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Carbonic anhydrase (1 or 2) simple or double mutants-Comparative transcriptome analysis between carbonic anhydrase mutants and wild type Arabidopsis

ABSTRACT: cea05-01_carbonic-anhydrase - carbonic anhydrase (1 or 2) simple or double mutants - Identification of genes differentially expressed in leaves of single CA1 and CA2 T-DNA insertional mutants and in the corresponding double mutant vs wild type - CA1 (At3g01500) and CA2 (At5g14740) T-DNA insertional mutant lines, the double (CA1+CA2) mutant and wild type Arabidopsis seeds were sown in soil in a phytotron. Leaves were harvested 40 days later for RNA extraction

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE36625 | GEO | 2012/03/25

SECONDARY ACCESSION(S): PRJNA156683

REPOSITORIES: GEO

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carbonic anhydrase (1 or 2) simple or double mutants-Comparative transcriptome analysis between carbonic anhydrase mutants and wild type Arabidopsis

Project description:cea05-01_carbonic-anhydrase - carbonic anhydrase (1 or 2) simple or double mutants - Identification of genes differentially expressed in leaves of single CA1 and CA2 T-DNA insertional mutants and in the corresponding double mutant vs wild type - CA1 (At3g01500) and CA2 (At5g14740) T-DNA insertional mutant lines, the double (CA1+CA2) mutant and wild type Arabidopsis seeds were sown in soil in a phytotron. Leaves were harvested 40 days later for RNA extraction 6 dye-swap - gene knock out

2012-03-25 | E-GEOD-36625 | biostudies-arrayexpress

Global transcriptomic changes following elevated carbon dioxide (5%) in the wild-type (WT) and mutant (M2; RNAi-knockdown line of carbonic anhydrase (CA2)) Nannochloropsis oceanica IMET1: Illumina sequencing

Project description:In Nannochloropsis oceanica IMET1, transcript knockdown of a cytosolic carbonic anhydrase (CA2; g2018) specifically inhibited by HC resulted in ~45%, ~30% and ~40% elevation of photosynthetic oxygen evolution rate, growth rate and biomass accumulation rate under high CO2 (5% ), respectively. This CA2-knockdown mutant is demonated as M2. To probe mechanistic links underlying the mutant (M2; RNAi-knockdown line of carbonic anhydrase (CA2)) phenotypes, temporal transcriptomic profiles are compared between RNAi-knockdown line of carbonic anhydrase (CA2) and WT, at 12 h and 24h under high CO2 (5%).

2020-10-28 | GSE115445 | GEO

Carbonic Anhydrase 2 is Required for Maintaining Metabolic Homeostasis of Human Articular Chondrocytes

Project description:Objective: To examine the role of carbonic anhydrase II (CA2 gene), the most efficient isoform in the CA family of zinc-containing metalloenzymes, in chondrocyte metabolism. Methods: CA2 expression was measured in human and mouse osteoarthritic cartilage. RNA-seq was performed in C28/I2 cells in which CA2 was silenced. Normal human chondrocytes were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen) following CA2 knockdown. Cell metabolism was studied by measuring extracellular lactate production, glucose consumption, intracellular ADP/ATP, intracellular pH, and ROS production. Glycolysis was measured using Seahorse XF96 analyzer. The effect of CA2 knockdown on anabolic and catabolic markers was measured following treatment with IL-1. Effects of CA2 was examined on cell proliferation, cell-cycle distribution, colony-formation, and migration. Results: CA2 was highly elevated in human and murine osteoarthritic cartilage. RNA-seq analysis revealed that processes related to glycolysis, apoptosis and TNF signaling were perturbed in cells lacking CA2. CA2 expression was 10-fold higher under hypoxia and its knockdown caused decreased extracellular lactate production, increased ADP/ATP ratio, impaired glycolysis, decreased glycolytic capacity and lowered the expression of glycolysis rate-limiting enzymes, but did not affect intracellular pH and ROS production. CA2 deficiency disturbed chondrocyte anabolic and catabolic equilibrium. CA2 knockdown suppressed chondrocyte migration and proliferation and induced cell-cycle arrest. Conclusion: This study unravels a novel role of CA2 in chondrocyte metabolism and inflammation. These findings indicate that CA2 is required for the maintenance of chondrocyte metabolic homeostasis. Future work is needed to further illuminate the mechanistic and functional role of CA2 in osteoarthritis development.

