scholarly journals Structural analysis of the role of the two conserved motifs of the ECF41 family sigma factor in the autoregulation of its own promoter in Azospirillum brasilense Sp245

2021 ◽  
Author(s):  
Ekta Pathak ◽  
AshutoshPrakash Dubey ◽  
Vijay Shankar Singh ◽  
Rajeev Mishra ◽  
Anil Kumar Tripathi
Keyword(s):  
Transcriptional Activation ◽  
Sigma Factor ◽  
Azospirillum Brasilense ◽  
Proximal Part ◽  
Dynamics Simulation ◽  
Promoter Activation ◽  
Terminal Sequence ◽  
Conserved Motifs ◽  
C Terminus

In Azospirillum brasilense, an extra-cytoplasmic function sigma factor (RpoE10) shows the characteristic 119 amino acid long C-terminal extension found in ECF41-type sigma factors, which possesses three conserved motifs (WLPEP, DGGGR, and NPDKV), one in the linker region between the sigma and sigma , and the other two in the SnoaL_2 domain of the C-terminal extension. Here, we have described the role of the two conserved motifs in the SnoaL_2 domain of RpoE10 in the inhibition and activation of its activity, respectively. Truncation of the distal part of the C-terminal sequence of the RpoE10 (including NPDKV but excluding the DGGGR motif) results in its promoter’s activation suggesting autoregulation. Further truncation of the C-terminal sequence up to its proximal part, including NPDKV and DGGGR motif, abolished promoter activation. Replacement of NPDKV motif with NAAAV in RpoE10 increased its ability to activate its promoter, whereas replacement of DGGGR motif led to reduced promoter activation. We have explored the dynamic modulation of sigma2 – sigma4 domains and the relevant molecular interactions mediated by the two conserved motifs of the SnoaL2 domain using molecular dynamics simulation. The analysis enabled us to explain that the NPDKV motif located distally in the C-terminus negatively impacts transcriptional activation. In contrast, the DGGGR motif found proximally of the C-terminal extension is required to activate RpoE1

scholarly journals A Lys49 PhospholipaseA2, Isolated fromBothrops asperSnake Venom, Induces Lipid Droplet Formation in Macrophages Which Depends on Distinct Signaling Pathways and the C-Terminal Region

2013 ◽  
Vol 2013 ◽  
pp. 1-14 ◽  
Author(s):  
Karina Cristina Giannotti ◽  
Elbio Leiguez ◽  
Vanessa Moreira ◽  
Neide Galvão Nascimento ◽  
Bruno Lomonte ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Protein Kinases ◽  
Inflammatory Responses ◽  
Critical Role ◽  
Terminal Sequence ◽  
Pharmacological Interventions ◽  
C Terminus ◽  
Inflammatory Mechanism ◽  
Terminal Loop

MT-II, a Lys49PLA2homologue devoid of catalytic activity fromB. aspervenom, stimulates inflammatory events in macrophages. We investigated the ability of MT-II to induce formation of lipid droplets (LDs), key elements of inflammatory responses, in isolated macrophages and participation of protein kinases and intracellular PLA2s in this effect. Influence of MT-II on PLIN2 recruitment and expression was assessed, and the effects of some synthetic peptides on LD formation were further evaluated. At noncytotoxic concentrations, MT-II directly activated macrophages to form LDs. This effect was reproduced by a synthetic peptide corresponding to the C-terminal sequence 115–129 of MT-II, evidencing the critical role of C-terminus for MT-II-induced effect. Moreover, MT-II induced expression and recruitment of PLIN2. Pharmacological interventions with specific inhibitors showed that PKC, PI3K, ERK1/2, and iPLA2, but notP38MAPKor cPLA2, signaling pathways are involved in LD formation induced by MT-II. This sPLA2homologue also induced synthesis of PGE2that colocalized to LDs. In conclusion, MT-II is able to induce formation of LDs committed to PGE2formation in a process dependent on C-terminal loop engagement and regulated by distinct protein kinases and iPLA2. LDs may constitute an important inflammatory mechanism triggered by MT-II in macrophages.

