scholarly journals Environment of the Active Site Region of RseP, an Escherichia coli Regulated Intramembrane Proteolysis Protease, Assessed by Site-directed Cysteine Alkylation

2006 ◽  
Vol 282 (7) ◽  
pp. 4553-4560 ◽  
Author(s):  
Kayo Koide ◽  
Saki Maegawa ◽  
Koreaki Ito ◽  
Yoshinori Akiyama
Keyword(s):  
Escherichia Coli ◽  
Stress Responses ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Lipid Phase ◽  
Intramembrane Proteolysis ◽  
Regulated Intramembrane Proteolysis ◽  
Eukaryotic Organisms ◽  
Alkylating Reagents ◽  
Active Site Region

Regulated intramembrane proteolysis (RIP) plays crucial roles in both prokaryotic and eukaryotic organisms. Proteases for RIP cleave transmembrane regions of substrate membrane proteins. However, the molecular mechanisms for the proteolysis of membrane-embedded transmembrane sequences are largely unknown. Here we studied the environment surrounding the active site region of RseP, an Escherichia coli S2P ortholog involved in the σE pathway of extracytoplasmic stress responses. RseP has two presumed active site motifs, HEXXH and LDG, located in membrane-cytoplasm boundary regions. We examined the reactivity of cysteine residues introduced within or in the vicinity of these two active site motifs with membrane-impermeable thiol-alkylating reagents under various conditions. The active site positions were inaccessible to the reagents in the native state, but many of them became partially modifiable in the presence of a chaotrope, while requiring simultaneous addition of a chaotrope and a detergent for full modification. These results suggest that the active site of RseP is not totally embedded in the lipid phase but located within a proteinaceous structure that is partially exposed to the aqueous milieu.

scholarly journals Multiple Drug-Induced Stress Responses Inhibit Formation of Escherichia coli Biofilms

2020 ◽  
Vol 86 (21) ◽  
Author(s):  
Nataliya A. Teteneva ◽  
Sergey V. Mart’yanov ◽  
María Esteban-López ◽  
Jörg Kahnt ◽  
Timo Glatter ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Responses ◽  
Biofilm Formation ◽  
Molecular Mechanisms ◽  
Drug Repurposing ◽  
Bacterial Attachment ◽  
Bacterial Biofilm ◽  
Multiple Drug ◽  
Content Type ◽  
Approved Drugs

ABSTRACT In most ecosystems, bacteria exist primarily as structured surface-associated biofilms that can be highly tolerant to antibiotics and thus represent an important health issue. Here, we explored drug repurposing as a strategy to identify new antibiofilm compounds, screening over 1,000 compounds from the Prestwick Chemical Library of approved drugs for specific activities that prevent biofilm formation by Escherichia coli. Most growth-inhibiting compounds, which include known antibacterial but also antiviral and other drugs, also reduced biofilm formation. However, we also identified several drugs that were biofilm inhibitory at doses where only a weak effect or no effect on planktonic growth could be observed. The activities of the most specific antibiofilm compounds were further characterized using gene expression analysis, proteomics, and microscopy. We observed that most of these drugs acted by repressing genes responsible for the production of curli, a major component of the E. coli biofilm matrix. This repression apparently occurred through the induction of several different stress responses, including DNA and cell wall damage, and homeostasis of divalent cations, demonstrating that biofilm formation can be inhibited through a variety of molecular mechanisms. One tested drug, tyloxapol, did not affect curli expression or cell growth but instead inhibited biofilm formation by suppressing bacterial attachment to the surface. IMPORTANCE The prevention of bacterial biofilm formation is one of the major current challenges in microbiology. Here, by systematically screening a large number of approved drugs for their ability to suppress biofilm formation by Escherichia coli, we identified a number of prospective antibiofilm compounds. We further demonstrated different mechanisms of action for individual compounds, from induction of replicative stress to disbalance of cation homeostasis to inhibition of bacterial attachment to the surface. Our work demonstrates the potential of drug repurposing for the prevention of bacterial biofilm formation and suggests that also for other bacteria, the activity spectrum of antibiofilm compounds is likely to be broad.

scholarly journals Irreversible Inactivations of Escherichia coli Alkaline Phosphatase with Active Site Directed Alkylating Reagents.

