Allogenic hematopoietic stem cell transplantation in an Iranian patient with osteopetrosis caused by carbonic anhydrase II deficiency: A case report.

BACKGROUND: Osteopetrosis is a group of geneticall heterogeneous disorders resulting from impaired osteoclast function and bone resorption. The identification of specific genetic mutations can yield important prognostic and therapeutic implications. Herein, we present the diagnosis and successful application of hematopoietic stem cell transplantation (HSCT) in a patient with osteopetrosis caused by carbonic anhydrase II deficiency (Intermediate osteopetrosis). CASE PRESENTATION: Herein, we describe a 2.5-year-old male patient born to consanguineous parents who presented at 8-month-old with hydrocephaly, brain shunt, and developmental delay. Later at 9 months old, he was found to have eye disorder such as nystagmus, fracture of the elbow, abnormal skeletal survey, normal cell blood count (CBC), and severe hypocellularity in the bone marrow. Further evaluation showed renal tubular acidosis type 2. Whole-exome sequencing revealed a pathogenic homozygous variant in intron 2 of the carbonic anhydrase 2 gene (CA2) gene (c.232 + 1 G>T). The diagnosis of intermediate autosomal recessive osteopetrosis was established, and allogenic HSCT from his mother, a full-matched related donor (MRD), was planned. The conditioning regimen included Busulfan, Fludarabine, and Rabbit anti-thymocyte globulin. Cyclosporine and Mycophenolate Mofetil were used for graft-versus-host-disease prophylaxis. He Engrafted on day +13, and 95% chimerism was achieved. He is currently doing well without immunosuppressive therapy, now 12 months post HSCT, with normal calcium level and improving visual quality and FISH analysis revealed complete donor chimerism. DISCUSSION: HSCT could be a promising curative treatment for intermediate osteopetrosis and can provide long-term survival. Ongoing challenges in various aspects of HSCT remain to be addressed.

Kidney intercalated cells and the transcription factor FOXi1 drive cystogenesis in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is caused by mutations in either TSC1 or TSC2 genes and affects multiple organs, including kidney, lung, and brain. In the kidney, TSC presents with the enlargement of benign tumors (angiomyolipomata) and cysts, which eventually leads to kidney failure. The factors promoting cyst formation and tumor growth in TSC are incompletely understood. Here, we report that mice with principal cell-specific inactivation of Tsc1 develop numerous cortical cysts, which are overwhelmingly composed of hyperproliferating A-intercalated (A-IC) cells. RNA sequencing and confirmatory expression studies demonstrated robust expression of Forkhead Transcription Factor 1 (Foxi1) and its downstream targets, apical H+-ATPase and cytoplasmic carbonic anhydrase 2 (CAII), in cyst epithelia in Tsc1 knockout (KO) mice but not in Pkd1 mutant mice. In addition, the electrogenic 2Cl-/H+ exchanger (CLC-5) is significantly up-regulated and shows remarkable colocalization with H+-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Deletion of Foxi1, which is vital to intercalated cells viability and H+-ATPase expression, completely abrogated the cyst burden in Tsc1 KO mice, as indicated by MRI images and histological analysis in kidneys of Foxi1/Tsc1 double-knockout (dKO) mice. Deletion of CAII, which is critical to H+-ATPase activation, caused significant reduction in cyst burden and increased life expectancy in CAII/Tsc1 dKO mice vs. Tsc1 KO mice. We propose that intercalated cells and their acid/base/electrolyte transport machinery (H+-ATPase/CAII/CLC-5) are critical to cystogenesis, and their inhibition or inactivation is associated with significant protection against cyst generation and/or enlargement in TSC.

Carbonic Anhydrase 2 Deletion Delays the Growth of Kidney Cysts Whereas Foxi1 Deletion Completely Abrogates Cystogenesis in TSC.