Role of the N-terminus in human 4-hydroxyphenylpyruvate dioxygenase activity

2019 ◽  
Vol 167 (3) ◽  
pp. 315-322
Author(s):  
An-Ning Feng ◽  
Chih-Wei Huang ◽  
Chi-Huei Lin ◽  
Yung-Lung Chang ◽  
Meng-Yuan Ni ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Dynamics Simulation ◽  
Wild Type ◽  
Catalytic Function ◽  
C Terminus ◽  
N Terminus ◽  
Key Enzyme ◽  
The Stability

Abstract 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism, catalysing the oxidation of 4-hydroxyphenylpyruvate to homogentisate. Genetic deficiency of this enzyme causes type III tyrosinaemia. The enzyme comprises two barrel-shaped domains formed by the N- and C-termini, with the active site located in the C-terminus. This study investigated the role of the N-terminus, located at the domain interface, in HPPD activity. We observed that the kcat/Km decreased ∼8-fold compared with wild type upon removal of the 12 N-terminal residues (ΔR13). Interestingly, the wild-type level of activity was retained in a mutant missing the 17 N-terminal residues, with a kcat/Km 11-fold higher than that of the ΔR13 mutant; however, the structural stability of this mutant was lower than that of wild type. A 2-fold decrease in catalytic efficiency was observed for the K10A and E12A mutants, indicating synergism between these residues in the enzyme catalytic function. A molecular dynamics simulation showed large RMS fluctuations in ΔR13 suggesting that conformational flexibility at the domain interface leads to lower activity in this mutant. These results demonstrate that the N-terminus maintains the stability of the domain interface to allow for catalysis at the active site of HPPD.

scholarly journals Role of the Sc C Terminus in Transcriptional Activation and E(spl) Repressor Recruitment

2004 ◽  
Vol 280 (2) ◽  
pp. 1299-1305 ◽  
Author(s):  
Nikolaos Giagtzoglou ◽  
Konstantinos A. Koumbanakis ◽  
John Fullard ◽  
Ioanna Zarifi ◽  
Christos Delidakis
Keyword(s):  
Transcriptional Activation ◽  
C Terminus

scholarly journals Multiple Mechanistically Distinct Functions of SAGA at the PHO5 Promoter

2003 ◽  
Vol 23 (10) ◽  
pp. 3468-3476 ◽  
Author(s):  
Slobodan Barbaric ◽  
Hans Reinke ◽  
Wolfram Hörz
Keyword(s):  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Promoter Activation ◽  
Sant Domain ◽  
Promoter Recruitment ◽  
Synthetic Phenotype ◽  
Pho5 Promoter ◽  
Histone Acetyltransferase Activity

ABSTRACT Our previous studies have shown that the rate of chromatin remodeling and consequently the rate of PHO5 activation are strongly decreased in the absence of Gcn5 histone acetyltransferase activity. Using chromatin immunoprecipitation, we demonstrate that SAGA is physically recruited to the PHO5 promoter. Recruitment is dependent on the specific activator Pho4 and occurs only under inducing conditions. Spt3, another subunit of SAGA, also plays a role in PHO5 activation but has a function that is completely different from that of Gcn5. An SPT3 deletion severely compromises the PHO5 promoter and reduces the extent of transcriptional activation by diminishing the binding of the TATA binding protein to the promoter without, however, affecting the rate or the extent of chromatin remodeling. A gcn5 spt3 double mutant shows a synthetic phenotype almost as severe as that observed for an spt7 or spt20 mutant. The latter two mutations are known to prevent the assembly of the complex and consequently lead to the loss of all SAGA functions. The absence of the Ada2 subunit causes a strong delay in chromatin remodeling and promoter activation that closely resembles the delay observed in the absence of Gcn5. A deletion of only the Ada2 SANT domain has exactly the same effect, strongly suggesting that Ada2 controls Gcn5 activity by virtue of its SANT domain. Finally, the Gcn5 bromodomain also contributes to but is not essential for Gcn5 function at the PHO5 promoter. Taken together, the results provide a detailed and differentiated description of the role of SAGA as a coactivator at the PHO5 promoter.

scholarly journals Surfaces of Spo0A and RNA Polymerase Sigma Factor A That Interact at the spoIIG Promoter in Bacillus subtilis

2004 ◽  
Vol 186 (1) ◽  
pp. 200-206 ◽  
Author(s):  
Amrita Kumar ◽  
Cindy Buckner Starke ◽  
Mark DeZalia ◽  
Charles P. Moran
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Dna Binding Protein ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Promoter Activation ◽  
Interacting Surfaces