1970 ◽  
Vol 24 ◽  
pp. 1025-1035 ◽  
Author(s):  
Hedvig Csopak ◽  
Georg Fölsch ◽  
K. Simons ◽  
L. Kääriäinen ◽  
S. E. Rasmussen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Active Site ◽  
Alkylating Reagents

Functional analysis of drought and salt tolerance mechanisms of mulberry RACK1 gene

2019 ◽  
Vol 39 (12) ◽  
pp. 2055-2069 ◽  
Author(s):  
Changying Liu ◽  
Panpan Zhu ◽  
Wei Fan ◽  
Yang Feng ◽  
Min Kou ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
G Proteins ◽  
Stress Responses ◽  
Molecular Mechanisms ◽  
Guanine Nucleotide Binding Protein ◽  
Expression Levels ◽  
Drought And Salt Tolerance ◽  
Drought And Salt Stresses ◽  
Guanine Nucleotide Binding ◽  
Eukaryotic Organisms

Abstract The receptor for activated C kinase 1 (RACK1) protein acts as a central hub for the integration of many physiological processes in eukaryotic organisms. Plant RACK1 is implicated in abiotic stress responses, but the underlying molecular mechanisms of stress adaptation remain largely unknown. Here, the overexpression of the mulberry (Morus alba L.) RACK1 gene in Arabidopsis decreased tolerance to drought and salt stresses and MaRACK1 overexpression changed expression levels of genes in response to stress and stimuli. We developed a simple and efficient transient transformation system in mulberry, and the mulberry seedlings transiently expressing MaRACK1 were hypersensitive to drought and salt stresses. The expression levels of guanine nucleotide-binding protein (G-protein) encoding genes in mulberry and Arabidopsis were not affected by MaRACK1 overexpression. The interactions between RACK1 and G-proteins were confirmed, and the RACK1 proteins from mulberry and Arabidopsis could not interact with their respective G-proteins, which indicated that RACK1 may regulate stress responses independently of G-proteins. Additionally, MaRACK1 may regulate drought and salt stress tolerances by interacting with a fructose 1, 6-bisphosphate aldolase. Our findings provide new insights into the mechanisms underlying RACK1 functions in abiotic stress responses and important information for their further characterization.

scholarly journals Structure of peptide from active site region of Escherichia coli L-asparaginase.

1977 ◽  
Vol 252 (6) ◽  
pp. 2072-2076
Author(s):  
R G Peterson ◽  
F F Richards ◽  
R E Handschumacher
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Active Site Region

scholarly journals TGF-β inhibits alveolar protein transport by promoting shedding, regulated intramembrane proteolysis, and transcriptional downregulation of megalin

2017 ◽  
Vol 313 (5) ◽  
pp. L807-L824 ◽  
Author(s):  
Luciana C. Mazzocchi ◽  
Christine U. Vohwinkel ◽  
Konstantin Mayer ◽  
Susanne Herold ◽  
Rory E. Morty ◽  
...  
Keyword(s):  
Cell Surface ◽  
Molecular Mechanisms ◽  
Protein Transport ◽  
Transforming Growth Factor ◽  
Direct Interaction ◽  
Alveolar Space ◽  
Intramembrane Proteolysis ◽  
Subsequent Increase ◽  
Regulated Intramembrane Proteolysis ◽  
Alveolar Edema