Tuberous sclerosis complex (TSC) presents with renal cysts and benign tumors, which eventually lead to kidney failure. The factors promoting kidney cyst formation in TSC are poorly understood. Inactivation of carbonic anhydrase 2 (Car2) significantly reduced, whereas, deletion of Foxi1 completely abrogated the cyst burden in Tsc1 KO mice. In these studies, we contrasted the ontogeny of cyst burden in Tsc1/Car2 dKO mice vs. Tsc1/Foxi1 dKO mice. Compared to Tsc1 KO, the Tsc1/Car2 dKO mice showed few small cysts at 47 days of age. However, by 110 days, the kidneys showed frequent and large cysts with overwhelming numbers of A-intercalated cells in their linings. The magnitude of cyst burden in Tsc1/Car2 dKO mice correlated with the expression levels of Foxi1 and was proportional to mTORC1 activation. This is in stark contrast to Tsc1/Foxi1 dKO mice, which showed a remarkable absence of kidney cysts at both 47 and 110 days of age. RNA-seq data pointed to profound upregulation of Foxi1 and kidney-collecting duct-specific H+-ATPase subunits in 110-day-old Tsc1/Car2 dKO mice. We conclude that Car2 inactivation temporarily decreases the kidney cyst burden in Tsc1 KO mice but the cysts increase with advancing age, along with enhanced Foxi1 expression.

Design of a Superpositively Charged Enzyme: Human Carbonic Anhydrase II Variant with Ferritin Encapsulation and Immobilization.

Supercharged proteins exhibit high solubility and other desirable properties, but no engineered superpositively charged enzymes have previously been made. Superpositively charged variants of proteins such as green fluorescent protein have been efficiently encapsulated within Archaeoglobus fulgidus thermophilic ferritin (AfFtn). Encapsulation by supramolecular ferritin can yield systems with a variety of sequestered cargo. To advance applications in enzymology and green chemistry, we sought a general method for supercharging an enzyme that retains activity and is compatible with AfFtn encapsulation. The zinc metalloenzyme human carbonic anhydrase II (hCAII) is an attractive encapsulation target based on its hydrolytic activity and physiologic conversion of carbon dioxide to bicarbonate. A computationally designed variant of hCAII contains positively charged residues substituted at 19 sites on the protein's surface, resulting in a shift of the putative net charge from -1 to +21. This designed hCAII(+21) exhibits encapsulation within AfFtn without the need for fusion partners or additional reagents. The hCAII(+21) variant retains esterase activity comparable to the wild type and spontaneously templates the assembly of AfFtn 24mers around itself. The AfFtn-hCAII(+21) host-guest complex exhibits both greater activity and thermal stability when compared to hCAII(+21). Upon immobilization on a solid support, AfFtn-hCAII(+21) retains enzymatic activity and exhibits an enhancement of activity at elevated temperatures.

Molecular cloning and transcriptional regulation of two γ-carbonic anhydrase genes in the green macroalga Ulva prolifera.

Ulva prolifera O.F. Müller (Ulvophyceae, Chlorophyta) is well known as a typical green-tide forming macroalga which has caused the world's largest macroalgal blooms in the Yellow Sea of China. In this study, two full-length γ-carbonic anhydrase (γ-CA) genes (UpγCA1 and UpγCA2) were cloned from U. prolifera. UpγCA1 has three conserved histidine residues, which act as an active site for binding a zinc metal ion. In UpγCA2, two of the three histidine residues were replaced by serine and arginine, respectively. The two γ-CA genes are clustered together with other γ-CAs in Chlorophyta with strong support value (100% bootstrap) in maximum likelihood (ML) phylogenetic tree. Quantitative real-time PCR (qRT-PCR) analysis showed that stressful environmental conditions markedly inhibited transcription levels of these two γ-CA genes. Low pH value (pH 7.5) significantly increased transcription level of UpγCA2 not UpγCA1 at 12 h, whereas high pH value (pH 8.5) significantly inhibited the transcription of these two γ-CA genes at 6 h. These findings enhanced our understanding on transcriptional regulation of γ-CA genes in response to environmental factors in U. prolifera.