ABSTRACT In Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two classes of promoters, those used by RNA polymerase containing the primary sigma factor, σA (e.g., spoIIG), and those used by RNA polymerase containing the secondary sigma factor, σH (e.g., spoIIA). Several single amino acid substitutions in region 4 of σA define positions in σA that are specifically required for Spo0A-dependent promoter activation. Similarly, several single amino acid substitutions in Spo0A define positions in Spo0A that are required for σA-dependent promoter activation but not for other functions of Spo0A. It is unknown whether these amino acids in Spo0A interact directly with those in region 4 of σA or whether they interact with another subunit of RNA polymerase to effect promoter activation. Here we report the identification of a new amino acid in region 4 of σA, arginine at position 355 (R355), that is involved in Spo0A-dependent promoter activation. To further investigate the role of R355, we used the coordinates of Spo0A and sigma region 4, each in complex with DNA, to build a model for the interaction of σA and Spo0A at the spoIIG promoter. We tested the model by examining the effects of amino acid substitutions in the putative interacting surfaces of these molecules. As predicted by the model, we found genetic evidence for interaction of R355 of σA with glutamine at position 221 of Spo0A. These results appear to define the surfaces of Spo0A and σA that directly interact during activation of the spoIIG promoter.

Role of molecular chaperones in steroid receptor action

2004 ◽  
Vol 40 ◽  
pp. 41-58 ◽  
Author(s):  
William B Pratt ◽  
Mario D Galigniana ◽  
Yoshihiro Morishima ◽  
Patrick J M Murphy
Keyword(s):  
Transcriptional Activation ◽  
Protein Trafficking ◽  
Steroid Receptors ◽  
Transcriptional Regulatory ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Receptor Action ◽  
Binding Cleft ◽  
Hsp 90

Unliganded steroid receptors are assembled into heterocomplexes with heat-shock protein (hsp) 90 by a multiprotein chaperone machinery. In addition to binding the receptors at the chaperone site, hsp90 binds cofactors at other sites that are part of the assembly machinery, as well as immunophilins that connect the assembled receptor-hsp90 heterocomplexes to a protein trafficking pathway. The hsp90-/hsp70-based chaperone machinery interacts with the unliganded glucocorticoid receptor to open the steroid-binding cleft to access by a steroid, and the machinery interacts in very dynamic fashion with the liganded, transformed receptor to facilitate its translocation along microtubular highways to the nucleus. In the nucleus, the chaperone machinery interacts with the receptor in transcriptional regulatory complexes after hormone dissociation to release the receptor and terminate transcriptional activation. By forming heterocomplexes with hsp90, the chaperone machinery stabilizes the receptor to degradation by the ubiquitin-proteasome pathway of proteolysis.

Factor Xa Enhances the Binding of Tissue Factor Pathway Inhibitor to Acidic Phospholipids

1996 ◽  
Vol 75 (05) ◽  
pp. 796-800 ◽  
Author(s):  
Sanne Valentin ◽  
Inger Schousboe
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Microtitre Plate ◽  
C Terminus ◽  
Microtitre Plate Assay ◽  
Kunitz Domain ◽  
Tissue Factor Pathway ◽  
Acidic Phospholipids

SummaryIn the present study, the interaction between tissue factor pathway inhibitor (TFPI) and phospholipids has been characterized using a microtitre plate assay. TFPI was shown to bind calcium-independently to an acidic phospholipid surface composed of phosphatidylserine, but not a surface composed of the neutral phosphatidylcholine. The interaction was demonstrated to be dependent on the presence of the TFPI C-terminus. The presence of heparin (1 U/ml, unfractionated) was able to significantly reduce the binding of TFPI to phospholipid. The interaction of TFPI with phosphatidylserine was significantly decreased in the presence of calcium, but this was counteracted, and even enhanced, following complex formation of TFPI with factor Xa prior to incubation with the phospholipid surface. Moreover, a TFPI variant, not containing the third Kunitz domain and the C-terminus, was unable to bind to phospholipid. However, following the formation of a TFPI/factor Xa-complex this TFPI variant was capable of interacting with the phospholipid surface. This indicates that the role of factor Xa as a TFPI cofactor, at least in part, is to mediate the binding of TFPI to the phospholipid surface.

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