scholarly journals Thr176 regulates the activity of the mouse nuclear receptor CAR and is conserved in the NR1I subfamily members PXR and VDR

2005 ◽  
Vol 388 (2) ◽  
pp. 623-630 ◽  
Author(s):  
Akiko UEDA ◽  
Kenji MATSUI ◽  
Yukio YAMAMOTO ◽  
Lars C. PEDERSEN ◽  
Tatsuya SUEYOSHI ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Receptor ◽  
Pregnane X Receptor ◽  
Activation Function ◽  
Constitutive Activity ◽  
Charged Amino Acids ◽  
Binding Surface ◽  
Hydrophobic Amino Acids ◽  
Constitutively Active

The mouse nuclear receptor CAR (constitutively active receptor) is a transcription factor that is activated by phenobarbital-type inducers such as TCPOBOP {1,4 bis[2-(3,5-dichloropyridyloxy)]benzene} in liver in vivo. However, CAR is constitutively active in cell-based transfection assays, the molecular mechanism for which has not been elucidated yet. In the model structure of CAR, Thr176 constitutes a part of the ligand-binding surface, but its side chain is not directed toward the surface, instead it forms a hydrogen bond with Thr350 in the AF2 (activation function 2) domain of CAR. Thr350 is known to regulate CAR activity [Ueda, Kakizaki, Negishi, and Sueyoshi (2002) Mol. Pharmacol. 61, 1284–1288]. Thr176 was mutated to various amino acids to examine whether this interaction played a role in conferring the constitutive activity. Hydrophobic and positively charged amino acids at position 176 abrogated the constitutive activity, whereas polar and negatively charged amino acids retained it. When one of the small hydrophobic amino acids, such as alanine or valine, was substituted for threonine, the mutants were fully activated by TCPOBOP. The co-activator SRC-1 (steroid receptor co-activator-1) regulated the activity changes associated with the mutations. Thr248 and Ser230 are the Thr176-corresponding residues in human pregnane X receptor and mouse vitamin D3 receptor respectively, interacting directly with the conserved threonine in the AF2 domains. Thr248 and Ser230 also regulated the ligand-dependent activity of these receptors by augmenting binding of the receptors to SRC-1. Thr176, Thr248 and Ser230 are conserved residues in the NR1I (nuclear receptor 1I) subfamily members and determine their activity.

scholarly journals Interaction of wild-type signal sequences and their charged variants with model and natural membranes

1993 ◽  
Vol 293 (1) ◽  
pp. 43-49 ◽  
Author(s):  
N M Rao ◽  
R Nagaraj
Keyword(s):  
Amino Acids ◽  
Synthetic Peptides ◽  
Model Membranes ◽  
Signal Peptides ◽  
Wild Type ◽  
Signal Sequences ◽  
Hydrophobic Region ◽  
Phospholipases A2 ◽  
Charged Amino Acids

The interaction of synthetic peptides corresponding to wild-type signal sequences, and their mutants having charged amino acids in the hydrophobic region, with model and natural membranes has been studied. At high peptide concentrations, i.e. low lipid/peptide ratios, the signal peptides cause release of carboxyfluorescein (CF) from model membranes with lipid compositions corresponding to those of translocation-competent as well as translocation-incompetent membranes. Interestingly, mutant sequences, which were non-functional in vivo, caused considerable release of CF compared with the wild-type sequences. Both wild-type and mutant signal sequences perturb model membranes even at lipid/peptide ratios of 1000:1, as indicated by the activities of phospholipases A2, C and D. These studies indicate that such mutant signals are non-functional not because of their inability to interact with membranes, but due to defective targeting to the membrane. The signal peptides inhibit phospholipase C activity in microsomes, uncouple oxidative phosphorylation in mitochondria and increase K+ efflux from erythrocytes, and one of the mutant sequences is a potent degranulator of the mast cells. Both wild-type and mutant signal sequences have the ability to perturb vesicles of various lipid compositions. With respect to natural membranes, the peptides do not show any bias towards translocation-competent membranes.

scholarly journals Activities in Pit-1 Determine Whether Receptor Interacting Protein 140 Activates or Inhibits Pit-1/Nuclear Receptor Transcriptional Synergy

