Stability of structured Kaposi's sarcoma-associated herpesvirus ORF57 protein is regulated by protein phosphorylation and homodimerization. Majerciak, V; Pripuzova, N; Chan, C; Temkin, N; Specht, SI; Zheng, ZM Journal of virology
89
3256-74
2015
Show Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 plays an essential role in KSHV lytic infection by promoting viral gene expression at the posttranscriptional level. Using bioinformatic and biochemical approaches, we determined that ORF57 contains two structurally and functionally distinct domains: a disordered nonstructural N-terminal domain (amino acids [aa] 1 to 152) and a structured α-helix-rich C-terminal domain (aa 153 to 455). The N-terminal domain mediates ORF57 interaction with several RNA-protein complexes essential for ORF57 to function. The N-terminal phosphorylation by cellular casein kinase II (CKII) at S21, T32, and S43, and other cellular kinases at S95 and S97 residues in proximity of the caspase-7 cleavage site, 30-DETD-33, inhibits caspase-7 digestion of ORF57. The structured C-terminal domain mediates homodimerization of ORF57, and the critical region for this function was mapped carefully to α-helices 7 to 9. Introduction of point mutations into α-helix 7 at ORF57 aa 280 to 299, a region highly conserved among ORF57 homologues from other herpesviruses, inhibited ORF57 homodimerization and led to proteasome-mediated degradation of ORF57 protein. Thus, homodimerization of ORF57 via its C terminus prevents ORF57 from degrading and allows two structure-free N termini of the dimerized ORF57 to work coordinately for host factor interactions, leading to productive KSHV lytic infection and pathogenesis.KSHV is a human oncogenic virus linked to the development of several malignancies. KSHV-mediated oncogenesis requires both latent and lytic infection. The KSHV ORF57 protein is essential for KSHV lytic replication, as it regulates the expression of viral lytic genes at the posttranscriptional level. This report provides evidence that the structural conformation of the ORF57 protein plays a critical role in regulation of ORF57 stability. Phosphorylation by CKII on the identified serine/threonine residues at the N-terminal unstructured domain of ORF57 prevents its digestion by caspase-7. The C-terminal domain of ORF57, which is rich in α-helices, contributes to homodimerization of ORF57 to prevent proteasome-mediated protein degradation. Elucidation of the ORF57 structure not only enables us to better understand ORF57 stability and functions but also provides an important tool for us to modulate ORF57's activity with the aim to inhibit KSHV lytic replication. | | 25568207
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Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum. Wagner, JC; Platt, RJ; Goldfless, SJ; Zhang, F; Niles, JC Nature methods
11
915-8
2014
Show Abstract
Malaria is a major cause of global morbidity and mortality, and new strategies for treating and preventing this disease are needed. Here we show that the Streptococcus pyogenes Cas9 DNA endonuclease and single guide RNAs (sgRNAs) produced using T7 RNA polymerase (T7 RNAP) efficiently edit the Plasmodium falciparum genome. Targeting the genes encoding native knob-associated histidine-rich protein (kahrp) and erythrocyte binding antigen 175 (eba-175), we achieved high (≥ 50-100%) gene disruption frequencies within the usual time frame for generating transgenic parasites. | | 25108687
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Functional Characterization of ABCB4 Mutations Found in Low Phospholipid-Associated Cholelithiasis (LPAC). Kim, TH; Park, HJ; Choi, JH The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology
17
525-30
2013
Show Abstract
Multidrug resistance 3 (MDR3) is expressed on the canalicular membrane of the hepatocytes and plays an important role in protecting the liver from bile acids. Altered ABCB4 gene expression can lead to a rare hepatic disease, low phospholipid-associated cholelithiasis (LPAC). In this study, we characterized 3 ABCB4 mutations in LPAC patients using various in vitro assay systems. We first measured the ability of each mutant to transport paclitaxel and then the mechanisms by which these mutations might change MDR3 transport activity were determined using immunoblotting, cell surface protein biotinylation, and immunofluorescence. Through a membrane vesicular transport assay, we observed that the uptake of paclitaxel was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Both mutants showed significantly decreased total and cell surface MDR3 expression. These data suggest two missense mutations of ABCB4 may alter function of MDR3 and ultimately can be determined as LPAC-causing mutations. | Western Blotting | 24381502
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An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum. Wagner, JC; Goldfless, SJ; Ganesan, SM; Lee, MC; Fidock, DA; Niles, JC Malaria journal
12
373
2013
Show Abstract
