scholarly journals Molecular analysis of scabrous mutant alleles from Drosophila melanogaster indicates a secreted protein with two functional domains.

Genetics ◽  
1995 ◽  
Vol 141 (2) ◽  
pp. 607-617
Author(s):  
X Hu ◽  
E C Lee ◽  
N E Baker
Keyword(s):  
Protein Interactions ◽  
Neural Development ◽  
Protein Function ◽  
Blood Clotting ◽  
Dominant Negative ◽  
Wild Type ◽  
Amino Terminal ◽  
Amino Terminal Domain ◽  
Amino Acid Segment

Abstract Mutations at the scabrous locus (sca) affect cell-cell signaling during neural development. Twenty-one mutant alleles of scabrous have been analyzed. Many synthesize no sca protein. In others, a defective protein is arrested intracellularly. Two mutants in which protein is not arrested must affect sca protein function outside the cell. Both affect the fibrinogen related domain (FReD), a 200-amino acid segment conserved in fibrinogen, tenascins, and other proteins. In fibrinogen, this region is involved in protein interactions and is altered in human mutations affecting blood clotting. In sca(UM2), an invariant Asp residue is replaced by Asn. In sca(MSKF) allele has dominant negative properties, indicating that the truncated amino-terminal portion interferes with the function of so me other gene product. These mutations show that the conserved FReD is essential for wild-type sca function, but suggest that the amino-terminal domain also interacts with other proteins, but other neural mutations were without effect. Models for the role of a two-domain protein in neural development are discussed.

scholarly journals A 12-Amino-Acid Segment, Present in Type s2 but Not Type s1 Helicobacter pylori VacA Proteins, Abolishes Cytotoxin Activity and Alters Membrane Channel Formation

2001 ◽  
Vol 183 (22) ◽  
pp. 6499-6508 ◽  
Author(s):  
Mark S. McClain ◽  
Ping Cao ◽  
Hideki Iwamoto ◽  
Arlene D. Vinion-Dubiel ◽  
Gabor Szabo ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Amino Acid ◽  
Dominant Negative ◽  
Membrane Channels ◽  
Wild Type ◽  
Amino Terminal ◽  
Protein Toxin ◽  
H Pylori ◽  
Amino Acid Segment ◽  
Negative Phenotype

ABSTRACT Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the “middle” region (i.e., m1 and m2) and in the 5′ end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, PCl/PNa = 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Δ6–27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Δ6–27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pyloriisolates suggests that type s2 VacA proteins retain an important biological activity.

scholarly journals The role of the amino-terminal domain in the interaction of unliganded peroxisome proliferator-activated receptor γ-2 with nuclear receptor co-repressor

2010 ◽  
Vol 45 (3) ◽  
pp. 133-145 ◽  
Author(s):  
Sadako Suzuki ◽  
Shigekazu Sasaki ◽  
Hiroshi Morita ◽  
Yutaka Oki ◽  
Daisuke Turiya ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Nuclear Receptor ◽  
Thyroid Hormone Receptor ◽  
Dominant Negative ◽  
Peroxisome Proliferator ◽  
Wild Type ◽  
Peroxisome Proliferator Activated Receptor ◽  
Amino Terminal

Peroxisome proliferator-activated receptor γ-2 (PPARG2) is a ligand-dependent transcriptional factor involved in the pathogenesis of insulin resistance. In the presence of a ligand, PPARG2 associates with co-activators, while it recruits co-repressors (CoRs) in the absence of a ligand. It has been reported that the interaction of liganded PPARG2 with co-activators is regulated by the amino-terminal A/B domain (NTD) via inter-domain communication. However, the role of the NTD is unknown in the case of the interaction between unliganded PPARG2 and CoRs. To elucidate this, total elimination of the influence of ligands is required, but the endogenous ligands of PPARG2 have not been fully defined. PPARG1-P467L, a naturally occurring mutant of PPARG1, was identified in a patient with severe insulin resistance. Reflecting its very low affinity for various ligands, this mutant does not have transcriptional activity in the PPAR response element, but exhibits dominant negative effects (DNEs) on liganded wild-type PPARG2-mediated transactivation. Using the corresponding PPARG2 mutant, PPARG2-P495L, we evaluated the role of the NTD in the interaction between unliganded PPARG2 and CoRs. Interestingly, the DNE of PPARG2-P495L was increased by the truncation of its NTD. NTD deletion also enhanced the DNE of a chimeric receptor, PT, in which the ligand-binding domain of PPARG2 was replaced with that of thyroid hormone receptor β-1. Moreover, NTD deletion facilitated the in vitro binding of nuclear receptor CoR with wild-type PPARG2, mutant P495L, and the PT chimera (PPARG2-THRB). Inter-domain communication in PPARG2 regulates not only ligand-dependent transactivation but also ligand-independent silencing.

