Inhibition of intravacuolar acidification by antisense RNA decreases osteoclast differentiation and bone resorption in vitro.
(25/1306)
The role of proton transport and production in osteoclast differentiation was studied in vitro by inhibiting the transcription/translation of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) by antisense RNA molecules. Antisense RNAs targeted against CA II, or the 16 kDa or 60 kDa subunit of V-ATPase were used to block the expression of the specific proteins. A significant decrease in bone resorption rate and TRAP-positive osteoclast number was seen in rat bone marrow cultures and fetal mouse metacarpal cultures after antisense treatment. Intravacuolar acidification in rat bone marrow cells was also significantly decreased after antisense treatment. The CA II antisense RNA increased the number of TRAP-positive mononuclear cells, suggesting inhibition of osteoclast precursor fusion. Antisense molecules decreased the number of monocytes and macrophages, but increased the number of granulocytes in marrow cultures. GM-CSF, IL-3 and IL-6 were used to stimulate haematopoietic stem cell differentiation. The 16 kDa V-ATPase antisense RNA abolished the stimulatory effect of GM-CSF, IL-3 and IL-6 on TRAP-positive osteoclast formation, but did not affect the formation of monocytes and macrophages after IL-3 treatment, or the formation of granulocytes after IL-6 treatment. These results suggest that CA II and V-ATPase are needed, not only for the actual resorption, but also for osteoclast formation in vitro. (+info)
Transcriptional regulation of the MN/CA 9 gene coding for the tumor-associated carbonic anhydrase IX. Identification and characterization of a proximal silencer element.
(26/1306)
The MN/CA 9 (MN) gene encodes a tumor-associated isoenzyme of the carbonic anhydrase family. Functional characterization of the 3. 5-kilobase pair MN 5' upstream region by deletion analysis led to the identification of the -173 to +31 fragment as the MN promoter. In vitro DNase I footprinting revealed the presence of five protected regions (PRs) within the MN promoter. Detailed deletion analysis of the promoter identified PR1 and PR2 (numbered from the transcription start) as the most critical for transcriptional activity. PR4 negatively affected transcription, since its deletion led to increased promoter activity and was confirmed to function as a promoter-, position-, and orientation-independent silencer element. Mutational analysis indicated that the direct repeat AGGGCacAGGGC is required for efficient repressor binding. Two components of the repressor complex (35 and 42 kDa) were found to be in direct contact with PR4 by UV cross-linking. Increased cell density, known to induce MN expression, did not affect levels of PR4 binding in HeLa cells. Significantly reduced repressor level seems to be responsible for MN up-regulation in the case of tumorigenic CGL3 as compared with nontumorigenic CGL1 HeLa x normal fibroblast hybrid cells. (+info)
Complementary role of two fragments of domain V of 23 S ribosomal RNA in protein folding.
(27/1306)
We have shown that the domain V of bacterial 23 S rRNA could fold denatured proteins to their active state. This segment of 23 S rRNA could further be split into two parts. One part containing mainly the central loop of domain V could bind denatured human carbonic anhydrase I stably. This association could be reversed by adding the other part of domain V. The released enzyme was directed in such a way by the central loop of domain V that it could now fold by itself to active form. This agrees with our earlier observation that proteins fold within the cell posttranslationally, a process that is completed after release of the newly synthesized polypeptide from the ribosome (Chattopadhyay, S., Pal, S., Chandra, S., Sarkar, D., and DasGupta, C. (1999) Biochim. Biophys. Acta 1429, 293-298). (+info)
Structural mapping of an aggregation nucleation site in a molten globule intermediate.
(28/1306)
Protein aggregation plays an important role in biotechnology and also causes numerous diseases. Human carbonic anhydrase II is a suitable model protein for studying the mechanism of aggregation. We found that a molten globule state of the enzyme formed aggregates. The intermolecular interactions involved in aggregate formation were localized in a direct way by measuring excimer formation between each of 20 site-specific pyrene-labeled cysteine mutants. The contact area of the aggregated protein was very specific, and all sites included in the intermolecular interactions were located in the large beta-sheet of the protein, within a limited region between the central beta-strands 4 and 7. This substructure is very hydrophobic, which underlines the importance of hydrophobic interactions between specific beta-sheet containing regions in aggregate formation. (+info)
Proton transfer to residues of basic pK(a) during catalysis by carbonic anhydrase.