A novel nickel-modified nano-magnetite for isolation of histidine-tagged proteins expressed in Escherichia coli.

Nano-magnetite with superparamagnetism could be coated by some organic compounds or by nano Au or Pt via surface modifications with multi-step reactions for the applications of isolating histidine-tagged (His-tagged) proteins. Introducing active sites of binding histidine onto the surface of nano-magnetite was the ultimate task. However, multi-step treatments might result in departure of the coatings from the surface of the nano-magnetite, which led to loss of active sites. In this work, we reported a convenient and efficient way of treating nano-magnetites and applied them in isolating His-tagged proteins. Carboxylates were introduced on the surface of home-made nano-magnetite directly via ultrasonic mixing with sodium bitartrate rather than complicated surface modifications, which was proved by thermogravimetric analyses. Ni2+ was, therefore, caught by the carboxylates of the coating via the coordinate interaction, demonstrated by X-ray photoelectron spectra. The coated magnetic nanoparticles with the bonded Ni2+ were successfully employed to selectively bind and separate recombinant His-tagged proteins directly from the mixture of Escherichia coli cell lysate, and showed wonderful affinity for His-tagged proteins with the saturated adsorption amount being 556 mg g-1. Additionally, such functionalized nano-magnetite manifested the excellent recyclability in isolating His-tagged proteins.

A novel homozygous nonsense mutation in the CA2 gene (c.368G>A, p.W123X) linked to carbonic anhydrase II deficiency syndrome in a Chinese family.

BACKGROUND: Carbonic anhydrase II deficiency syndrome is an autosomal recessive osteopetrosis with renal tubular acidosis and cerebral calcifications. We tried to detect the causative mutation for carbonic anhydrase II deficiency syndrome in a five-generation Chinese family. MATERIALS AND METHODS: Genomic DNA was extracted from whole blood of the proband, his grandmother, parents, aunt, uncle and sister. The exomes were sequenced by whole exon sequencing followed by genetic analysis and Sanger sequencing validation. Then, physical and chemical properties studies and structure analysis were performed on mutated protein. Finally, Minigene model of vector plasmids for wild type and mutant type was constructed and transfected into human embryonic kidney 293T cells to further explore the expression change of CA2 transcript and protein after mutation. RESULTS: Sequencing and genetic analysis have revealed the homozygous nonsense mutation of CA2 gene (c.368G > A, p.W123X) in the exon 4 of chromosome 8 of the proband, while it was not found in his grandmother, parents, aunt, uncle and sister. Furthermore, Sanger sequencing in the proband and his parents validated the mutation. Properties and structure of mutated CA2 proteins changed after mutation, especially in change of protein modification and hindrance of zinc ions binding, which may lead to decreased protein expression level of CA2. CONCLUSIONS: We found a new homozygous nonsense mutation in CA2 gene (c.368G > A, p.W123X), which may be valuable in the early diagnosis and therapy of carbonic anhydrase II deficiency syndrome.

3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics.

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.

Developmental loss, but not pharmacological suppression, of renal carbonic anhydrase 2 results in pyelonephritis susceptibility.

Carbonic anhydrase II knockout (Car2-/-) mice have depleted numbers of renal intercalated cells, which are increasingly recognized to be innate immune effectors. We compared pyelonephritis susceptibility following reciprocal renal transplantations between Car2-/- and wild-type mice. We examined the effect of pharmacological CA suppression using acetazolamide in an experimental murine model of urinary tract infection. Car2-/- versus wild-type mice were compared for differences in renal innate immunity. In our transplant scheme, mice lacking CA-II in the kidney had increased pyelonephritis risk. Mice treated with acetazolamide had lower kidney bacterial burdens at 6 h postinfection, which appeared to be due to tubular flow from diuresis because comparable results were obtained when furosemide was substituted for acetazolamide. Isolated Car2-/- kidney cells enriched for intercalated cells demonstrated altered intercalated cell innate immune gene expression, notably increased calgizzarin and insulin receptor expression. Intercalated cell number and function along with renal tubular flow are determinants of pyelonephritis risk.

