scholarly journals Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin ActivityIn Vivo

2019 ◽  
Vol 39 (13) ◽  
Author(s):  
Wi S. Lai ◽  
Deborah J. Stumpo ◽  
Melissa L. Wells ◽  
Artiom Gruzdev ◽  
Stephanie N. Hicks ◽  
...  
Keyword(s):  
Binding Domain ◽  
Content Type ◽  
Target Mrna ◽  
Binding Domains ◽  
Inflammatory Protein ◽  
Carboxyl Terminal ◽  
The Stability ◽  
Inflammatory Syndrome

ABSTRACTTristetraprolin (TTP) is an anti-inflammatory protein that modulates the stability of certain cytokine/chemokine mRNAs. After initial high-affinity binding to AU-rich elements in 3′ untranslated regions of target mRNAs, mediated through its tandem zinc finger (TZF) domain, TTP promotes the deadenylation and ultimate decay of target transcripts. These transcripts and their encoded proteins accumulate abnormally in TTP knockout (KO) mice, leading to a severe inflammatory syndrome. To assess the importance of the highly conserved C-terminal CNOT1 binding domain (CNBD) of TTP to the TTP deficiency phenotype in mice, we created a mouse model in which TTP lacked its CNBD. CNBD deletion mice exhibited a less severe phenotype than the complete TTP KO mice. In macrophages, the stabilization of target transcripts seen in KO mice was partially normalized in the CNBD deletion mice. In cell-free experiments, recombinant TTP lacking its CNBD could still activate target mRNA deadenylation by purified recombinantSchizosaccharomyces pombeCCR4/NOT complexes, although to a lesser extent than full-length TTP. Thus, TTP lacking its CNBD can still act to promote target mRNA instabilityin vitroandin vivo. These data have implications for TTP family members throughout the eukarya, since species from all four kingdoms contain proteins with linked TZF and CNOT1 binding domains.

scholarly journals The 5′ NAD Cap of RNAIII Modulates Toxin Production in Staphylococcus aureus Isolates

2019 ◽  
Vol 202 (6) ◽  
Author(s):  
Hector Gabriel Morales-Filloy ◽  
Yaqing Zhang ◽  
Gabriele Nübel ◽  
Shilpa Elizabeth George ◽  
Natalya Korn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Secondary Structure ◽  
Quorum Sensing ◽  
Opportunistic Pathogen ◽  
Toxin Production ◽  
Dual Function ◽  
Content Type ◽  
The Stability

ABSTRACT Nicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5′ ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here, we report the occurrence of several NAD-RNAs in the opportunistic pathogen Staphylococcus aureus. Most prominently, RNAIII, a central quorum-sensing regulator of this bacterium’s physiology, was found to be 5′ NAD capped in a range from 10 to 35%. NAD incorporation efficiency into RNAIII was found to depend in vivo on the −1 position of the P3 promoter. An increase in RNAIII’s NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects seem to be caused neither by changes in RNAIII’s secondary structure nor by a different translatability upon NAD attachment, as indicated by unaltered patterns in in vitro chemical probing and toeprinting experiments. Even though we did not observe any effect of this modification on RNAIII’s secondary structure or translatability in vitro, additional unidentified factors might account for the modulation of exotoxins in vivo. Ultimately, the study constitutes a step forward in the discovery of new roles of the NAD molecule in bacteria. IMPORTANCE Numerous organisms, including bacteria, are endowed with a 5′ NAD cap in specific RNAs. While the presence of the 5′ NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5′ NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating the bacterium’s virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.

Phytochemical screening and potential of natural dye colourant from pomegranate (Punica granatum L.)