Disruption of the alveolar-capillary barrier is a hallmark of acute respiratory distress syndrome (ARDS) that leads to the accumulation of protein-rich edema in the alveolar space, often resulting in comparable protein concentrations in alveolar edema and plasma and causing deleterious remodeling. Patients who survive ARDS have approximately three times lower protein concentrations in the alveolar edema than nonsurvivors; thus the ability to remove excess protein from the alveolar space may be critical for a positive outcome. We have recently shown that clearance of albumin from the alveolar space is mediated by megalin, a 600-kDa transmembrane endocytic receptor and member of the low-density lipoprotein receptor superfamily. In the currents study, we investigate the molecular mechanisms by which transforming growth factor-β (TGF-β), a key molecule of ARDS pathogenesis, drives downregulation of megalin expression and function. TGF-β treatment led to shedding and regulated intramembrane proteolysis of megalin at the cell surface and to a subsequent increase in intracellular megalin COOH-terminal fragment abundance resulting in transcriptional downregulation of megalin. Activity of classical protein kinase C enzymes and γ-secretase was required for the TGF-β-induced megalin downregulation. Furthermore, TGF-β-induced shedding of megalin was mediated by matrix metalloproteinases (MMPs)-2, -9, and -14. Silencing of either of these MMPs stabilized megalin at the cell surface after TGF-β treatment and restored normal albumin transport. Moreover, a direct interaction of megalin with MMP-2 and -14 was demonstrated, suggesting that these MMPs may function as novel sheddases of megalin. Further understanding of these mechanisms may lead to novel therapeutic approaches for the treatment of ARDS.

scholarly journals DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

2015 ◽  
Vol 112 (50) ◽  
pp. E6862-E6871 ◽  
Author(s):  
Andrey Parshin ◽  
Anthony L. Shiver ◽  
Jookyung Lee ◽  
Maria Ozerova ◽  
Dina Schneidman-Duhovny ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Active Site ◽  
Amino Acid Starvation ◽  
Cross Linking ◽  
Molecular Function ◽  
Secondary Channel ◽  
Global Response ◽  
Microbial Life ◽  
Active Site Region

Sensing and responding to nutritional status is a major challenge for microbial life. In Escherichia coli, the global response to amino acid starvation is orchestrated by guanosine-3′,5′-bisdiphosphate and the transcription factor DksA. DksA alters transcription by binding to RNA polymerase and allosterically modulating its activity. Using genetic analysis, photo–cross-linking, and structural modeling, we show that DksA binds and acts upon RNA polymerase through prominent features of both the nucleotide-access secondary channel and the active-site region. This work is, to our knowledge, the first demonstration of a molecular function for Sequence Insertion 1 in the β subunit of RNA polymerase and significantly advances our understanding of how DksA binds to RNA polymerase and alters transcription.

scholarly journals C-terminal domain phosphatase-like 1 (CPL1) is involved in floral transition in Arabidopsis

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Chen Yuan ◽  
Jingya Xu ◽  
Qianqian Chen ◽  
Qinggang Liu ◽  
Yikai Hu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Stress Responses ◽  
Molecular Mechanisms ◽  
Floral Transition ◽  
Mirna Biogenesis ◽  
Terminal Domain ◽  
Delayed Flowering ◽  
Eukaryotic Organisms

Abstract Background RNA polymerase II plays critical roles in transcription in eukaryotic organisms. C-terminal Domain Phosphatase-like 1 (CPL1) regulates the phosphorylation state of the C-terminal domain of RNA polymerase II subunit B1, which is critical in determining RNA polymerase II activity. CPL1 plays an important role in miRNA biogenesis, plant growth and stress responses. Although cpl1 mutant showes delayed-flowering phenotype, the molecular mechanism behind CPL1’s role in floral transition is still unknown. Results To study the role of CPL1 during the floral transition, we first tested phenotypes of cpl1-3 mutant, which harbors a point-mutation. The cpl1-3 mutant contains a G-to-A transition in the second exon, which results in an amino acid substitution from Glu to Lys (E116K). Further analyses found that the mutated amino acid (Glu) was conserved in these species. As a result, we found that the cpl1-3 mutant experienced delayed flowering under both long- and short-day conditions, and CPL1 is involved in the vernalization pathway. Transcriptome analysis identified 109 genes differentially expressed in the cpl1 mutant, with 2 being involved in floral transition. Differential expression of the two flowering-related DEGs was further validated by qRT-PCR. Conclusions Flowering genetic pathways analysis coupled with transciptomic analysis provides potential genes related to floral transition in the cpl1-3 mutant, and a framework for future studies of the molecular mechanisms behind CPL1’s role in floral transition.

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