RNA-seq analysis of chondrocyte transcriptome reveals genetic heterogeneity in LG/J and SM/J murine strains.

OBJECTIVE: To investigate the transcriptomic differences in chondrocytes obtained from LG/J (large, healer) and SM/J (small, non-healer) murine strains in an attempt to discern the molecular pathways implicated in cartilage regeneration and susceptibility to osteoarthritis (OA). DESIGN: We performed RNA-sequencing on chondrocytes derived from LG/J (n = 16) and SM/J (n = 16) mice. We validated the expression of candidate genes and compared single nucleotide polymorphisms (SNPs) between the two mouse strains. We also examined gene expression of positional candidates for ear pinna regeneration and long bone length quantitative trait loci (QTLs) that display differences in cartilaginous expression. RESULTS: We observed a distinct genetic heterogeneity between cells derived from LG/J and SM/J mouse strains. We found that gene ontologies representing cell development, cartilage condensation, and regulation of cell differentiation were enriched in LG/J chondrocytes. In contrast, gene ontologies enriched in the SM/J chondrocytes were mainly related to inflammation and degeneration. Moreover, SNP analysis revealed that multiple validated genes vary in sequence between LG/J and SM/J in coding and highly conserved noncoding regions. Finally, we showed that most QTLs have 20-30% of their positional candidates displaying differential expression between the two mouse strains. CONCLUSIONS: While the enrichment of pathways related to cell differentiation, cartilage development and cartilage condensation infers superior healing potential of LG/J strain, the enrichment of pathways related to cytokine production, immune cell activation and inflammation entails greater susceptibility of SM/J strain to OA. These data provide novel insights into chondrocyte transcriptome and aid in identification of the quantitative trait genes and molecular differences underlying the phenotypic differences associated with individual QTLs.

Cooperative protein-solvent tuning of proton transfer energetics: carbonic anhydrase as a case study.

We investigate the coupling between the proton transfer (PT) energetics and the protein-solvent dynamics using the intra-molecular PT in wild type (wt) human carbonic anhydrase II and its ten-fold faster mutant Y7F/N67Q as a test case. We calculate the energy variation upon PT, and from that we also calculate the PT reaction free energy, making use of a hybrid quantum mechanics/molecular dynamics approach. In agreement with the experimental data, we obtain that the reaction free energy is basically the same in the two systems. Yet, we show that the instantaneous PT energy is on average lower in the mutant possibly contributing to the faster PT rate. Analysis of the contribution to the PT energetics of the solvent and of each protein residue, also not in the vicinity of the active site, provides evidence for electrostatic tuning of the PT energy arising from the combined effect of the solvent and the protein environment. These findings open up a way to the more general task of the rational design of mutants with either enhanced or reduced PT rate.

Fast Microsecond Dynamics of the Protein-Water Network in the Active Site of Human Carbonic Anhydrase II Studied by Solid-State NMR Spectroscopy.

Protein-water interactions have widespread effects on protein structure and dynamics. As such, the function of many biomacromolecules can be directly related to the presence and exchange of water molecules. While the presence of structural water sites can be easily detected by X-ray crystallography, the dynamics within functional water-protein network architectures is largely elusive. Here we use solid-state NMR relaxation dispersion measurements with a focus on those active-site residues in the enzyme human carbonic anhydrase II (hCAII) that constitute the evolutionarily conserved water pocket, key for CAs' enzymatic catalysis. Together with chemical shifts, peak broadening, and results of molecular dynamics (MD) and DFT shift calculations, the relaxation dispersion data suggest the presence of a widespread fast µs-time-scale dynamics in the pocket throughout the protein-water network. This process is abrogated in the presence of an inhibitor which partially disrupts the network. The time scale of the protein-water pocket motion coincides both with the estimated residence time of Zn-bound water/OH- in the pocket showing the longest lifetimes in earlier magnetic relaxation dispersion experiments as well as with the rate-limiting step of catalytic turnover. As such, the reorganization of the water pocket:enzyme architecture might constitute an element of importance for enzymatic activity of this and possibly other proteins.

