Effect of mutation of a calmodulin binding site on Ca2+ regulation of inositol trisphosphate receptors

2001 ◽  
Vol 360 (2) ◽  
pp. 395-400 ◽  
Author(s):  
Xianchao ZHANG ◽  
Suresh K. JOSEPH
Keyword(s):  
Splice Variants ◽  
Binding Motif ◽  
Type I ◽  
Channel Activity ◽  
Wild Type ◽  
Binding Assays ◽  
Ip3 Receptor ◽  
Calmodulin Binding ◽  
Mutant Receptors

Several studies have shown that calmodulin (CaM) modulates d-myo-inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel activity and ligand binding to IP3Rs. It has been proposed that CaM may act as the Ca2+ sensor for mediating Ca2+ inhibition of IP3R channel activity. However, the functional role of CaM binding sites and the mechanism by which CaM regulates IP3R activities remains unclear. Tryptophan at position 1577 of type I IP3R has been shown to be part of a motif that is responsible for CaM binding to IP3Rs and we have mutated this residue to alanine in the long (neuronal) and short (peripheral) SII splice variants of the type I IP3R. CaM–Sepharose binding assays using COS-7 cell lysates confirmed that the W1577A mutant in both splice variants completely eliminated CaM binding. Functional measurements of IP3-mediated 45Ca2+ fluxes indicated that there was no change in the IP3 sensitivity of the channel induced by the W1577A mutation. Such measurements also indicated that the W1577A mutants of both splice variants have a dependence on external [Ca2+] that was indistinguishable from the corresponding wild-types. Although subtle differences in the Ca2+ and CaM sensitivity of [3H]IP3 binding were noted between wild-type and mutant receptors, our data suggest that the CaM binding motif involving the W1577A locus does not play a role in Ca2+ regulation of IP3R channel activity.

The role of N-terminal and C-terminal Arg residues from BK on interaction with kinin B2 receptor

2016 ◽  
Vol 397 (4) ◽  
pp. 305-314 ◽  
Author(s):  
Rafael Filippelli-Silva ◽  
Renan P. Martin ◽  
Eliete S. Rodrigues ◽  
Clovis R. Nakaie ◽  
Laerte Oliveira ◽  
...  
Keyword(s):  
Type B ◽  
Wild Type ◽  
Binding Assays ◽  
Physiological Processes ◽  
Intracellular Calcium Mobilization ◽  
Stomach Fundus ◽  
Residue Numbers ◽  
Mutant Receptors ◽  
G Protein Coupled

Abstract Bradykinin (BK) is a nonapeptide important for several physiological processes such as vasodilatation, increase in vascular permeability and release of inflammatory mediators. BK performs its actions by coupling to and activating the B2 receptor, a family A G-protein coupled receptor. Using a strategy which allows systematical monitoring of BK R1 and R9 residues and B2 receptor acidic residues Glu5.35(226) and Asp6.58(298), our study aims at clarifying the BK interaction profile with the B2 receptor [receptor residue numbers are normalized according to Ballesteros and Weinstein, Methods Neurosci. 25 (1995), pp. 366–428) followed by receptor sequence numbering in brackets]. N- and C-terminal analogs of BK (-A1, -G1, -K1, -E1 and BK-A9) were tested against wild type B2, Glu5.35(226)Ala and Asp6.58(298)Ala B2 mutant receptors for their affinity and capability to elicit responses by mechanical recordings of isolated mice stomach fundus, measuring intracellular calcium mobilization, and competitive fluorimetric binding assays. BK showed 2- and 15-fold decreased potency for Glu5.35(226) and Asp6.58(298) B2 mutant receptors, respectively. In B2-Glu5.35(226)Ala BK analogs showed milder reduction in evaluated parameters. On the other hand, in the B2-Asp6.58(298)Ala mutant, no N-terminal analog was able to elicit any response. However, the BK-A9 analog presented higher affinity parameters than BK in the latter mutant. These findings provide enough support for defining a novel interaction role of BK-R9 and Asp6.58(298) receptor residues.

scholarly journals Induction of Dendritic Cell Production of Type I and Type III Interferons by Wild-Type and Vaccine Strains of Measles Virus: Role of Defective Interfering RNAs

2013 ◽  
Vol 87 (14) ◽  
pp. 7816-7827 ◽  
Author(s):  
R. Shivakoti ◽  
M. Siwek ◽  
D. Hauer ◽  
K. L. W. Schultz ◽  
D. E. Griffin
Keyword(s):  
Dendritic Cell ◽  
Measles Virus ◽  
Type I ◽  
Wild Type ◽  
Cell Production ◽  
Type Iii