2022-03-17 | GSE184062 | GEO

Carbonic anhydrase 2 facilitates sorafenib resistance by counteracting MCT4-mediated intracellular pH dysregulation in hepatocellular carcinoma

Project description:Sorafenib, the first targeted therapy for hepatocellular carcinoma (HCC), has been utilized in clinics over a decade. However, its effectiveness is severely hindered by the acquired drug resistance, the mechanisms of which remain largely elusive. In this study, we identify that carbonic anhydrase 2 (CA2) is a key regulator of sorafenib resistance. Mechanistically, sorafenib treatment decreases intracellular pH (pHi) by suppressing monocarboxylate transporter 4 (MCT4) expression, while high levels of CA2 counteract MCT4-mediated pHi dysregulation upon sorafenib treatment, maintaining pHi homeostasis to facilitate cell survival and sorafenib resistance. Targeting CA2 re-sensitizes resistant HCC cells to sorafenib both in vitro and in vivo. Importantly, analysis of clinical samples demonstrates a strong correlation between CA2 expression levels and the therapeutic efficacy of sorafenib in HCC patients. Our findings highlight the significance of CA2 in facilitating sorafenib resistance and propose targeting CA2 as a potential strategy for overcoming sorafenib resistance in HCC patients.

2024-10-24 | PXD056884 | Pride

Carbonic anhydrases, EPF2 peptide and a novel protease, CRSP, mediate CO2 control of stomatal development

Project description:Environmental stimuli, including elevated CO2, regulate stomatal development1-3 but the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. Diverse plant species show a decrease in stomatal density in response to the continuing rise of atmospheric CO2 4. To date, one mutant, hic5, defective in cell wall wax biosynthesis, has been identified that exhibits a de-regulation of this CO2-controlled stomatal development response. Here we show that recently isolated Arabidopsis thaliana carbonic anhydrase double mutant plants6 exhibit an inversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We have characterized the mechanisms mediating this response and demonstrate extracellular signaling in the regulation of CO2-controlled stomatal development by carbonic anhydrases. Transcriptomic RNA-Seq analyses show that the extracellular pro-peptide gene EPF2 7,8, but not EPF1 9, is induced at elevated CO2 in wild type, but not ca1ca4 mutant leaves. Moreover, EPF2 is essential for CO2 control of stomatal development. Using cell wall proteomic and CO2-dependent transcriptome analyses, we have identified a novel, CO2-induced extracellular protease, CRSP (CO2 Response Secreted Protease), as a mediator of CO2 controlled stomatal development. Our results identify mechanisms and genes that function in the repression of stomatal development in leaves during atmospheric CO2 elevation, including the CA1/CA4 carbonic anhydrases and the secreted protease CRSP that cleaves the pro-peptide EPF2, which in turn represses stomatal development.

2014-08-22 | PXD000956 | Pride

RNA-Seq analysis of Oryza sativa, ssp. japonica, cv. Kitaake leaves from plants lacking carbonic anhydrase in the chloroplast

Project description:The aim of this experiment was to assess the impact on the transcriptome of relocalisaiton of carbonic anhydrase activity from the chloroplast to the cytosol in Oryza sativa, ssp. japonica, cv. Kitaake. Four genome edited lines and one wil-type control line were analysed.

2024-06-10 | E-MTAB-14111 | biostudies-arrayexpress

Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II.

Project description:Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties.

| S-EPMC3378272 | biostudies-literature

Chemical rescue of enzymes: proton transfer in mutants of human carbonic anhydrase II.

Project description:In human carbonic anhydrase II (HCA II), the mutation of position 64 from histidine to alanine (H64A) disrupts the rate limiting proton transfer (PT) event, resulting in a reduction of the catalytic activity of the enzyme as compared to the wild-type. Potential of mean force (PMF) calculations utilizing the multistate empirical valence bond (MS-EVB) methodology for H64A HCA II yields a PT free energy barrier significantly higher than that found in the wild-type enzyme. This high barrier, determined in the absence of exogenous buffer and assuming no additional ionizable residues in the PT pathway, indicates the likelihood of alternate enzyme pathways that utilize either ionizable enzyme residues (self-rescue) and/or exogenous buffers (chemical rescue). It has been shown experimentally that the catalytic activity of H64A HCA II can be chemically rescued to near wild-type levels by the addition of the exogenous buffer 4-methylimidazole (4MI). Crystallographic studies have identified two 4MI binding sites, yet site-specific mutations intended to disrupt 4MI binding have demonstrated these sites to be nonproductive. In the present work, MS-EVB simulations show that binding of 4MI near Thr199 in the H64A HCA II mutant, a binding site determined by NMR spectroscopy, results in a viable chemical rescue pathway. Additional viable rescue pathways are also identified where 4MI acts as a proton transport intermediary from the active site to ionizable residues on the rim of the active site, revealing a probable mode of action for the chemical rescue pathway.

| S-EPMC4120857 | biostudies-literature