Carbonic anhydrase 2 (CAII) supports tumor blood endothelial cell survival under lactic acidosis in the tumor microenvironment.

BACKGROUND: Tumor endothelial cells (TECs) perform tumor angiogenesis, which is essential for tumor growth and metastasis. Tumor cells produce large amounts of lactic acid from glycolysis; however, the mechanism underlying the survival of TECs to enable tumor angiogenesis under high lactic acid conditions in tumors remains poorly understood. METHODOLOGY: The metabolomes of TECs and normal endothelial cells (NECs) were analyzed by capillary electrophoresis time-of-flight mass spectrometry. The expressions of pH regulators in TECs and NECs were determined by quantitative reverse transcription-PCR. Cell proliferation was measured by the MTS assay. Western blotting and ELISA were used to validate monocarboxylate transporter 1 and carbonic anhydrase 2 (CAII) protein expression within the cells, respectively. Human tumor xenograft models were used to access the effect of CA inhibition on tumor angiogenesis. Immunohistochemical staining was used to observe CAII expression, quantify tumor microvasculature, microvessel pericyte coverage, and hypoxia. RESULTS: The present study shows that, unlike NECs, TECs proliferate in lactic acidic. TECs showed an upregulated CAII expression both in vitro and in vivo. CAII knockdown decreased TEC survival under lactic acidosis and nutrient-replete conditions. Vascular endothelial growth factor A and vascular endothelial growth factor receptor signaling induced CAII expression in NECs. CAII inhibition with acetazolamide minimally reduced tumor angiogenesis in vivo. However, matured blood vessel number increased after acetazolamide treatment, similar to bevacizumab treatment. Additionally, acetazolamide-treated mice showed decreased lung metastasis. CONCLUSION: These findings suggest that due to their effect on blood vessel maturity, pH regulators like CAII are promising targets of antiangiogenic therapy. Video Abstract.

Carbonic anhydrase 2 inhibits epithelial-mesenchymal transition and metastasis in hepatocellular carcinoma.

Carbonic anhydrase 2 (CA2) plays vital role in the regulation of ion transport and pH balance and is involved in many biological processes; however, its role in cancer remains obscure. In this study, we identified a novel function of CA2 in facilitating hepatocellular carcinoma (HCC) metastasis. CA2 expression was elevated in Na+-K+-ATPase α1 (ATP1A1)-downregulated HCC cells and was inversely correlated with that of ATP1A1 in HCC. ATP1A1 acted as an oncoprotein whereas CA2 overexpression inhibited cell migration and invasion by reversing epithelial-mesenchymal transition (EMT) in HCC. CA2 downregulation promoted HCC metastasis and invasion whereas ATP1A1 downregulation inhibited HCC metastasis. Because of the opposing effects of CA2 and ATP1A1 in HCC, we examined the role of their correlation in HCC metastasis. CA2 attenuated ATP1A1-triggered tumor growth in vivo and ATP1A1-induced metastasis in vitro. Taken together, the present results suggest that CA2 serves as a suppressor of HCC metastasis and EMT and is correlated with favorable overall survival (OS) in HCC patients.

Targeted deletion of the Ncoa7 gene results in incomplete distal renal tubular acidosis in mice.