1997 ◽  
Vol 11 (9) ◽  
pp. 1332-1341 ◽  
Author(s):  
F. Max Chuang ◽  
Brian L. West ◽  
John D. Baxter ◽  
Fred Schaufele
Keyword(s):  
Amino Acids ◽  
Nuclear Receptor ◽  
Thyroid Hormone Receptor ◽  
Activation Function ◽  
Activation Functions ◽  
Nuclear Receptor Coactivator ◽  
Interacting Protein ◽  
Synergistic Interactions ◽  
Synergistic Activation ◽  
Context Specific

Abstract Pituitary-specific transcription of the evolutionarily related rat (r) GH and PRL genes involves synergistic interactions between Pit-1 and other promoter-binding factors including nuclear receptors. We show that Pit-1/thyroid hormone receptor (TR) and Pit-1/estrogen receptor (ER) synergistic activation of the rGH and rPRL promoters are globally similar. Both synergies depend upon the same activation functions in Pit-1 and also require activation function-2 conserved in TR and ER. The activation function-2 binding protein, RIP140, previously thought to be a nuclear receptor coactivator, strongly inhibits both Pit-1/TR and Pit-1/ER synergy. RIP140 inhibition is profoundly influenced, in a promoter-specific fashion, by a synergism-selective function in Pit-1: deletion of Pit-1 amino acids 72–100 switches RIP140 to an activator of Pit-1/ER and Pit-1/TR synergy at the rPRL promoter but not at the rGH promoter. Pit-1 amino acids 101–125 are required for RIP140 inhibition or activation again only at the rPRL promoter. Therefore, functions within one factor can determine the activity of a coactivator binding to its synergistic partner. This promoter context-specific synergistic interplay between transcription factors and coactivators is likely an essential determinant of cell-specific transcriptional regulation.

scholarly journals Antimicrobial and Chemoattractant Activity, Lipopolysaccharide Neutralization, Cytotoxicity, and Inhibition by Serum of Analogs of Human Cathelicidin LL-37

2005 ◽  
Vol 49 (7) ◽  
pp. 2845-2850 ◽  
Author(s):  
Cristina D. Ciornei ◽  
Thorgerdur Sigurdardóttir ◽  
Artur Schmidtchen ◽  
Mikael Bodelsson
Keyword(s):  
Amino Acids ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Antimicrobial Properties ◽  
Neutrophil Granulocytes ◽  
Beneficial Effects ◽  
Hydrophobic Amino Acids ◽  
Conventional Antibiotics

ABSTRACT Antimicrobial peptides have been evaluated in vitro and in vivo as alternatives to conventional antibiotics. Apart from being antimicrobial, the native human cathelicidin-derived peptide LL-37 (amino acids [aa] 104 to 140 of the human cathelicidin antimicrobial peptide) also binds and neutralizes bacterial lipopolysaccharide (LPS) and might therefore have beneficial effects in the treatment of septic shock. However, clinical trials have been hampered by indications of toxic effects of LL-37 on mammalian cells and evidence that its antimicrobial effects are inhibited by serum. For the present study, LL-37 was compared to two less hydrophobic fragments obtained by N-terminal truncation, named 106 (aa 106 to 140) and 110 (aa 110 to 140), and to a previously described more hydrophobic variant, the 18-mer LLKKK, concerning antimicrobial properties, lipopolysaccharide neutralization, toxicity against human erythrocytes and cultured vascular smooth muscle cells, chemotactic activity, and inhibition by serum. LL-37, fragments 106 and 110, and the 18-mer LLKKK inhibited the growth of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans in a radial diffusion assay, inhibited lipopolysaccharide-induced vascular nitric oxide production, and attracted neutrophil granulocytes similarly. While fragments 106 and 110 caused less hemolysis and DNA fragmentation in cultured cells than did LL-37, the 18-mer LLKKK induced severe hemolysis. The antibacterial effect of fragments 106 and 110 was not affected by serum, while the effect of LL-37 was reduced. We concluded that the removal of N-terminal hydrophobic amino acids from LL-37 decreases its cytotoxicity as well as its inhibition by serum without negatively affecting its antimicrobial or LPS-neutralizing action. Such LL-37-derived peptides may thus be beneficial for the treatment of patients with sepsis.

scholarly journals Toll-like receptor-4 synthesis is regulated by JNK signaling by three glucocorticoids isoforms and by three interferons isoforms, also its advantages in activities depending on the Containment from Arg, proline and hydrophobic amino acids in its compositions

