Cathepsin B is an intracellular lysosomal cysteine peptidase belonging to the papain superfamily (Turk et al., 1996). It is expressed in all mammalian tissues. It is responsible for degrading internalized proteins and by this it contributes to the amino acid supply of the cell. It is accepted that cathepsin B is involved in the pathogenesis of various diseases such as inflammatory processes, bone disorders and first of all cancer. During tumor progression, the enzyme is secreted and associated with the plasma membrane of metastatic cells. Cathepsin B is responsible for the degradation of the basal lamina during initial stages of invasion of colon, mamma, pancreas and prostate carcinoma cells [Campo et al., 1994; Schwartz, 1995; Mai et al., 2000b; Sinha et al., 2002]. In these cases, cathepsin B is secreted and associated with the plasma membrane of infiltrating cells and seems to be responsible for degradation of extracellular matrix (ECM) components, in particular laminin, fibrinogen, fibronectin, collagen IV and a variety of proteoglycans.
The human cathepsin B gene is located on chromosome 8p22. cathepsins B, like MMPs and serine proteases, are upregulated and down regulated in number of diseases. Its expression is altered by various ways, one mechanism by which its expression changes is through binding of transcription factors on the promoter region of this gene. There is little information available on the promoter region of this gene. Therefore present study was undertaken to clone the human cathepsin B promoter.
Cathepsin B promoter has the putative binding sites for SP1 ,AP-1, and Nf-kappa B (Sandra Bien, et al 2004) similarly cathepsin s promoter has the binding sites for transcription factors like IRF 1,IRF2 ap1 and sp1 .These transcription factors are activated by specific stimulus during pathological conditions e.g AP-1 is activated by mitogen activated protein kinase pathway which in turn is stimulated by (PCBs) poly chlorinated biphenyls these are persistent organic pollutants SP family of transcription factors are activated by valporate drug used in the treatment of mania and bipolar disorders ,NF-kaap B is activated by lactate dithiocarbamate and diethyldithiocarbamate curcumin inhibits NF kappa B .One of the well known chemical acetaldehyde is known to activate number of transcription factors.
Lysosomal Cathepsins
Lysosomal cysteine cathepsins belong, besides papain and related plant peptidases,to the papain superfamily (Barrett et al.,1988). All peptidases of the papain superfamily share the motif of two globular domains, which build the active site cleft consisting of the amino acids cysteine and histidine. Most papain-like cathepsins work as endopeptidases. All cathepsins are synthesized at the endoplasmic reticulum and then further transported to the Golgi apparatus. In the cis-Golgi, mannose residues are phosphorylated and then they are detected in the trans-Golgi by a mannose-6-receptor thereby, cathepsins are sorted into lysosomes and not into secretory vesicles.
In the lysosomes the maturation into the active enzyme occurs by either autocatalytic processing or by cleaving of the propeptide by other peptidases like cathepsin D or legumain (Kirschke et al.,1995; Brix, 2005). From their discovery in the first half of twenty century lysosomal cysteine proteases have come a long way: from being the enzymes non selectively degrading proteins in lysosomes to being those responsible for a number of important cellular and pathological processes. Among these proteases cathepsin l and b are major contributors of these processes their activity is regulated both at protein level as well as at mRNA level their expression also depends on external environment.
Preparation of Competent Cell
DH5α strain of E. coli was used as host for performing all the transformations. Procedure used for the preparation of competent cells was a variation of Cohen et al., 1972. At first, single, fresh colony of the host cells was inoculated into 5 ml sterile LB (Luria-Bertaini) medium and grown overnight at 37°C whilst shaking at 150 rpm in a shaker incubator (Lab Line Instruments Inc., Illinois, and USA). 200 μl of this overnight cell culture was aseptically transferred to 25 ml of LB medium in a 100 ml conical flask and incubated at 37°C whilst shaking at 150 rpm till OD (A600) of the culture reaches 0.3-0.4 (usually after about 3 hours). Then the cell suspension was transferred to a 50 ml centrifuge tube, chilled on ice for 5-10 minutes and bacterial cells were pelleted down by centrifugation at 5000Xg for 10 minutes at 4°C in a Sorvall RC5B centrifuge (DuPont Instruments, USA).