We recently reported that nuclear receptor coactivator 7 (Ncoa7) is a vacuolar proton pumping ATPase (V-ATPase) interacting protein whose function has not been defined. Ncoa7 is highly expressed in the kidney and partially colocalizes with the V-ATPase in collecting duct intercalated cells (ICs). Here, we hypothesized that targeted deletion of the Ncoa7 gene could affect V-ATPase activity in ICs in vivo. We tested this by analyzing the acid-base status, major electrolytes, and kidney morphology of Ncoa7 knockout (KO) mice. We found that Ncoa7 KO mice, similar to Atp6v1b1 KOs, did not develop severe distal renal tubular acidosis (dRTA), but they exhibited a persistently high urine pH and developed hypobicarbonatemia after acid loading with ammonium chloride. Conversely, they did not develop significant hyperbicarbonatemia and alkalemia after alkali loading with sodium bicarbonate. We also found that ICs were larger and with more developed apical microvilli in Ncoa7 KO compared with wild-type mice, a phenotype previously associated with metabolic acidosis. At the molecular level, the abundance of several V-ATPase subunits, carbonic anhydrase 2, and the anion exchanger 1 was significantly reduced in medullary ICs of Ncoa7 KO mice, suggesting that Ncoa7 is important for maintaining high levels of these proteins in the kidney. We conclude that Ncoa7 is involved in IC function and urine acidification in mice in vivo, likely through modulating the abundance of V-ATPase and other key acid-base regulators in the renal medulla. Consequently, mutations in the NCOA7 gene may also be involved in dRTA pathogenesis in humans.

Loss of the Chloroplast Transit Peptide from an Ancestral C3 Carbonic Anhydrase Is Associated with C4 Evolution in the Grass Genus Neurachne.

Neurachne is the only known grass lineage containing closely related C3, C3-C4 intermediate, and C4 species, making it an ideal taxon with which to study the evolution of C4 photosynthesis in the grasses. To begin dissecting the molecular changes that led to the evolution of C4 photosynthesis in this group, the complementary DNAs encoding four distinct ß-carbonic anhydrase (CA) isoforms were characterized from leaf tissue of Neurachne munroi (C4), Neurachne minor (C3-C4), and Neurachne alopecuroidea (C3). Two genes (CA1 and CA2) each encode two different isoforms: CA1a/CA1b and CA2a/CA2b. Transcript analyses found that CA1 messenger RNAs were significantly more abundant than transcripts from the CA2 gene in the leaves of each species examined, constituting ∼99% of all ß-CA transcripts measured. Localization experiments using green fluorescent protein fusion constructs showed that, while CA1b is a cytosolic CA in all three species, the CA1a proteins are differentially localized. The N. alopecuroidea and N. minor CA1a isoforms were imported into chloroplasts of Nicotiana benthamiana leaf cells, whereas N. munroi CA1a localized to the cytosol. Sequence analysis indicated an 11-amino acid deletion in the amino terminus of N. munroi CA1a relative to the C3 and C3-C4 proteins, suggesting that chloroplast targeting of CA1a is the ancestral state and that loss of a functional chloroplast transit peptide in N. munroi CA1a is associated with the evolution of C4 photosynthesis in Neurachne spp. Remarkably, this mechanism is homoplastic with the evolution of the C4-associated CA in the dicotyledonous genus Flaveria, although the actual mutations in the two lineages differ.

Role of MCT1 and CAII in skeletal muscle pH homeostasis, energetics, and function: in vivo insights from MCT1 haploinsufficient mice.

The purpose of this study was to investigate the effects of a partial suppression of monocarboxylate transporter (MCT)-1 on skeletal muscle pH, energetics, and function (MCT1+/- mice). Twenty-four MCT1+/- and 13 wild-type (WT) mice were subjected to a rest-exercise-recovery protocol, allowing assessment of muscle energetics (by magnetic resonance spectroscopy) and function. The study included analysis of enzyme activities and content of protein involved in pH regulation. Skeletal muscle of MCT1+/- mice had lower MCT1 (-61%; P < 0.05) and carbonic anhydrase (CA)-II (-54%; P < 0.05) contents. Although intramuscular pH was higher in MCT1+/- mice at rest (P < 0.001), the mice showed higher acidosis during the first minute of exercise (P < 0.01). Then, the pH time course was similar among groups until exercise completion. MCT1+/- mice had higher specific peak (P < 0.05) and maximum tetanic (P < 0.01) forces and lower fatigability (P < 0.001) when compared to WT mice. We conclude that both MCT1 and CAII are involved in the homeostatic control of pH in skeletal muscle, both at rest and at the onset of exercise. The improved muscle function and resistance to fatigue in MCT1+/- mice remain unexplained.-Chatel, B., Bendahan, D., Hourdé, C., Pellerin, L., Lengacher, S., Magistretti, P., Fur, Y. L., Vilmen, C., Bernard, M., Messonnier, L. A. Role of MCT1 and CAII in skeletal muscle pH homeostasis, energetics, and function: in vivo insights from MCT1 haploinsufficient mice.