2021 ◽  
Vol 1 (2) ◽  
Author(s):  
Ashraf Marzouk El Tantawi
Keyword(s):  
Amino Acids ◽  
Collagen Synthesis ◽  
Fatty Acyl ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Jnk Signaling ◽  
Gamma Subunits ◽  
Hydrophobic Amino Acids ◽  
Oxidative Processes

Toll-like receptor-4 (TLR4), synthesis is regulated by JNK signaling , by three glucocorticoids isoforms, and by the three interferons isoforms, also depending on avaliablities of LPS & on long fatty acids chains with Arg and proline availabilities For performing and running the mitochondrial oxidative processes for producing fatty-acyl-CoA-synthetase followed by fatty-acyl-CoA-synthase followed by fatty-acyl-CoA-phospholipase productions for linear TLR4 active beta-subunits which will be transformed into TLR4-alpha upon phospholipase effects , which will follow phosphorylation process (alpha-oxidations) for generate Guanosine triphosphate cyclohydrolase (GTP-Chase) subunits which supposed to contain specific hydrophobic amino acids including Arg, Tyr, leu, proline…. Etc, which is the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, which is essential for inducible iNOS from fatty-acyl-CoA-synthase upon the nitric oxidesynthase (NO-S) regulations effects. Proline can accelerate anabolic oxidative processes by OPA1 enzymes and provides site-specific flexibility for collagen synthesis in vivo, and also plays an necessary important roles in TLR4 and TGF-gamma/beta/& alpha synthesis and activities, where the presence of proline in IFN-gamma, in TLR4 genes and in IFN-beta will accelerate oxidative OPA1 anabolic processes and direct the flow of biological processes to proliferations of plasma-membranes , collagen synthesis and blood platelets biosynthesis. Vitamin E & K-dependent protein C are the key components of anticoagulant serine protease, And therefore vit E and vit k are providing specific advantages to TLR4 synthesis and modulated first in vivo as proper fatty-acyl-CoAsynthetase subunits (gamma-subunits) then modified fatty-acyl-CoA-synthase subunits upon synthase effects on gamma-subunits for producing IL-beta upon which will promote linear TLR4 upon both synthase and phospholipase effects for starting proliferation stepes started by catalyzing Arg for producing GTP-Chase and citrulline which the main basis for Erythropoietin productions , for Plasma membrane synthesis… etc. Both IFN-beta and glucocorticoid-beta are designed anti-inflammatory subunits are depending on each others and on the activities of OPA1-synthase enzyme for producing the long fatty acyl-CoA-synthase (Beta-subunit) with specific compositions and sequences from amino acids which can determine their advantages in immunity functions eg their containment of tyr, proline, Arg, gly.. etc, where, Both glucocorticoid-beta and IFN-beta are able to recover each others in their different tissues.

Molecular basis of the complex formation between the two calcium-binding proteins S100A8 (MRP8) and S100A9 (MRP14)

2005 ◽  
Vol 386 (5) ◽  
pp. 429-434 ◽  
Author(s):  
Nadja Leukert ◽  
Clemens Sorg ◽  
Johannes Roth
Keyword(s):  
Amino Acids ◽  
Complex Formation ◽  
Calcium Binding ◽  
Site Directed Mutagenesis ◽  
Dimer Interface ◽  
Yeast Two Hybrid System ◽  
Hydrophobic Amino Acids ◽  
Functional Relevance ◽  
First Time

Abstract S100 proteins form characteristic homo- and/or heterodimers that play a role in calcium-mediated signaling. We characterized the formation of the human S100A8/S100A9 heterodimer using the yeast two-hybrid system. Employing site-directed mutagenesis we found that distinct hydrophobic amino acids of helix I/I′ are located at a crucial site of the S100A8/S100A9 dimer interface, whereas conserved residues within helix IV/IV′ are not important for heterodimerization. Furthermore, amino acids Y16 and F68 prevent homodimerization of human S100A8. These data demonstrate for the first time the functional relevance of distinct hydrophobic amino acids for human S100A8/S100A9 complex formation in vivo.

scholarly journals Topogenesis in membranes of the NTB–VPg protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain

2004 ◽  
Vol 85 (2) ◽  
pp. 535-545 ◽  
Author(s):  
Aiming Wang ◽  
Sumin Han ◽  
Hélène Sanfaçon
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Proteinase K ◽  
Hydrophobic Region ◽  
C Terminus ◽  
Microsomal Membranes ◽  
Hydrophobic Amino Acids ◽  
Definition Of

The putative NTP-binding protein (NTB) of Tomato ringspot nepovirus (ToRSV) contains a hydrophobic region at its C terminus consisting of two adjacent stretches of hydrophobic amino acids separated by a few amino acids. In infected plants, the NTB–VPg polyprotein (containing the domain for the genome-linked protein) is associated with endoplasmic reticulum-derived membranes that are active in ToRSV replication. Recent results from proteinase K protection assays suggested a luminal location for the VPg domain in infected plants, providing support for the presence of a transmembrane domain at the C terminus of NTB. In this study, we have shown that NTB–VPg associates with canine microsomal membranes in the absence of other viral proteins in vitro and adopts a topology similar to that observed in vivo in that the VPg is present in the lumen. Truncated proteins containing 60 amino acids at the C terminus of NTB and the entire VPg exhibited a similar topology, confirming that this region of the protein contains a functional transmembrane domain. Deletion of portions of the C-terminal hydrophobic region of NTB by mutagenesis and introduction of glycosylation sites to map the luminal regions of the protein revealed that only the first stretch of hydrophobic amino acids traverses the membrane, while the second stretch of hydrophobic amino acids is located in the lumen. Our results provide additional evidence supporting the hypothesis that the NTB–VPg polyprotein acts as a membrane-anchor for the replication complex.

Characterization of the hydrophobic properties of amino acids on the basis of their partition and distribution coefficients in the 1-octanol-water system

1991 ◽  
Vol 56 (10) ◽  
pp. 2030-2041 ◽  
Author(s):  
Josef Chmelík ◽  
Jiří Hudeček ◽  
Karol Putyera ◽  
Jiří Makovička ◽  
Vítěz Kalous ◽  
...  
Keyword(s):  
Amino Acids ◽  
Water System ◽  
Distribution Coefficients ◽  
Published Data ◽  
Hydrophobic Properties ◽  
Charged Amino Acids ◽  
Hydrophobic Amino Acids ◽  
Partition Process

The hydrophobic properties of amino acid side chains were characterized on the basis of the partition process in the 1-octanol-water system. The partition coefficients were calculated from the published data and the distribution coefficients were determined experimentally on the basis of a double partition process utilizing the fact that the amino acids pass almost completely into the aqueous phase in the partition process. When the volumes of water and 1-octanol are suitably selected, this fact permits avoidance of the difficulties associated with the determination of amino acids in 1-octanol, where their solubilities are very low. Our scale is the only complete experimental scale based on the partition process of amino acids in the 1-octanol-water system. It follows from comparison of the calculated and the experimental data with the values published for the distribution coefficients of N-acetyl amides of amino acids that the best agreement was achieved for hydrophobic amino acids, while greater differences were observed for hydrophilic amino acids. These differences, expressed as the logarithm of the distribution coefficients, correspond to an average of 0.08 for nonpolar amino acids and 0.30 for acidic and basic amino acids: expressed as relative deviations, these values correspond to 2-10% for nonpolar amino acids, and 5-30% for charged amino acids.

scholarly journals The Escherichia coli Ada Protein Can Interact with Two Distinct Determinants in the ς70 Subunit of RNA Polymerase According to Promoter Architecture: Identification of the Target of Ada Activation at the alkAPromoter

1999 ◽  
Vol 181 (5) ◽  
pp. 1524-1529 ◽  
Author(s):  
Paolo Landini ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Rna Polymerase ◽  
Promoter Architecture ◽  
Charged Amino Acids ◽  
Identify Amino Acid ◽  
Ada Protein ◽  
Positively Charged

