strains. Whole genome resequencing revealed frequent mutations in genes controlling N-acetylglucosamine catabolism (nagC, nagA), cell shape (mrdA, mreB), osmoprotectant uptake (proV), and motility (fimA). Possible epistatic interactions between nagC, nagA, fimA, and proV deletions were also detected when reconstructed as defined mutations. Biofilm formation under osmotic stress was found to be decreased in most mutant isolates, coupled with perturbations in indole secretion. Transcriptional analysis also revealed significant changes in ompACGL porin expression and increased transcription of sulfonate uptake systems in the evolved mutants. These findings expand our current knowledge of the osmotic stress phenotype and will be useful for the rational engineering of osmotic tolerance into industrial strains in the future. PMID:24727267

  10. Microarray Analysis of Rice d1 (RGA1) Mutant Reveals the Potential Role of G-Protein Alpha Subunit in Regulating Multiple Abiotic Stresses Such as Drought, Salinity, Heat, and Cold

    PubMed Central

    Jangam, Annie P.; Pathak, Ravi R.; Raghuram, Nandula

    2016-01-01

    The genome-wide role of heterotrimeric G-proteins in abiotic stress response in rice has not been examined from a functional genomics perspective, despite the availability of mutants and evidences involving individual genes/processes/stresses. Our rice whole transcriptome microarray analysis (GSE 20925 at NCBI GEO) using the G-alpha subunit (RGA1) null mutant (Daikoku 1 or d1) and its corresponding wild type (Oryza sativa Japonica Nipponbare) identified 2270 unique differentially expressed genes (DEGs). Out of them, we mined for all the potentially abiotic stress-responsive genes using Gene Ontology terms, STIFDB2.0 and Rice DB. The first two approaches produced smaller subsets of the 1886 genes found at Rice DB. The GO approach revealed similar regulation of several families of stress-responsive genes in RGA1 mutant. The Genevestigator analysis of the stress-responsive subset of the RGA1-regulated genes from STIFDB revealed cold and drought-responsive clusters. Meta data analysis at Rice DB revealed large stress-response categories such as cold (878 up/810 down), drought (882 up/837 down), heat (913 up/777 down), and salt stress (889 up/841 down). One thousand four hundred ninety-eight of them are common to all the four abiotic stresses, followed by fewer genes common to smaller groups of stresses. The RGA1-regulated genes that uniquely respond to individual stresses include 111 in heat stress, eight each in cold only and drought only stresses, and two genes in salt stress only. The common DEGs (1498) belong to pathways such as the synthesis of polyamine, glycine-betaine, proline, and trehalose. Some of the common DEGs belong to abiotic stress signaling pathways such as calcium-dependent pathway, ABA independent and dependent pathway, and MAP kinase pathway in the RGA1 mutant. Gene ontology of the common stress responsive DEGs revealed 62 unique molecular functions such as transporters, enzyme regulators, transferases, hydrolases, carbon and protein metabolism

  11. Analysis of p53 mutants for transcriptional activity.

    PubMed Central

    Raycroft, L; Schmidt, J R; Yoas, K; Hao, M M; Lozano, G

    1991-01-01

    The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active. Images PMID:1944276

  12. Liver tumor formation by a mutant retinoblastoma protein in the transgenic mice is caused by an upregulation of c-Myc target genes

    SciTech Connect

    Wang, Bo; Hikosaka, Keisuke; Sultana, Nishat; Sharkar, Mohammad Tofael Kabir; Noritake, Hidenao; Kimura, Wataru; Wu, Yi-Xin; Kobayashi, Yoshimasa; Uezato, Tadayoshi; Miura, Naoyuki

