scholarly journals Camelpox virus encodes a schlafen-like protein that affects orthopoxvirus virulence

2007 ◽  
Vol 88 (6) ◽  
pp. 1667-1676 ◽  
Author(s):  
Caroline Gubser ◽  
Rory Goodbody ◽  
Andrea Ecker ◽  
Gareth Brady ◽  
Luke A. J. O'Neill ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Sequence Similarity ◽  
Small Animal ◽  
Wild Type ◽  
Recombinant Viruses ◽  
C Terminus ◽  
N Terminus ◽  
Type V

Camelpox virus (CMLV) gene 176R encodes a protein with sequence similarity to murine schlafen (m-slfn) proteins. In vivo, short and long members of the m-slfn family inhibited T-cell development, whereas in vitro, only short m-slfns caused arrest of fibroblast growth. CMLV 176 protein (v-slfn) is most closely related to short m-slfns; however, when expressed stably in mammalian cells, v-slfn did not inhibit cell growth. v-slfn is a predominantly cytoplasmic 57 kDa protein that is expressed throughout infection. Several other orthopoxviruses encode v-slfn proteins, but the v-slfn gene is fragmented in all sequenced variola virus and vaccinia virus (VACV) strains. Consistent with this, all 16 VACV strains tested do not express a v-slfn detected by polyclonal serum raised against the CMLV protein. In the absence of a small animal model to study CMLV pathogenesis, the contribution of CMLV v-slfn to orthopoxvirus virulence was studied via its expression in an attenuated strain of VACV. Recombinant viruses expressing wild-type v-slfn or v-slfn tagged at its C terminus with a haemagglutinin (HA) epitope were less virulent than control viruses. However, a virus expressing v-slfn tagged with the HA epitope at its N terminus had similar virulence to controls, implying that the N terminus has an important function. A greater recruitment of lymphocytes into infected lung tissue was observed in the presence of wild-type v-slfn but, interestingly, these cells were less activated. Thus, v-slfn is an orthopoxvirus virulence factor that affects the host immune response to infection.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals A Novel Type V CRISPR System with Potential for Genome Editing in the Liver

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1862-1862
Author(s):  
Gregory J. Cost ◽  
Morayma Temoche-Diaz ◽  
Janet Mei ◽  
Cristina N. Butterfield ◽  
Christopher T. Brown ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genome Editing ◽  
Mammalian Cells ◽  
Conflicts Of Interest ◽  
Attractive Alternative ◽  
Vitro Assay ◽  
Crispr System ◽  
Type V

Abstract RNA guided CRISPR genome editing systems can make specific changes to the genomes of mammalian cells and have the potential to treat a range of diseases including those that can be addressed by editing hepatocytes. Attempts to edit the liver in vivo have relied almost exclusively on the Cas9 nucleases derived from the bacteria S treptococcus pyogenes or Staphylococcus aureus to which humans are commonly exposed. Pre-existing immunity to both these proteins has been reported in humans which raises concerns about their in vivo application. In silico analysis of a large metagenomics database followed by testing in mammalian cells in culture identified MG29-1, a novel CRISPR system which is a member of the Type V family but exhibits only 41 % amino acid identity to Francisella tularensis Cas12a/cpf1. MG29-1 is a 1280 amino acid RNA programmable nuclease that utilizes a single guide RNA comprised of a 22 nucleotide (nt) constant region and a 20 to 25 nt spacer, recognizes the PAM KTTN (predicted frequency 1 in 16 bp) and generates staggered cuts. MG29-1 was derived from a sample taken from a hydrothermal vent and it is therefore unlikely that humans will have developed pre-existing immunity to this protein. A screen for sgRNA targeting serum albumin in the mouse liver cell line Hepa1-6 identified 6 guides that generated more than 80% INDELS. The MG29-1 system was optimized for in vivo delivery by screening chemical modifications to the guide that improve stability in mammalian cell lysates while retaining or improving editing activity. Two lead guide chemistries were evaluated in mice using MG29-1 mRNA and sgRNA packaged in lipid nanoparticles (LNP). Three days after a single IV administration on-target editing was evaluated in the liver by Sanger sequencing. The sgRNA that was the most stable in the in vitro assay generated INDELS that ranged from 20 to 25% while a sgRNA with lower in vitro stability failed to generate detectable INDELs. The short sgRNA and small protein size compared to spCas9 makes MG29-1 an attractive alternative to spCas9 for in vivo editing applications. Evaluation of the potential of MG29-1 to perform gene knockouts and gene additions via non-homologous end joining is ongoing. Disclosures No relevant conflicts of interest to declare.

scholarly journals Genetic Analysis of theEscherichia coliFtsZ·ZipA Interaction in the Yeast Two-hybrid System

2001 ◽  
Vol 276 (15) ◽  
pp. 11980-11987 ◽  
Author(s):  
Steven A. Haney ◽  
Elizabeth Glasfeld ◽  
Cynthia Hale ◽  
David Keeney ◽  
Zhizhen He ◽  
...  
Keyword(s):  
Hybrid System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Conserved Sequence ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Two Hybrid

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division inEscherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZD373G) identified eight different changes at two residues within this sequence.In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZD373Gfailed to interact. Two mutant proteins examined restored this interaction significantly.In vivo, the alleles tested are significantly more toxic than the wild typeftsZand cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZD373G, no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

Rhogtpase RAC2 Is Required for In Vivo Transformation Activity by P210 Bcr-Abl Fusion Oncogene.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 717-717
Author(s):  
Nithya Krishnan ◽  
Jeff R. Bailey ◽  
Victoria Summey-Harner ◽  
Claudio Brunstein ◽  
Catherine M. Verfaillie ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Philadelphia Chromosome ◽  
Protein Domain ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Empty Vector