CO2/bicarbonate modulates cone photoreceptor ROS-GC1 and restores its CORD6-linked catalytic activity.

This study with recombinant reconstituted system mimicking the cellular conditions of the native cones documents that photoreceptor ROS-GC1 is modulated by gaseous CO2. Mechanistically, CO2 is sensed by carbonic anhydrase (CAII), generates bicarbonate that, in turn, directly targets the core catalytic domain of ROS-GC1, and activates it to increased synthesis of cyclic GMP. This, then, functions as a second messenger for the cone phototransduction. The study demonstrates that, in contrast to the Ca2+-modulated phototransduction, the CO2 pathway is Ca2+-independent, yet is linked with it and synergizes it. It, through R787C mutation in the third heptad of the signal helix domain of ROS-GC1, affects cone-rod dystrophy, CORD6. CORD6 is caused firstly by lowered basal and GCAP1-dependent ROS-GC1 activity and secondly, by a shift in Ca2+ sensitivity of the ROS-GC1/GCAP1 complex that remains active in darkness. Remarkably, the first but not the second defect disappears with bicarbonate thus explaining the basis for CORD6 pathological severity. Because cones, but not rods, express CAII, the excessive synthesis of cyclic GMP would be most acute in cones.

Synthesis, biological evaluation and computational studies of novel iminothiazolidinone benzenesulfonamides as potent carbonic anhydrase II and IX inhibitors.

A series of iminothiazolidinone-sulfonamide hybrids (2a-k) was synthesized by heterocyclization of sulfanilamide thioureas with methyl bromoacetate and characterized by spectroscopic techniques, mass and elemental analysis. The synthesized derivatives were screened against four relevant human (h) isoforms of carbonic anydrases (CAs, EC 4.2.1.1) I, II, IV and IX. These enzymes are involved in a variety of diseases, including glaucoma, retinitis pigmentosa, epilepsy, arthritis, and tumors. Derivatives 2a-2k exhibited the best inhibitory activity against the cytosolyc hCA II (KIs are reaching the sub-nanomolar range, 0.41-37.8 nM) and against the tumor-associated isoform hCA IX (KIs are spanning between 24.3 and 368.3 nM). The binding mode of the reported iminothiazolidinone benzenesulfonamides within hCA II and IX catalytic clefts was investigated by docking studies.

Toll-like Receptor 3 Agonist, Polyinosinic-polycytidylic Acid, Upregulates Carbonic Anhydrase II in Human Keratinocytes.

Carbonic anhydrases are ubiquitously expressed enzymes that reversibly hydrate carbon dioxide to bicarbonate and protons. While the main function of carbonic anhydrases is to regulate pH and osmotic balance, their involvement in other physiological processes remains to be explored. This study analysed changes in mRNA and protein levels of carbonic anhydrase II in human primary keratinocytes treated with various toll-like receptor agonists and cytokines. A significant upregulation of carbonic anhydrase II at the mRNA and protein levels was observed upon treatment with polyinosinic-polycytidylic acid, a toll-like receptor 3 agonist. Furthermore, in agreement with the increased expression of carbonic anhydrase II in atopic dermatitis skin, carbonic anhydrase II was upregulated by the Th2 cytokines interleukins -4 and -13. In conclusion, these results suggest a potential role of carbonic anhydrase II in Th2-dependent and toll-like receptor 3-induced pathways in inflammatory skin conditions.