2016 ◽  
Vol 45 (1) ◽  
pp. 38-44 ◽  
Author(s):  
S. Mohajer ◽  
R.M. Taha ◽  
S.Z. Azmi
Keyword(s):  
Polyvinyl Alcohol ◽  
Antioxidant Activities ◽  
Punica Granatum ◽  
Natural Dye ◽  
Coating System ◽  
Content Type ◽  
Ic50 Values ◽  
The Stability

Purpose – The purpose of this paper is to identify the most dominant pigment of pomegranate explants for natural color coatings and detect the presence of phytochemical constituents and comparison of the antioxidant activities. Design/methodology/approach – Extracts of leaf, stem, peel and seed of in vitro and in vivo growth cultures were prepared for phytochemical constituent and antioxidant activity. The supernatant from 95 per cent methanol was mixed with 15 per cent polyvinyl alcohol (PVA) with the ratio of 1:1 to form a coating system. Findings – Although glycosides was not found in this species, tests for tannins and flavonoids were positive in all samples. The IC50 values were also comparable to commercial antioxidant ascorbic acid with 34.92 per cent inhibition. Chlorophyll a and b were detected in stem and leaf using UV-photospectrometer in 420 and 645 nm wavelengths ranges. The effects of heat and salt on the stability of natural dye colorants mixed with polyvinyl alcohol to form a basic coating system were indicated negatively in in vivo and in vitro growth cultures. Originality/value – The paper shows that further improvement with co-pigmentations may give a notable mixture from pomegranate extraction for the paint materials or nail varnish. It was also indicated that pomegranate contains some compounds such as polyphenolics that can donate electron/hydrogen easily.

scholarly journals Overexpression of human fibroblast caldesmon fragment containing actin-, Ca++/calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments.

1994 ◽  
Vol 125 (2) ◽  
pp. 359-368 ◽  
Author(s):  
K S Warren ◽  
J L Lin ◽  
D D Wamboldt ◽  
J J Lin
Keyword(s):  
Cho Cells ◽  
Actin Filaments ◽  
Actin Binding ◽  
Expression Vectors ◽  
Binding Domains ◽  
Microfilament Bundles ◽  
The Stability ◽  
Triton X

Fibroblast caldesmon is a protein postulated to participate in the modulation of the actin cytoskeleton and the regulation of actin-based motility. The cDNAs encoding the NH2-terminal (aa.1-243, CaD40) and COOH-terminal (aa.244-538, CaD39) fragments of human caldesmon were subcloned into expression vectors and we previously reported that bacterially produced CaD39 protein retains its actin-binding properties as well as its ability to enhance low M(r) tropomyosin (TM) binding to actin and to inhibit TM-actin-activated HMM ATPase activity in vitro (Novy, R. E., J. R. Sellers, L.-F. Liu, and J. J.-C. Lin. 1993. Cell Motil. Cytoskeleton. 26:248-261). Bacterially produced CaD40 does not bind actin. To study the in vivo effects of CaD39 expression on the stability of actin filaments in CHO cells, we isolated and characterized stable CHO transfectants which express varying amounts of CaD39. We found that expression of CaD39 in CHO cells stabilized microfilament bundles as well as endogenous TM. CaD39-expressing clones displayed an increased resistance to cytochalasin B and Triton X-100 treatments and yielded increased amounts of TM-containing actin filaments in microfilament isolation procedures. In addition, analysis of these clones with immunoblotting and indirect immunofluorescence microscopy with anti-TM antibody revealed that stabilized endogenous TM and enhanced TM-containing microfilament bundles parallel increased amounts of CaD39 expression. The increased TM observed corresponded to a decrease in TM turnover rate and did not appear to be due to increased synthesis of endogenous TM. Additionally, the phenomenon of stabilized TM did not occur in stable CHO clones expressing CaD40. Therefore, it is likely that CaD39 can enhance TM's binding to F-actin in vivo, thus reducing TM's rate of turnover and stabilizing actin microfilament bundles.

scholarly journals Mechanism for Stabilizing mRNAs Involved in Methanol-Dependent Methanogenesis of Cold-Adaptive Methanosarcina mazei zm-15