ABSTRACT The methylated form of the Ada protein (meAda) activates transcription from the Escherichia coli ada,aidB, and alkA promoters with different mechanisms. In this study we identify amino acid substitutions in region 4 of the RNA polymerase subunit ς70 that affect Ada-activated transcription at alkA. Substitution to alanine of residues K593, K597, and R603 in ς70 region 4 results in decreased Ada-dependent binding of RNA polymerase to thealkA promoter in vitro and impairs alkAtranscription both in vivo and in vitro, suggesting that these residues define a determinant for meAda-ς70interaction. In a previous study (P. Landini, J. A. Bown, M. R. Volkert, and S. J. W. Busby, J. Biol. Chem. 273:13307–13312, 1998), we showed that a set of negatively charged amino acids in ς70 region 4 is involved inmeAda-ς70 interaction at the adaand aidB promoters. However, the alanine substitutions of positively charged residues K593, K597, and R603 do not affectmeAda-dependent transcription at ada andaidB. Unlike the ς70 amino acids involved in the interaction with meAda at the ada andaidB promoters, K593, K597, and R603 are not conserved in ςS, an alternative ς subunit of RNA polymerase mainly expressed during the stationary phase of growth. WhilemeAda is able to promote transcription by the ςS form of RNA polymerase (EςS) atada and aidB, it fails to do so atalkA. We propose that meAda can activate transcription at different promoters by contacting distinct determinants in ς70 region 4 in a manner dependent on the location of the Ada binding site.

scholarly journals On the substrate recognition and negative regulation of SPAK, a kinase modulating Na+-K+-2Cl− cotransport activity

2010 ◽  
Vol 299 (3) ◽  
pp. C614-C620 ◽  
Author(s):  
Kenneth B. Gagnon ◽  
Eric Delpire
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Catalytic Domain ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Xenopus Laevis Oocytes ◽  
Threonine Residue ◽  
In Vivo Experiments ◽  
Constitutively Active

Threonines targeted by Ste20-related proline-alanine-rich kinase (SPAK) for phosphorylation have been identified in Na+-K+-2Cl− cotransporter type 1 (NKCC1), NKCC2, and Na+-Cl− cotransporter (NCC). However, what constitutes the substrate recognition of the kinase is still unknown. Using site-directed mutagenesis and functional measurement of NKCC1 activity in Xenopus laevis oocytes, we determined that SPAK recognizes two threonine residues separated by four amino acids. Addition or removal of a single residue abrogated SPAK activation of NKCC1. Although both threonines are followed by hydrophobic residues, in vivo experiments have determined that SPAK activation of the cotransporter only requires a hydrophobic residue after the first threonine. Interestingly, downstream of the second threonine residue, we have identified a conserved aspartic acid residue which is critical for NKCC1 function. Mouse SPAK activity requires phosphorylation of two specific residues by WNK [with no lysine (K)] kinases: a threonine (T243) in the catalytic domain and a serine (S383) in the regulatory domain. We found that mutating the threonine residue into a glutamic acid (T243E) combined with mutation of the serine into an aspartic acid (S383D) rendered SPAK constitutively active. Surprisingly, alanine substitution of S383 or mutation of residues surrounding this residue also resulted in a constitutively active kinase. Interestingly, deletion of amino acids 356–398 identified another serine residue in the catalytic domain (S321) as another putative target of WNK phosphorylation. We found that WNK4 is capable of stimulating the deletion mutant when S321 is present, but not when S321 is mutated into an alanine.

The VES KM: a pathway for protein folding in vivo

2020 ◽  
Vol 92 (1) ◽  
pp. 179-191
Author(s):  
Leonor Cruzeiro
Keyword(s):  
Amino Acids ◽  
Protein Folding ◽  
Protein Structures ◽  
Kinetic Mechanism ◽  
Thermal Bath ◽  
Charged Amino Acids ◽  
Nascent Chain ◽  
Dynamics Simulations ◽  
Positively Charged

AbstractWhile according to the thermodynamic hypothesis, protein folding reproducibility is ensured by the assumption that the native state corresponds to the minimum of the free energy in normal cellular conditions, here, the VES kinetic mechanism for folding in vivo is described according to which the nascent chain of all proteins is helical and the first and structure defining step in the folding pathway is the bending of that initial helix around a particular amino acid site. Molecular dynamics simulations are presented which indicate both the viability of this mechanism for folding and its limitations in the presence of a Markovian thermal bath. An analysis of a set of protein structures formed only of helices and loops suggests that bending sites are correlated with regions bounded, on the N-side, by positively charged amino acids like Lysine and Histidine and on the C-side by negatively charged amino acids like Aspartic acid.

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