(29/1306)
The maximal velocity in the hydration of CO(2) catalyzed by the carbonic anhydrases in well-buffered solutions is limited by an intramolecular proton transfer from zinc-bound water to acceptor groups of the enzyme and hence to buffer in solution. Stopped-flow spectrophotometry was used to accumulate evidence that this maximal velocity is affected by residues of basic pK(a), near 8 to above 9, in catalysis of the hydration of CO(2) by carbonic anhydrases III, IV, V, and VII. A mutant of carbonic anhydrase II containing the replacement His-64-->Ala, which removes the prominent histidine proton shuttle (with pK(a) near 7), allows better observation of these basic groups. We suggest this feature of catalysis is general for the human and animal carbonic anhydrases and is due to residues of basic pK(a), predominantly lysines and tyrosines more distant from the zinc than His-64, that act as proton acceptors. These groups supplement the well-studied proton transfer from zinc-bound water to His-64 in the most efficient of the carbonic anhydrases, isozymes II, IV, and VII. (+info)
Structure of the carbonic anhydrase VI (CA6) gene: evidence for two distinct groups within the alpha-CA gene family.
(30/1306)
The secreted carbonic anhydrase (CA VI) is believed to be one of the oldest mammalian CAs in evolutionary terms. To elucidate its gene structure and compare it with other members of the alpha-CA family, we cloned genomic fragments encoding the bovine CA6 gene and determined its exon/intron organization. The gene spans approx. 25 kb and consists of eight exons and seven introns. Exon 1 encodes the 5' untranslated region, the signal peptide and the N-terminus of the mature enzyme. Exon 8 encodes the 3' untranslated region and the C-terminal extension that is unique to CA VI. Exons 2-7 encode the CA domain, which shows significant sequence similarity to other CAs. Two distinct groups exist in the alpha-CA family on the basis of a comparison of the known gene structures. One group consists of the cytoplasmic (CA I, II, III and VII) and mitochondrial (CA V) members. The other group consists of the membrane-bound (CA IV and IX) and secreted (CA VI) members. In particular, the seven exon/intron boundaries in the CA domain of the CA6 gene are conserved in the CA9 gene, which encodes the multidomain protein CA IX that is overexpressed in tumours and has transforming potential. (+info)
Regional expression and androgen regulation of carbonic anhydrase IV and II in the adult rat epididymis.
(31/1306)
Carbonic anhydrase (CA) is implicated in the acidification of epididymal fluid and thereby in the regulation of sperm maturation and motility. Among the CA isoenzymes, CA IV and II have been shown to be present in the rat epididymal duct epithelium. In the present study, we examined the expression and androgen regulation of CA IV and II mRNAs along the epididymal duct. Northern blot analysis revealed the presence of CA II mRNA in all regions of the epididymis with the strongest signal in the corpus region, while CA IV mRNA was expressed predominantly in the corpus epididymidis. Three days after bilateral castration, CA IV and II mRNAs were decreased by 80-90% in the corpus epididymidis. Testosterone (T) replacement maintained the expression of CA mRNAs at 50-60% of the control levels, indicating that circulating androgens alone are not sufficient to recover the CA expression in the corpus region. However, unilateral castration did not affect the mRNA levels of CA IV and II, suggesting that factors in testicular fluid do not play a major role in the regulation of CA expression in the corpus epididymidis. Immunoblot analysis showed that CA IV protein levels decreased 3 days after castration, while T administration maintained the protein expression virtually at the precastration levels. These data demonstrate that mRNAs for CA IV and II are predominantly expressed in the corpus region of the rat epididymis and can be regulated by androgens in that region. The present data suggest that the regulation of CA expression in the corpus epididymidis by androgens contributes to the known androgen effects on epididymal acidification. (+info)
Carbonic anhydrase IX antigen differentiates between preneoplastic malignant lesions in non-small cell lung carcinoma.
(32/1306)
The MaTu interval (MN)/carbonic anhydrase (CA) IX tumour-associated antigen is a protein that is normally expressed in the gut and belongs to the carbonic anhydrase enzyme family (CA IX). It has been detected in tumour cell lines and in some solid tumours including cervical, oesophageal and clear cell renal carcinoma. This study determined MN/CA IX expression in 65 primary non-small cell lung cancer resected with curative intent and in 38 bronchial preneoplastic lesions, carcinoma in situ or microinvasive carcinoma as well as in normal bronchial tissue. The presence of MN/CA IX was detected using immunohistochemistry and Western blot analysis, whenever frozen material was available. Immunostaining was positive in 52/65 (80%) of the tumour samples. The staining was more often focal than diffuse. The percentage of stained cells in positive tumours was highly variable, ranging 1-85%. The pattern of immunostaining was predominantly cytoplasmic with a membranous reinforcement (87%). The intensity was mainly strong (69%). The presence of the protein in the tumour was confirmed by Western blot analysis in the eight samples tested. All the morphologically normal epithelia, except in close vicinity of tumours in some cases, as well as the preneoplastic bronchial lesions (basal cell hyperplasia, metaplasia and dysplasia) were immunonegative for MN/CA IX expression. In contrast, carcinoma in situ and microinvasive epithelioma showed the presence of MN-immunopositive tumoural cells in 5/7 and 4/5 of the samples, respectively. These data suggest that MN/CA IX is a useful marker for the differentiation between preneoplastic lesions and bronchial non-small cell lung cancer in the lung. (+info)