2013 ◽  
Vol 80 (4) ◽  
pp. 1291-1298 ◽  
Author(s):  
Yi Cao ◽  
Jie Li ◽  
Na Jiang ◽  
Xiuzhu Dong
Keyword(s):  
Half Life ◽  
Content Type ◽  
Methanosarcina Mazei ◽  
Reverse Transcription Quantitative Pcr ◽  
Aceticlastic Methanogenesis ◽  
The Stability ◽  
Zoige Wetland ◽  
Mrna Half Life

ABSTRACTMethylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats,Methanosarcina mazeizm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances ofmtaA1,mtaB1, andmtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, whileackAandptamRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. Thein vivohalf-lives ofmtaA1andmtaC1B1mRNAs were similar in 30°C and 15°C cultures. However, thepta-ackAmRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5′ untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified formtaA1andmtaC1B1mRNAs, while only a 27-nt 5′ UTR was present in thepta-ackAtranscript. Removal of the 5′ UTRs significantly reduced thein vitrohalf-lives ofmtaA1andmtaC1B1mRNAs. Remarkably, fusion of themtaA1ormtaC1B15′ UTRs topta-ackAmRNA increased itsin vitrohalf-life at both 30°C and 15°C. These results demonstrate that the large 5′ UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptiveM. mazeizm-15.

scholarly journals A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

2020 ◽  
Vol 168 (5) ◽  
pp. 515-533
Author(s):  
Jose M González de Cózar ◽  
Maria Carretero-Junquera ◽  
Grzegorz L Ciesielski ◽  
Sini M Miettinen ◽  
Markku Varjosalo ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Shotgun Proteomics ◽  
Binding Domain ◽  
The Novel ◽  
Binding Model ◽  
Interaction Partners ◽  
The Stability ◽  
Deletion Series

Abstract In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, while loss of the novel HBD led to an ∼90% drop in Kcat with a decreased KM, and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite-binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo cross-linking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, we were not able to document direct and stable interactions with mtSSB when the proteins were co-overexpressed in S2 cells, and functional interactions between them in vitro were minor.

scholarly journals MBD3L1 Is a Transcriptional Repressor That Interacts with Methyl-CpG-binding Protein 2 (MBD2) and Components of the NuRD Complex

2004 ◽  
Vol 279 (50) ◽  
pp. 52456-52464 ◽  
Author(s):  
Chun-Ling Jiang ◽  
Seung-Gi Jin ◽  
Gerd P. Pfeifer
Keyword(s):  
Transcriptional Repression ◽  
Binding Protein ◽  
Transcriptional Repressor ◽  
Binding Domain ◽  
Nurd Complex ◽  
Methylated Dna ◽  
Binding Domains ◽  
Mouse Cells

Methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) are transcriptional repressors that contain methyl-CpG binding domains and are components of a CpG-methylated DNA binding complex named MeCP1. Methyl-CpG-binding protein 3-like 1 (MBD3L1) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG binding domain. MBD3L1 interacts with MBD2 and MBD3in vitroand in yeast two-hybrid assays. Gel shift experiments with a CpG-methylated DNA probe indicate that recombinant MBD3L1 can supershift an MBD2-methylated DNA complex.In vivo, MBD3L1 associates with and colocalizes with MBD2 but not with MBD3 and is recruited to 5-methylcytosine-rich pericentromeric heterochromatin in mouse cells. In glutathioneS-transferase pull-down assays MBD3L1 is found associated with several known components of the MeCP1·NuRD complex, including HDAC1, HDAC2, MTA2, MBD2, RbAp46, and RbAp48, but MBD3 is not found in the MBD3L1-bound fraction. MBD3L1 enhances transcriptional repression of methylated DNA by MBD2. The data are consistent with a role of MBD3L1 as a methylation-dependent transcriptional repressor that may interchange with MBD3 as an MBD2-interacting component of the NuRD complex. MBD3L1 knockout mice were created and were found to be viable and fertile, indicating that MBD3L1 may not be essential or there is functional redundancy (through MBD3) in this pathway. Overall, this study reveals additional complexities in the mechanisms of transcriptional repression by the MBD family proteins.