scholarly journals Role of Amino-Terminal Domain in the Assembly Mechanism of Kainate-Subtype Glutamate Receptor Ion Channels

2014 ◽  
Vol 106 (2) ◽  
pp. 151a
Author(s):  
Sagar Chittori ◽  
Janesh Kumar ◽  
Suvendu Lomash ◽  
Huaying Zhao ◽  
Peter Schuck ◽  
...  
Keyword(s):  
Ion Channels ◽  
Glutamate Receptor ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Assembly Mechanism ◽  
Amino Terminal Domain

scholarly journals Interactions between Integrase and Excisionase in the Phage Lambda Excisive Nucleoprotein Complex

2002 ◽  
Vol 184 (18) ◽  
pp. 5200-5203 ◽  
Author(s):  
Eun Hee Cho ◽  
Richard I. Gumport ◽  
Jeffrey F. Gardner
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Mobility Shift ◽  
Host Chromosome ◽  
Amino Terminal ◽  
Nucleoprotein Complex ◽  
Carboxyl Terminal ◽  
Α Helix ◽  
Gel Mobility Shift ◽  
Amino Terminal Domain

ABSTRACT Bacteriophage lambda site-specific recombination comprises two overall reactions, integration into and excision from the host chromosome. Lambda integrase (Int) carries out both reactions. During excision, excisionase (Xis) helps Int to bind DNA and introduces a bend in the DNA that facilitates formation of the proper excisive nucleoprotein complex. The carboxyl-terminal α-helix of Xis is thought to interact with Int through direct protein-protein interactions. In this study, we used gel mobility shift assays to show that the amino-terminal domain of Int maintained cooperative interactions with Xis. This finding indicates that the amino-terminal arm-type DNA binding domain of Int interacts with Xis.

AMP kinase is not required for the GLUT4 response to exercise and denervation in skeletal muscle

2004 ◽  
Vol 287 (4) ◽  
pp. E739-E743 ◽  
Author(s):  
Burton F. Holmes ◽  
David B. Lang ◽  
Morris J. Birnbaum ◽  
James Mu ◽  
G. Lynis Dohm
Keyword(s):  
Skeletal Muscle ◽  
Dominant Negative ◽  
Treadmill Running ◽  
Wild Type ◽  
Α Subunit ◽  
Ampk Activity ◽  
Denervated Muscles ◽  
Amp Activated Protein Kinase ◽  
Response To Exercise

An acute bout of exercise increases muscle GLUT4 mRNA in mice, and denervation decreases GLUT4 mRNA. AMP-activated protein kinase (AMPK) activity in skeletal muscle is also increased by exercise, and GLUT4 mRNA is increased in mouse skeletal muscle after treatment with AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside(AICAR). These findings suggest that AMPK activation might be responsible for the increase in GLUT4 mRNA expression in response to exercise. To investigate the role of AMPK in GLUT4 regulation in response to exercise and denervation, transgenic mice with a mutated AMPK α-subunit (dominant negative; AMPK-DN) were studied. GLUT4 did not increase in AMPK-DN mice that were treated with AICAR, demonstrating that muscle AMPK is inactive. Exercise (two 3-h bouts of treadmill running separated by 1 h of rest) increased GLUT4 mRNA in both wild-type and AMPK-DN mice. Likewise, denervation decreased GLUT4 mRNA in both wild-type and AMPK-DN mice. GLUT4 mRNA was also increased by AICAR treatment in both the innervated and denervated muscles. These data demonstrate that AMPK is not required for the response of GLUT4 mRNA to exercise and denervation.

Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC

2002 ◽  
Vol 283 (2) ◽  
pp. E338-E345 ◽  
Author(s):  
Masatoshi Tsuru ◽  
Hideki Katagiri ◽  
Tomoichiro Asano ◽  
Tetsuya Yamada ◽  
Shigeo Ohno ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Dominant Negative ◽  
Transport Activity ◽  
Wild Type ◽  
Atypical Pkc ◽  
Endogenous Expression ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Pkc Ζ

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-α, novel PKC-δ, and atypical PKC isoforms of PKC-λ and PKC-ζ, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-α and PKC-λ/ζ, but not of PKC-δ, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-α and exogenous PKC-δ but not atypical PKC-λ/ζ. Insulin also activated the overexpressed PKC-δ but not PKC-α. Expression of the wild-type PKC-α or PKC-δ resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-α expression, which inhibited the PMA activation of PKC-α, decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-δ but not of PKC-α. These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-λ/ζ was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.

scholarly journals A Dominant-Negative fur Mutation in Bradyrhizobium japonicum

2004 ◽  
Vol 186 (5) ◽  
pp. 1409-1414 ◽  
Author(s):  
Heather P. Benson ◽  
Kristin LeVier ◽  
Mary Lou Guerinot
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Iron Uptake ◽  
Outer Membrane Proteins ◽  
Dominant Negative ◽  
Ferric Uptake Regulator ◽  
Null Mutant ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Fur Protein

ABSTRACT In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified a fur mutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of the fur gene repress iron uptake systems and starve for iron, whereas fur mutants fail to repress iron uptake systems and survive. The B. japonicum fur mutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron. Unexpectedly, a wild-type copy of the fur gene cannot complement the fur mutant. Expression of the fur mutant allele in wild-type cells leads to a fur phenotype. Unlike a B. japonicum fur-null mutant, the strain carrying the dominant-negative fur mutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.

Functional Analysis of Human Smad1: Role of the Amino-Terminal Domain

1999 ◽  
Vol 258 (2) ◽  
pp. 366-373 ◽  
Author(s):  
Ren-He Xu ◽  
Robert J. Lechleider ◽  
Hsiu-Ming Shih ◽  
Chen-Fei Hao ◽  
Dvora Sredni ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain

The Critical Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPalpha Mutations.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2213-2213
Author(s):  
J. Pulikkan ◽  
A. Peer Zada ◽  
M. Geletu ◽  
V. Dengler ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Pcr Analysis ◽  
Wild Type ◽  
Rt Pcr ◽  
Promoter Assay ◽  
Type C ◽  
Acute Myeloid

Abstract CCAAT enhancer binding protein alpha (C/EBPα) is a myeloid specific transcription factor that coordinates cellular differentiation and cell cycle arrest. Loss of C/EBPα expression or function in leukemic blasts contributes to a block in myeloid cell differentiation. C/EBPα is mutated in around 9% of acute myeloid leukemia (AML). The mutations reported in C/EBPα are frame shift mutations and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. The role of peptidyl-prolyl cis/trans isomerase, Pin1 in tumorogenesis and its overexpression in many cancers led us to investigate its role in acute myeloid leukemia with C/EBPα mutation. Here we show that Pin1 is upregulated in patients with acute myeloid leukemia by affymetrix analysis. By quantitative Real-Time RT-PCR analysis, we show C/EBPα-p30 could induce Pin1 transcription, while the wild type C/EBPα downregulates Pin1 expression. Luciferase promoter assay for the Pin1 promoter shows that wild type C/EBPα is able to block Pin1 promoter activity. Mean while, C/EBPα-p30 couldn’t block Pin1 promotor activity. By silencing Pin1 by RNA Interference as well as with inhibitor against Pin1 (PiB) we could show myeloid differentiation in human CD34+ cord blood cells as well as in Kasumi-6 cells as assessed by FACS analysis with granulocytic markers. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We report that Pin1 increases the transcriptional activity of the oncogene c-jun. We also show that c-jun blocks the DNA binding and transactivation of C/EBPα protein as assessed by gel shift assay and promoter assay respectively. We have previously shown that c-jun expression is high in AML patients with C/EBPα mutation and c-jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that inhibition of Pin1 by the inhibitor PiB downregulates c-jun mRNA expression. In conclusion, inhibition of Pin1 leads to granulocytic differentiation. Our results show Pin1 as a novel target in treating AML patients with C/EBPα mutation.

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