Abstract Bcr-Abl, the translocation product of the Philadelphia chromosome implicated in human chronic myelogenous leukemia (CML), is a kinase affecting hematopoietic stem cell (HSC) behavior with respect to proliferation, apoptosis, adhesion and migration. Rho GTPases, particularly the Rac subfamily, have been shown to regulate these same cell functions in normal HSC and also regulate gene expression in many mammalian cells. BCR contains a “GTPase-activating protein” domain and a guanine nucleotide exchange domain, the latter or which is preserved in p210 Bcr-Abl. Since HSC functions regulated by Bcr-Abl and Rac are similar, we studied the potential involvement of Rac activation in Bcr-Abl signaling cascade. Human CML samples demonstrate baseline activation of Rac proteins that is reversed by in vitro treatment with STI571. To study the specific involvement of Rac2, we used a gene targeted mouse model with Rac2 null bone marrow. Using retovirus-mediated gene transfer, we introduced p210 Bcr-Abl in the MSCV vector into wild-type or Rac2−/− HSC/P and studied the behavior of these cells in vitro and in vivo. Irradiated recipient mice injected with LDBM cells transduced with p210 developed a uniformly fatal myeloproliferative syndrome (Median survival: 45 days, N=12), while mice injected with p210 transduced Rac2−/− LDBM cells (N=12, 2 independent exp.) had 100% survival and no development of leukocytosis, splenomegaly or organ infiltration of hematopoietic cells. These data suggest that Rac GTPases are critical for the transformation of HSC by Bcr-Abl and provide an additional therapeutic target for intervention in CML. WILD TYPE Rac 2 −/− Empty Vector MSCV-p210 Empty vector MSCV-p210 *p < 0.01 vs WT-MIEG3, **p< 0.01 vs WT-p210 bcr-abl. Proliferation (CPM) Medium 562 ± 278 16,207± 1605* 819.7 ± 363 3,135.5 ± 498** SCF (100ng/ml) 856 ± 187 23,226 ± 2203* 853.7 ± 524 3,756.8 ± 207** Cytokines (SCF, GCSF, MGDF) 8011± 1412 42,711± 13393* 4833 ±1019 3,614.5 ± 1982** Migration (%) Fibronectin 7 ± 0.4 38 ± 1.9* 0.4 ± 0.0 0.8 ± 0.1** SDF-1α 30 ±2.8 13 ±1.1* 0.5 ± 0.0 0.6 ± 0.0** Adhesion (% ) Fibronectin 76± 2.9 40 ±3* 4 ±0.4 10 ±0.1 **

scholarly journals The multiple facets of the Golgi reassembly stacking proteins

2011 ◽  
Vol 433 (3) ◽  
pp. 423-433 ◽  
Author(s):  
Fabian P. Vinke ◽  
Adam G. Grieve ◽  
Catherine Rabouille
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Mitotic Checkpoint ◽  
Biochemical Properties ◽  
C Terminus ◽  
Unconventional Secretion ◽  
Golgi Ribbon

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.

scholarly journals Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins

2000 ◽  
Vol 182 (3) ◽  
pp. 637-646 ◽  
Author(s):  
Sabine Enz ◽  
Susanne Mahren ◽  
Uwe H. Stroeher ◽  
Volkmar Braun
Keyword(s):  
Nitrilotriacetic Acid ◽  
Ferric Citrate ◽  
Binding Assays ◽  
Surface Binding ◽  
Citrate Transport ◽  
C Terminus ◽  
N Terminus ◽  
Surface Signaling

ABSTRACT In Escherichia coli, transcription of the ferric citrate transport genes fecABCDE is controlled by a novel signal transduction mechanism that starts at the cell surface. Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm where FecI, the sigma factor, is activated. Interaction between the signaling proteins was demonstrated by utilizing two methods. In in vitro binding assays, FecR that was His tagged at the N terminus [(His)10-FecR] and bound to a Ni-nitrilotriacetic acid agarose column was able to retain FecA, and FecR that was His tagged at the C terminus [FecR-(His)6] retained FecI on the column. An N-terminally truncated, induction-negative but transport-active FecA protein did not bind to (His)10-FecR. The in vivo assay involved the determination of the FecA, FecR, and FecI interacting domains with the bacterial two-hybrid Lex-based system. FecA1–79 interacts with FecR101–317 and FecR1–85 interacts with FecI1–173. These data clearly support a model that proposes interaction of the periplasmic N terminus of FecA with the periplasmic C-terminal portion of FecR and interaction of the cytoplasmic N terminus of FecR with FecI, which results in FecI activation.

scholarly journals Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi

2008 ◽  
Vol 82 (10) ◽  
pp. 4955-4964 ◽  
Author(s):  
B. Costes ◽  
G. Fournier ◽  
B. Michel ◽  
C. Delforge ◽  
V. Stalin Raj ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Thymidine Kinase ◽  
Artificial Chromosome ◽  
Wild Type ◽  
Bac Clone ◽  
Recombinant Viruses ◽  
Koi Herpesvirus ◽  
Cyprinus Carpio Koi

ABSTRACT Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

scholarly journals Functional Characterization of the Interaction of Ste50p with Ste11p MAPKKK inSaccharomyces cerevisiae

1999 ◽  
Vol 10 (7) ◽  
pp. 2425-2440 ◽  
Author(s):  
Cunle Wu ◽  
Ekkehard Leberer ◽  
David Y. Thomas ◽  
Malcolm Whiteway
Keyword(s):  
Protein Kinase ◽  
Functional Characterization ◽  
Hog Pathway ◽  
C Terminus ◽  
Extracellular Signal ◽  
Osmotic Stress Response ◽  
N Terminus

The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as theSchizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

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