The construction of plasmid vectors for transgene expression in the malaria parasite, Plasmodium falciparum, presents major technical hurdles. Traditional molecular cloning by restriction and ligation often yields deletions and re-arrangements when assembling low-complexity (A + T)-rich parasite DNA. Furthermore, the use of large 5'- and 3'- untranslated regions of DNA sequence (UTRs) to drive transgene transcription limits the number of expression cassettes that can be incorporated into plasmid vectors.To address these challenges, two high fidelity cloning strategies, namely yeast homologous recombination and the Gibson assembly method, were evaluated for constructing P. falciparum vectors. Additionally, some general rules for reliably using the viral 2A-like peptide to express multiple proteins from a single expression cassette while preserving their proper trafficking to various subcellular compartments were assessed.Yeast homologous recombination and Gibson assembly were found to be effective strategies for successfully constructing P. falciparum plasmid vectors. Using these cloning methods, a validated family of expression vectors that provide a flexible starting point for user-specific applications was created. These vectors are also compatible with traditional cloning by restriction and ligation, and contain useful combinations of commonly used features for enhancing plasmid segregation and site-specific integration in P. falciparum. Additionally, application of a 2A-like peptide for the synthesis of multiple proteins from a single expression cassette, and some rules for combinatorially directing proteins to discrete subcellular compartments were established.A set of freely available, sequence-verified and functionally validated parts that offer greater flexibility for constructing P. falciparum vectors having expanded expression capacity is provided. | Western Blotting | 24160265
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Metastasis of aggressive amoeboid sarcoma cells is dependent on Rho/ROCK/MLC signaling. Kosla, J; Paňková, D; Plachý, J; Tolde, O; Bicanová, K; Dvořák, M; Rösel, D; Brábek, J Cell communication and signaling : CCS
11
51
2013
Show Abstract
Although there is extensive evidence for the amoeboid invasiveness of cancer cells in vitro, much less is known about the role of amoeboid invasiveness in metastasis and the importance of Rho/ROCK/MLC signaling in this process.We analyzed the dependence of amoeboid invasiveness of rat and chicken sarcoma cells and the metastatic activity of chicken cells on individual elements of the Rho/ROCK/MLC pathway. In both animal models, inhibition of Rho, ROCK or MLC resulted in greatly decreased cell invasiveness in vitro, while inhibition of extracellular proteases using a broad spectrum inhibitor did not have a significant effect. The inhibition of both Rho activity and MLC phosphorylation by dominant negative mutants led to a decreased capability of chicken sarcoma cells to metastasize. Moreover, the overexpression of RhoA in non-metastatic chicken cells resulted in the rescue of both invasiveness and metastatic capability. Rho and ROCK, unlike MLC, appeared to be directly involved in the maintenance of the amoeboid phenotype, as their inhibition resulted in the amoeboid-mesenchymal transition in analyzed cell lines.Taken together, these results suggest that protease-independent invasion controlled by elements of the Rho/ROCK/MLC pathway can be frequently exploited by metastatic sarcoma cells. | | 23899007
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Mechanistic insight into the pathology of polyalanine expansion disorders revealed by a mouse model for X linked hypopituitarism. Hughes, J; Piltz, S; Rogers, N; McAninch, D; Rowley, L; Thomas, P PLoS genetics
9
e1003290
2013
Show Abstract
Polyalanine expansions in transcription factors have been associated with eight distinct congenital human diseases. It is thought that in each case the polyalanine expansion causes misfolding of the protein that abrogates protein function. Misfolded proteins form aggregates when expressed in vitro; however, it is less clear whether aggregation is of relevance to these diseases in vivo. To investigate this issue, we used targeted mutagenesis of embryonic stem (ES) cells to generate mice with a polyalanine expansion mutation in Sox3 (Sox3-26ala) that is associated with X-linked Hypopituitarism (XH) in humans. By investigating both ES cells and chimeric mice, we show that endogenous polyalanine expanded SOX3 does not form protein aggregates in vivo but rather is present at dramatically reduced levels within the nucleus of mutant cells. Importantly, the residual mutant protein of chimeric embryos is able to rescue a block in gastrulation but is not sufficient for normal development of the hypothalamus, a region that is functionally compromised in Sox3 null embryos and individuals with XH. Together, these data provide the first definitive example of a disease-relevant PA mutant protein that is both nuclear and functional, thereby manifesting as a partial loss-of-function allele. | | 23505376
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Mutation in the catalytic subunit of DNA polymerase alpha influences transcriptional gene silencing and homologous recombination in Arabidopsis. Liu J, Ren X, Yin H, Wang Y, Xia R, Wang Y, Gong Z Plant J
61
36-45. Epub 2009 Sep 21.