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Fifty percent of the mutant Rb transgenic mice produced liver tumors. Black-Right-Pointing-Pointer In the tumor, Foxm1, Skp2, Bmi1 and AP-1 mRNAs were up-regulated. Black-Right-Pointing-Pointer No increase in expression of the Myc-target genes was observed in the non-tumorous liver. Black-Right-Pointing-Pointer Tumor formation depends on up-regulation of the Myc-target genes. -- Abstract: The retinoblastoma (Rb) tumor suppressor encodes a nuclear phosphoprotein that regulates cellular proliferation, apoptosis and differentiation. In order to adapt itself to these biological functions, Rb is subjected to modification cycle, phosphorylation and dephosphorylation. To directly determine the effect of phosphorylation-resistant Rb on liver development and function, we generated transgenic mice expressing phosphorylation-resistant human mutant Rb (mt-Rb) under the control of the rat hepatocyte nuclear factor-1 gene promoter/enhancer. Expression of mt-Rb in the liver resulted in macroscopic neoplastic nodules (adenomas) with {approx}50% incidence within 15 months old. Interestingly, quantitative reverse transcriptase-PCR analysis showed that c-Myc was up-regulated in the liver of mt-Rb transgenic mice irrespective of having tumor tissues or no tumor. In tumor tissues, several c-Myc target genes, Foxm1, c-Jun, c-Fos, Bmi1 and Skp2, were also up-regulated dramatically. We determined whether mt-Rb activated the Myc promoter in the HTP9 cells and demonstrated that mt-Rb acted as an inhibitor of wild-type Rb-induced repression on the Myc promoter. Our results suggest that continued upregulation of c-Myc target genes promotes the liver tumor formation after about 1 year of age.

  13. Pathogenesis and micro-anatomic characterization of a cell-adapted mutant foot-and-mouth disease virus in cattle: Impact of the Jumonji C-domain containing protein 6 (JMJD6) and route of inoculation.

    PubMed

    Lawrence, Paul; Pacheco, Juan; Stenfeldt, Carolina; Arzt, Jonathan; Rai, Devendra K; Rieder, Elizabeth

    2016-05-01

    A companion study reported Jumonji-C domain containing protein 6 (JMJD6) is involved in an integrin- and HS-independent pathway of FMDV infection in CHO cells. JMJD6 localization was investigated in animal tissues from cattle infected with either wild type A24-FMDV (A24-WT) or mutant FMDV (JMJD6-FMDV) carrying E95K/S96L and RGD to KGE mutations in VP1. Additionally, pathogenesis of mutant JMJD6-FMDV was investigated in cattle through aerosol and intraepithelial lingual (IEL) inoculation. Interestingly, JMJD6-FMDV pathogenesis was equivalent to A24-WT administered by IEL route. In contrast, JMJD6-FMDV aerosol-infected cattle did not manifest signs of FMD and animals showed no detectable viremia. Immunofluorescent microscopy of post-mortem tissue revealed JMJD6-FMDV exclusively co-localized with JMJD6(+) cells while A24-WT was occasionally found in JMJD6(+) cells. In vitro, chemical uptake inhibitors demonstrated JMJD6-FMDV entered cells via clathrin-coated pit endocytosis. In vivo, JMJD6-FMDV exhibited preference for JMJD6(+) cells, but availability of this alternative receptor likely depends on route of inoculation. PMID:26914509

  14. Pathogenesis and micro-anatomic characterization of a cell-adapted mutant foot-and-mouth disease virus in cattle: Impact of the Jumonji C-domain containing protein 6 (JMJD6) and route of inoculation.

    PubMed

    Lawrence, Paul; Pacheco, Juan; Stenfeldt, Carolina; Arzt, Jonathan; Rai, Devendra K; Rieder, Elizabeth

    2016-05-01

    A companion study reported Jumonji-C domain containing protein 6 (JMJD6) is involved in an integrin- and HS-independent pathway of FMDV infection in CHO cells. JMJD6 localization was investigated in animal tissues from cattle infected with either wild type A24-FMDV (A24-WT) or mutant FMDV (JMJD6-FMDV) carrying E95K/S96L and RGD to KGE mutations in VP1. Additionally, pathogenesis of mutant JMJD6-FMDV was investigated in cattle through aerosol and intraepithelial lingual (IEL)