Retrovirus-induced interference with collagen I gene expression in Mov13 fibroblasts is maintained in the absence of DNA methylation

1991 ◽  
Vol 11 (1) ◽  
pp. 47-54
Author(s):  
H Chan ◽  
S Hartung ◽  
M Breindl
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Chromatin Structure ◽  
Transcriptional Activity ◽  
Xenopus Laevis Oocytes ◽  
Regulatory Sequences ◽  
Type I ◽  
Wild Type ◽  
Col1a1 Gene

We have studied the role of DNA methylation in repression of the murine alpha 1 type I collagen (COL1A1) gene in Mov13 fibroblasts. In Mov13 mice, a retroviral provirus has inserted into the first intron of the COL1A1 gene and blocks its expression at the level of transcriptional initiation. We found that regulatory sequences in the COL1A1 promoter region that are involved in the tissue-specific regulation of the gene are unmethylated in collagen-expressing wild-type fibroblasts and methylated in Mov13 fibroblasts, confirming and extending earlier observations. To directly assess the role of DNA methylation in the repression of COL1A1 gene transcription, we treated Mov13 fibroblasts with the demethylating agent 5-azacytidine. This treatment resulted in a demethylation of the COL1A1 regulatory sequences but failed to activate transcription of the COL1A1 gene. Moreover, the 5-azacytidine treatment induced a transcription-competent chromatin structure in the retroviral sequences but not in the COL1A1 promoter. In DNA transfection and microinjection experiments, we found that the provirus interfered with transcriptional activity of the COL1A1 promoter in Mov13 fibroblasts but not in Xenopus laevis oocytes. In contrast, the wild-type COL1A1 promoter was transcriptionally active in Mov13 fibroblasts. These experiments showed that the COL1A1 promoter is potentially transcriptionally active in the presence of proviral sequences and that Mov13 fibroblasts contain the trans-acting factors required for efficient COL1A1 gene expression. Our results indicate that the provirus insertion in Mov13 can inactivate COL1A1 gene expression at several levels. It prevents the developmentally regulated establishment of a transcription-competent methylation pattern and chromatin structure of the COL1A1 domain and, in the absence of DNA methylation, appears to suppress the COL1A1 promoter in a cell-specific manner, presumably by assuming a dominant chromatin structure that may be incompatible with transcriptional activity of flanking cellular sequences.

Functional comparison of the role of dynamin 2 splice variants on GLUT-4 endocytosis in 3T3L1 adipocytes

2000 ◽  
Vol 278 (5) ◽  
pp. E825-E831 ◽  
Author(s):  
Aimee W. Kao ◽  
Chunmei Yang ◽  
Jeffrey E. Pessin
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Splice Variants ◽  
Wild Type ◽  
Cell Cytoplasm ◽  
Functional Specificity ◽  
Glut 4 ◽  
Green Fluorescent ◽  
Dynamin 2

Previously, we reported that expression of a dominant-interfering neuronal-specific dynamin 1 (K44A/dynamin 1) inhibited the plasma membrane internalization of GLUT-4 in 3T3L1 adipocytes (15). To investigate the role of the ubiquitously expressed isoform of dynamin, dynamin 2, on adipocyte GLUT-4 internalization, and to determine whether dynamin splice variants have functional specificity, we expressed each of the four dynamin 2 isoforms (aa, ab, ba, and bb) as either wild-type proteins or GTPase-defective mutants. When expressed as enhanced green fluorescent protein (EGFP) fusions, these isoforms were found to have overlapping subcellular distributions being localized throughout the cell cytoplasm, on punctate vesicles and in a perinuclear compartment. This distribution was qualitatively similar to that of endogenous dynamin 2 and overlapped with GLUT-4 in the basal state. Expression of wild-type dynamin 2 isoforms had no effect on the basal or insulin-stimulated distribution of GLUT-4; however, expression of the dominant-interfering dynamin 2 mutants inhibited GLUT-4 endocytosis. These data demonstrate that dynamin 2 is required for GLUT-4 endocytosis in 3T3L1 adipocytes and suggest that, relative to GLUT-4 trafficking, the dynamin 2 splice variants have overlapping functions and are probably not responsible for mediating distinct GLUT-4 budding events.