2010
Show Abstract
REPRESSOR OF SILENCING 1 (ROS1) encodes a DNA demethylase that actively removes DNA methylation. Mutation in ROS1 leads to transcriptional gene silencing of a T-DNA locus that contains two genes, RD29A-LUC and 35S-NPTII, originally expressed in the C24 wild type. These units have different silencing regulation mechanisms: the former mechanism is dependent on small interfering RNA (siRNA)-directed DNA methylation, but the latter is not. We studied the latter gene silencing mechanism by screening the suppressors of the ros1 mutant using the silenced 35S-NPTII as a selection marker gene. The polalpha/incurvata2 (icu2) gene was isolated as one ros1 suppressor because its mutation leads to the reactivation of the silenced 35S-NPTII gene. POLalpha/ICU2 encodes a catalytic subunit of DNA polymerase alpha. Mutation of POLalpha/ICU2 did not affect DNA methylation, but reduced histone H3 Lys9 dimethylation (H3K9me2) modification in the 35S promoter. The polalpha mutation also influences the development of the shoot apical meristem, and delays the G2/M phase with high expression of a G2/M marker gene CycB1;1:GUS. Furthermore, the frequency of homologous recombination is greater in the polalpha/icu2 mutant than in the C24 wild type. Our results suggest that DNA polymerase alpha is involved in mediating epigenetic states and in DNA homologous recombination in Arabidopsis. | | 19769574
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Missense mutations associated with Diamond-Blackfan anemia affect the assembly of ribosomal protein S19 into the ribosome. Angelini, M; Cannata, S; Mercaldo, V; Gibello, L; Santoro, C; Dianzani, I; Loreni, F Human molecular genetics
16
1720-7
2007
Show Abstract
RPS19 has been identified as the first gene associated with Diamond-Blackfan anemia (DBA), a rare congenital hypoplastic anemia that includes variable physical malformations. It is mutated in approximately 25% of the patients although doubts remain as to whether DBA clinical phenotype depends on the ribosomal function of RPS19 or on an extra-ribosomal role or on both. RPS19 mRNAs with mutations that introduce premature stop codons or eliminate it are rapidly turned over by the surveillance mechanisms possibly causing a decrease in the RPS19 protein level. A decrease in RPS19 level has been shown to cause a defect in the maturation of 18S ribosomal RNA. Less clear is the effect of missense mutations in RPS19. With the aim of analyzing the functional features of mutated RPS19, we prepared cDNA constructs expressing RPS19 containing 11 missense mutations and a trinucleotide insertion found in DBA patients. After transfection, we analyzed the following properties of the mutated proteins: (i) protein stability, (ii) subcellular localization and (iii) assembly into ribosomes. Our results indicate that some RPS19 mutations alter the capacity of the protein to localize in nucleolar structure and these mutated RPS19 are very unstable. Moreover, none of the mutated RPS19 analyzed in this study, including those proteins that appear localized into the nucleolus, is able to be assembled into mature ribosome. | | 17517689
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