scholarly journals Dectin Immunoadhesins and Pneumocystis Pneumonia

2013 ◽  
Vol 81 (9) ◽  
pp. 3451-3462 ◽  
Author(s):  
David M. Ricks ◽  
Kong Chen ◽  
Mingquan Zheng ◽  
Chad Steele ◽  
Jay K. Kolls
Keyword(s):  
Inflammatory Response ◽  
Opportunistic Pathogen ◽  
Pneumocystis Pneumonia ◽  
Carbohydrate Binding ◽  
Content Type ◽  
Specificity Of Binding ◽  
Inflammatory Syndrome ◽  
Dependent Mechanism

ABSTRACTThe opportunistic pathogenPneumocystis jiroveciiis a significant cause of disease in HIV-infected patients and others with immunosuppressive conditions.Pneumocystiscan also cause complications in treatment following antiretroviral therapy or reversal of immunosuppressive therapy, as the newly reconstituted immune system can develop a pathological inflammatory response to remaining antigens or a previously undetected infection. To target β-(1,3)-glucan, a structural component of thePneumocystiscell wall with immune-stimulating properties, we have developed immunoadhesins consisting of the carbohydrate binding domain of Dectin-1 fused to the Fc regions of the 4 subtypes of murine IgG (mIgG). These immunoadhesins bind β-glucan with high affinity, and precoating the surface of zymosan with Dectin-1:Fc can reduce cytokine production by macrophages in anin vitrostimulation assay. All Dectin-1:Fc variants showed specificity of binding to the asci ofPneumocystis murina, but effector activity of the fusion molecules varied depending on Fc subtype. Dectin-1:mIgG2a Fc was able to reduce the viability ofP. murinain culture through a complement-dependent mechanism, whereas previous studies have shown the mIgG1 Fc fusion to increase macrophage-dependent killing. In anin vivochallenge model, systemic expression of Dectin-1:mIgG1 Fc significantly reduced ascus burden in the lung. When administered postinfection in a model of immune reconstitution inflammatory syndrome (IRIS), both Dectin-1:mIgG1 and Dectin-1:mIgG2a Fc reduced hypoxemia despite minimal effects on fungal burden in the lung. Taken together, these data indicate that molecules targeting β-glucan may provide a mechanism for treatment of fungal infection and for modulation of the inflammatory response toPneumocystisand other pathogens.

scholarly journals A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis

2002 ◽  
Vol 13 (10) ◽  
pp. 3683-3695 ◽  
Author(s):  
Petra Björk ◽  
Göran Baurén ◽  
ShaoBo Jin ◽  
Yong-Guang Tong ◽  
Thomas R. Bürglin ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Ribosomal Subunit ◽  
Binding Domain ◽  
Dependent Manner ◽  
Binding Domains ◽  
40S Ribosomal Subunit ◽  
Rna Binding Domain

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20–30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

DNMT1 Cxxc Domain Can Functionally Substitute In An MLL Fusion Protein

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4193-4193
Author(s):  
Laurie E. Risner ◽  
Aravinda Kuntimaddi ◽  
John H. Bushweller ◽  
Nancy J. Zeleznik-Le
Keyword(s):  
Dna Binding ◽  
Binding Affinity ◽  
Fusion Proteins ◽  
Binding Domain ◽  
Cpg Dna ◽  
Domain Swap ◽  
Binding Domains ◽  
Dna Binding Affinity