Role of common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of murine mast cells

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2172-2180 ◽  
Author(s):  
Kotaro Suzuki ◽  
Hiroshi Nakajima ◽  
Norihiko Watanabe ◽  
Shin-ichiro Kagami ◽  
Akira Suto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Cytokine Receptor ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Wild Type ◽  
Peritoneal Mast Cells ◽  
The Common

Abstract The regulatory roles of the common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using γc-deficient (γc−) and Jak3-deficient (Jak3−) mice. Although the mast cells in γc− and Jak3− mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in γc− and Jak3− mice as compared with that in wild-type mice. Among γc-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow–derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from γc− and Jak3−mice. In addition, IL-4Rα, γc, and Jak3, but not IL-2Rβ or IL-7Rα, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Rα1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from γc− and Jak3− mice. These results indicate that γc- and Jak3-dependent signaling is essential for IL-4– and IL-9–induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a γc- and Jak3-dependent manner.

scholarly journals Two Outer Membrane Proteins Are Required for Maximal Type I Secretion of the Caulobacter crescentus S-Layer Protein

2004 ◽  
Vol 186 (23) ◽  
pp. 8000-8009 ◽  
Author(s):  
Michael C. Toporowski ◽  
John F. Nomellini ◽  
Peter Awram ◽  
John Smit
Keyword(s):  
Outer Membrane ◽  
Caulobacter Crescentus ◽  
Outer Membrane Proteins ◽  
Functional System ◽  
Type I ◽  
Wild Type ◽  
Residual Level ◽  
The Individual ◽  
Multicopy Vector

ABSTRACT Transport of RsaA, the crystalline S-layer subunit protein of Caulobacter crescentus, is mediated by a type I secretion mechanism. Two proteins have been identified that play the role of the outer membrane protein (OMP) component in the RsaA secretion machinery. The genes rsaF a and rsaF b were identified by similarity to the Escherichia coli hemolysin secretion OMP TolC by using the C. crescentus genome sequence. The rsaF a gene is located several kilobases downstream of the other transporter genes, while rsaF b is completely unlinked. An rsaF a knockout had ∼56% secretion compared to wild-type levels, while the rsaF b knockout reduced secretion levels to ∼79%. When expression of both proteins was eliminated, there was no RsaA secretion, but a residual level of ∼9% remained inside the cell, suggesting posttranslational autoregulation. Complementation with either of the individual rsaF genes by use of a multicopy vector, which resulted in 8- to 10-fold overexpression of the proteins, did not restore RsaA secretion to wild-type levels, indicating that both rsaF genes were required for full-level secretion. However, overexpression of rsaFa (with normal rsaF b levels) in concert with overexpression of rsaA resulted in a 28% increase in RsaA secretion, indicating a potential for significantly increasing expression levels of an already highly expressing type I secretion system. This is the only known example of type I secretion requiring two OMPs to assemble a fully functional system.

scholarly journals CRISPR-Cas9 Mediated RNase L Knockout Regulates Cellular Function of PK-15 Cells and Increases PRV Replication

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Chao Sui ◽  
Dandan Jiang ◽  
Xiangju Wu ◽  
Xiaoyan Cong ◽  
Feng Li ◽  
...  
Keyword(s):  
Cell Line ◽  
Gene Editing ◽  
Rna Degradation ◽  
Type I ◽  
Rnase L ◽  
Agarose Gel Electrophoresis ◽  
Biological Functions ◽  
Wild Type ◽  
Induction Of Apoptosis

Ribonuclease L (RNase L) is an important antiviral endoribonuclease regulated by type I IFN. RNase L is activated by viral infection and dsRNA. Because the role of swine RNase L (sRNase L) is not fully understood, in this study, we generated a sRNase L knockout PK-15 (KO-PK) cell line through the CRISPR/Cas9 gene editing system to evaluate the function of sRNase L. After transfection with CRISPR-Cas9 followed by selection using puromycin, sRNase L knockout in PK-15 cells was further validated by agarose gel electrophoresis, DNA sequencing, and Western blotting. The sRNase L KO-PK cells failed to trigger RNA degradation and induced less apoptosis than the parental PK-15 cells after transfected with poly (I: C). Furthermore, the levels of ISGs mRNA in sRNase L KO-PK cells were higher than those in the parental PK-15 cells after treated with poly (I: C). Finally, both wild type and attenuated pseudorabies viruses (PRV) replicated more efficiently in sRNase L KO-PK cells than the parental PK-15 cells. Taken together, these findings suggest that sRNase L has multiple biological functions including cellular single-stranded RNA degradation, induction of apoptosis, downregulation of transcript levels of ISGs, and antiviral activity against PRV. The sRNase L KO-PK cell line will be a valuable tool for studying functions of sRNase L as well as for producing PRV attenuated vaccine.