The pellet was thoroughly resuspended in 12.5 ml of ice cold CaCl2 (50mM) by vortexing and incubated on ice for 45mins-1hour after which the cell suspension was again centrifuged at 3000X g for 20 minutes at 4°C. The supernatant was discarded and the cell pellet was gently resuspended in 2.5 ml of ice-cold 50 mM CaCl2. These processed bacterial cells remained competent for 24 hours and therefore was either used for transformation within 24 hours or had to be stored at –70°C as 200 μl aliquots after adding sterile glycerol to a final concentration of 15% (v/v). For transformations, the frozen competent cell aliquots was thawed on ice and used immediately.
Transformation
Bacterial transformation is the process by which bacterial cells take up naked DNA molecules. In the present study, all transformations were performed according to the method of Mandel and Higa, 1970. At first, a suitable aliquot (2-5 μl) of the ligation mixture was gently mixed with a 200-μl aliquot of competent cells in a sterile microcentrifuge tube. After incubation on ice for 30-45 minutes, a brief heat shock at 42°C in a water bath for exactly 90 seconds was given to assist the entry of the plasmid DNA into the cells, followed by a quick chill on ice for 5 minutes.
Then 800 μl of LB broth was added to the mixture and the cells were allowed to rejuvenate at 37°C, whilst shaking at 150 rpm for an hour. This step allows the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid. 200-μl aliquot of cells was plated onto 100 ml LB agar plates containing 100 μg ampicillin/ml media. Then the plates were incubated for 12-16 hours at 37°C. The following day, colonies were picked, grown in LB broth containing 1 μl ampicillin/ml media and subjected to screening after isolation of the plasmid DNA.
Procedure
Procedure used for the preparation of competent cells was a variation of Cohen et al., 1972. At first, single, fresh colony of the host cells was inoculated into 5 ml sterile LB (Luria-Bertaini) medium and grown overnight at 37°C whilst shaking at 150 rpm in a shaker incubator (Lab Line Instruments Inc., Illinois, and USA). 200 μl of this overnight cell culture was aseptically transferred to 25 ml of LB medium in a 100 ml conical flask and incubated at 37°C whilst shaking at 150 rpm till OD (A600) of the culture reaches 0.3-0.4 (usually after about 3 hours). Then the cell suspension was transferred to a 50 ml centrifuge tube, chilled on ice for 5-10 minutes and bacterial cells were pelleted down by centrifugation at 5000Xg for 10 minutes at 4°C in a Sorvall RC5B centrifuge (DuPont Instruments, USA).
The pellet was thoroughly resuspended in 12.5 ml of ice cold CaCl2 (50mM) by vortexing and incubated on ice for 45mins-1hour after which the cell suspension was again centrifuged at 3000X g for 20 minutes at 4°C. The supernatant was discarded and the cell pellet was gently resuspended in 2.5 ml of ice-cold 50 mM CaCl2. These processed bacterial cells remained competent for 24 hours and therefore was either used for transformation within 24 hours or had to be stored at –70°C as 200 μl aliquots after adding sterile glycerol to a final concentration of 15% (v/v). For transformations, the frozen competent cell aliquots was thawed on ice and used immediately.
Conclusion
consists of 13 exons, and the gene encompasses approximately 27 kb. Homology search for cathepsin B nucleotide sequence with chromosome 8 nucleotide sequence was performed on the NCBI BLAST server at www.ncbi.nlm.nih.gov. The homologous sequence obtained was then used to design strategy for further work. The sequence Shown below is1.64 kb long and contains exon one and upstream region of cathepsin B gene on chromosome 8. The region from +520 is upstream region of exon one and intron 1 and downstream to this is upstream region of cathepsin B.