Abstract Abstract 4193 The MLL gene encodes a multi-domain protein that is involved in the maintenance of Hox gene expression during development and hematopoiesis, and was first identified through its involvement in chromosome translocations that cause leukemia. The CXXC domain of MLL, which is retained in leukemic MLL fusion proteins, is a cysteine rich DNA binding domain, with specificity for binding nonmethylated CpG-containing DNA, and is essential for MLL fusion proteins' oncogenic properties. We performed domain swap experiments in which CXXC domains from other proteins were swapped in to replace MLL's CXXC domain in the context of an oncogenic MLL fusion. CXXC domains from DNA methyltransferase 1 (DNMT1), CpG binding protein (CGBP), and methyl-CpG binding domain protein 1 (MBD1), as well as a methyl binding domain (MBD) from MBD1 were swapped into the MLL-AF9 fusion. These particular domains were chosen because their described CpG DNA binding capacity is either similar or different from that described for MLL. In vitro colony assays on isolated murine bone marrow progenitor cells infected with domain swapped or wild type MLL-AF9 fusion genes were performed in order to determine whether CpG binding domains from other proteins would affect the ability of MLL-AF9 to give an enhanced proliferative capacity to bone marrow progenitor cells. In vivo murine studies determined whether the different CpG binding domains alter the ability of MLL fusion proteins to cause leukemia. We predicted that the different CpG binding domains would change the strength or specificity of MLL binding to DNA, which would affect the ability of MLL-AF9 to cause leukemia. The results of both in vitro replating assays and in vivo leukemogenesis experiments have shown significant differences between the ability of various CpG DNA binding domains to function in the context of an MLL-AF9 fusion protein. MLL-AF9 containing the DNMT1 CXXC domain shows robust in vitro colony forming activity and in vivo leukemogenesis activity, similar to the oncogenic MLL-AF9 fusion. However, MLL-AF9 containing either the CXXC domain from CGBP or MBD1, or the MBD domain of MBD1 all show reduced colony forming ability and leukemogenicity in vivo. In vitro DNA binding experiments are currently being performed to directly measure and compare the DNA binding affinity of the CXXC domain from MLL to the other domain swap proteins. Preliminary data suggests that MLL CXXC has a stronger DNA binding affinity to non-methylated DNA compared to the other CXXC domains. Furthermore, the DNMT and CGBP CXXC domains both show lower affinity DNA binding compared to MLL CXXC, but they have different effects in MLL-AF9. This suggests that CXXC domain properties in addition to DNA binding affinity, perhaps including protein recruitment, also contribute to an MLL fusion protein's leukemogenic properties. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lysostaphin Lysibody Leads to Effective Opsonization and Killing of Methicillin-ResistantStaphylococcus aureusin a Murine Model

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Assaf Raz ◽  
Anna Serrano ◽  
Maneesha Thaker ◽  
Tricia Alston ◽  
Vincent A. Fischetti
Keyword(s):  
Binding Domain ◽  
Human Neutrophils ◽  
Carbohydrate Binding ◽  
Igg Antibody ◽  
Content Type ◽  
Bacterial Surface ◽  
Gram Positive Bacteria ◽  
Binding Domains ◽  
Surface Carbohydrates

ABSTRACTThe cell wall of Gram-positive bacteria contains abundant surface-exposed carbohydrate structures that are highly conserved. While these properties make surface carbohydrates ideal targets for immunotherapy, carbohydrates elicit a poor immune response that results primarily in low-affinity IgM antibodies. In a previous publication, we introduced the lysibody approach to address this shortcoming. Lysibodies are engineered molecules that combine a high-affinity carbohydrate-binding domain of bacterial or bacteriophage origin and an Fc effector portion of a human IgG antibody, thus directing effective immunity to conserved bacterial surface carbohydrates. Here, we describe the first example of a lysibody containing the binding domain from a bacteriocin, lysostaphin. We also describe the creation of five lysibodies with binding domains derived from phage lysins, directed againstStaphylococcus aureus. The lysostaphin and LysK lysibodies showed the most promise and were further characterized. Both lysibodies bound a range of clinically important staphylococcal strains, fixed complement on the staphylococcal surface, and induced phagocytosis ofS. aureusby macrophages and human neutrophils. The lysostaphin lysibody had superiorin vitroactivity compared to that of the LysK lysibody, as well as that of the previously characterized ClyS lysibody, and it effectively protected mice in a kidney abscess/bacteremia model. These results further demonstrate that the lysibody approach is a reproducible means of creating antibacterial antibodies that cannot be produced by conventional means. Lysibodies therefore are a promising solution for opsonic antibodies that may be used passively to both treat and prevent infection by drug-resistant pathogens.

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