scholarly journals Distinct Roles of Lymphotoxin α and the Type I Tumor Necrosis Factor (TNF) Receptor in the Establishment of Follicular Dendritic Cells from Non–Bone Marrow–derived Cells

1997 ◽  
Vol 186 (12) ◽  
pp. 1997-2004 ◽  
Author(s):  
Mitsuru Matsumoto ◽  
Yang-Xin Fu ◽  
Hector Molina ◽  
Guangming Huang ◽  
Jinho Kim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Tumor Necrosis ◽  
Type I ◽  
Follicular Dendritic Cells ◽  
Wild Type ◽  
Lymphotoxin Α ◽  
Deficient Mice ◽  
Necrosis Factor

In mice deficient in either lymphotoxin α (LT-α) or type I tumor necrosis factor receptor (TNFR-I), organized clusters of follicular dendritic cells (FDC) and germinal centers (GC) are absent from the spleen. We investigated the role of LT-α and TNFR-I in the establishment of spleen FDC and GC structure by using reciprocal bone marrow (BM) transfer. When LT-α–deficient mice were reconstituted with wild-type BM, FDC organization and the ability to form GC were restored, indicating that the LT-α–expressing cells required to establish organized FDC are derived from BM. The role of LT-α in establishing organized FDC structure was further investigated by the transfer of complement receptor 1 and 2 (CR1/2)–deficient BM cells into LT-α–deficient mice. Organized FDC were identified with both the FDC-M1 and anti-CR1 monoclonal antibodies in these BM-chimeric mice, indicating that these cells were derived from the LT-α–deficient recipient. Thus, expression of LT-α in the BM-derived cells, but not in the non–BM-derived cells, is required for the maturation of FDC from non-BM precursor cells. In contrast, when TNFR-I–deficient mice were reconstituted with wild-type BM, they showed no detectable FDC clusters or GC formation. This indicates that TNFR-I expression on non–BM-derived cellular components is necessary for the establishment of these lymphoid structures. TNFR-I–deficient BM was able to restore FDC organization and GC formation in LT-α–deficient mice, indicating that formation of these structures does not require TNFR-I expression on BM-derived cells. The data in this study demonstrate that FDC organization and GC formation are controlled by both LT-α–expressing BM-derived cells and by TNFR-I-expressing non–BM-derived cells.

scholarly journals Analyzing the Regulatory Role of the HigA Antitoxin within Mycobacterium tuberculosis

2010 ◽  
Vol 192 (17) ◽  
pp. 4348-4356 ◽  
Author(s):  
Amanda S. Fivian-Hughes ◽  
Elaine O. Davis
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Normal Growth ◽  
Global Gene Expression ◽  
Growth Conditions ◽  
Regulatory Role ◽  
Binding Motif ◽  
Binding Assays ◽  
Direct Role

ABSTRACT Bacterial chromosomally encoded type II toxin-antitoxin (TA) loci may be involved in survival upon exposure to stress and have been linked to persistence and dormancy. Therefore, understanding the role of the numerous predicted TA loci within the human pathogen Mycobacterium tuberculosis has become a topic of great interest. Antitoxin proteins are known to autoregulate TA expression under normal growth conditions, but it is unknown whether they have a more global role in transcriptional regulation. This study focuses on analyzing the regulatory role of the M. tuberculosis HigA antitoxin. We first show that the M. tuberculosis higBA locus is functional within its native organism, as higB, higA, and Rv1957 were successfully deleted from the genome together while the deletion of higA alone was not possible. The effects of higB-Rv1957 deletion on M. tuberculosis global gene expression were investigated, and a number of potential HigA-regulated genes were identified. Transcriptional fusion and protein-DNA-binding assays were utilized to confirm the direct role of HigA in Rv1954A-Rv1957 repression, and the M. tuberculosis HigA DNA-binding motif was defined as ATATAGG(N6)CCTATAT. As HigA failed to bind to the next-most-closely related motif within the M. tuberculosis genome, HigA may not directly regulate any other genes in addition to its own operon.

scholarly journals p53 functional impairment and high p21waf1/cip1 expression in human T- cell lymphotropic/leukemia virus type I-transformed T cells

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1551-1560 ◽  
Author(s):  
A Cereseto ◽  
F Diella ◽  
JC Mulloy ◽  
A Cara ◽  
P Michieli ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Virus Type ◽  
Cell Transformation ◽  
Leukemia Virus ◽  
Type I ◽  
Wild Type ◽  
Human T Cell ◽  
Wild Type P53

Abstract Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53- regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I- infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I- transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T- cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

Sign in / Sign up

Export Citation Format

Share Document