Analysis of a Truncated Form of Cathepsin H in Human Prostate Tumor Cells (2002)

Increased expression of proteases has been correlated with the malignant progression of a variety of tumors. We found a significant increase in cathepsin H expression in high-grade prostatic intraepithelial neoplasia and carcinoma of the prostate. Two forms of cathepsin H, the full-length form (CTSH) and a truncated form with a 12-amino acid deletion in its signal peptide region (CTSHΔ10–21), were identified by cDNA sequence analysis. This deletion occurred not at the genomic level but likely at the RNA processing level. Both forms are expressed in prostate tissues as well as LNCaP, PC-3, and DU-145 prostate cancer cell lines. The deletion within the signal peptide region affected the trafficking of cathepsin H. Fluorescence microscopy, subcellular fractionation, and activity data indicated that the truncated form was perinuclear and secreted and had a reduced lysosomal association as compared with the full-length cathepsin H. Furthermore, the truncated cathepsin H was enzymatically active. Therefore, an increase in overall cathepsin H expression, particularly in the truncated form with a high secretion propensity, may affect cell biological behaviors such as those associated with tumor progression. Increased expression of proteases has been correlated with the malignant progression of a variety of tumors. We found a significant increase in cathepsin H expression in high-grade prostatic intraepithelial neoplasia and carcinoma of the prostate. Two forms of cathepsin H, the full-length form (CTSH) and a truncated form with a 12-amino acid deletion in its signal peptide region (CTSHΔ10–21), were identified by cDNA sequence analysis. This deletion occurred not at the genomic level but likely at the RNA processing level. Both forms are expressed in prostate tissues as well as LNCaP, PC-3, and DU-145 prostate cancer cell lines. The deletion within the signal peptide region affected the trafficking of cathepsin H. Fluorescence microscopy, subcellular fractionation, and activity data indicated that the truncated form was perinuclear and secreted and had a reduced lysosomal association as compared with the full-length cathepsin H. Furthermore, the truncated cathepsin H was enzymatically active. Therefore, an increase in overall cathepsin H expression, particularly in the truncated form with a high secretion propensity, may affect cell biological behaviors such as those associated with tumor progression. Cathepsin H is a lysosomal cysteine protease and, like other members of the family, is involved in lysosomal protein degradation (1.Chapman H.A. Riese R.J. Shi G.P. Annu. Rev. Physiol. 1997; 59: 63-88Crossref PubMed Scopus (671) Google Scholar). It is synthesized as an inactive proenzyme (41 kDa) and then proteolytically processed to an active single chain, mature form (28 kDa) within the endosomes/lysosomes (2.Nishimura Y. Kato K. Biochem. Biophys. Res. Commun. 1987; 146: 159-164Crossref PubMed Scopus (22) Google Scholar). Cathepsin H is easily distinguished from other endolysosomal cysteine proteases by its unique aminopeptidase activity (3.Turk V. Turk B. Turk D. EMBO J. 2001; 20: 4629-4633Crossref PubMed Scopus (620) Google Scholar, 4.Takahashi T. Dehdarani A.H. Tang J. J. Biol. Chem. 1988; 263: 10952-10957Abstract Full Text PDF PubMed Google Scholar). The crystal structure data of mature cathepsin H (5.Guncar G. Podobnik M. Pungercar J. Strukelj B. Turk V. Turk D. Structure. 1998; 6: 51-61Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) have greatly clarified the mechanism of its limited endopeptidase activity on synthetic (1.Chapman H.A. Riese R.J. Shi G.P. Annu. Rev. Physiol. 1997; 59: 63-88Crossref PubMed Scopus (671) Google Scholar) and natural substrates (4.Takahashi T. Dehdarani A.H. Tang J. J. Biol. Chem. 1988; 263: 10952-10957Abstract Full Text PDF PubMed Google Scholar,6.Tsushima H. Ueki A. Matsuoka Y. Mihara H. Hopsu-Havu V.K. Int. J. Cancer. 1991; 48: 726-732Crossref PubMed Scopus (25) Google Scholar). Lysosomal cysteine proteases play a central role in a number of important cellular processes such as degradation of intracellular proteins, extracellular matrix remodeling, and apoptosis (7.Turk B. Turk D. Turk V. Biochim. Biophys. Acta. 2000; 1477: 98-111Crossref PubMed Scopus (676) Google Scholar, 8.Nitatori T. Sato N. Kominami E. Uchiyama Y. Adv. Exp. Med. Biol. 1996; 389: 177-185Crossref PubMed Scopus (25) Google Scholar). There is growing evidence that the expression of cathepsin H is increased in disease states including breast carcinoma (9.Gabrijelcic D. Svetic B. Spaic D. Skrk J. Budihna M. Dolenc I. Popovic T. Cotic V. Turk V. Eur. J. Clin. Chem. Clin. Biochem. 1992; 30: 69-74PubMed Google Scholar), melanoma (10.Kos J. Stabuc B. Schweiger A. Krasovec M. Cimerman N. Kopitar-Jerala N. Vrhovec I. Clin. Cancer Res. 1997; 3: 1815-1822PubMed Google Scholar), gliomas (11.Sivaparvathi M. Sawaya R. Gokaslan Z.L. Chintala S.K. Rao J.S. Chintala K.S. Cancer Lett. 1996; 104: 121-126Crossref PubMed Scopus (43) Google Scholar), and colorectal (12.del Re E.C. Shuja S. Cai J. Murnane M.J. Br. J. Cancer. 2000; 82: 1317-1326Crossref PubMed Scopus (42) Google Scholar) and prostate carcinoma (13.Friedrich B. Jung K. Lein M. Turk I. Rudolph B. Hampel G. Schnorr D. Loening S.A. Eur. J. Cancer. 1999; 35: 138-144Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In contrast, decreased cathepsin H expression has been reported in squamous cell carcinoma of the head and neck (14.Budihna M. Strojan P. Smid L. Skrk J. Vrhovec I. Zupevc A. Rudolf Z. Zargi M. Krasovec M. Svetic B. Kopitar-Jerala N. Kos J. Biol. Chem. Hoppe-Seyler. 1996; 377: 385-390Crossref PubMed Scopus (84) Google Scholar). Other studies have found a higher cathepsin H expression in well-differentiated pancreatic cancer cells compared with less well-differentiated cancer cells (15.Paciucci R. Berrozpe G. Tora M. Navarro E. Garcia de Herreros A. Real F.X. FEBS Lett. 1996; 385: 72-76Crossref PubMed Scopus (30) Google Scholar). However, the role of cathepsin H in tumor progression is not well understood. A possible function of cathepsin H in tumor progression is its ability to degrade fibrinogen and fibronectin, suggesting that, along with other proteases, cathepsin H may be involved in the destruction of extracellular matrix components leading to cancer proliferation, migration, and metastasis (6.Tsushima H. Ueki A. Matsuoka Y. Mihara H. Hopsu-Havu V.K. Int. J. Cancer. 1991; 48: 726-732Crossref PubMed Scopus (25) Google Scholar). Recently, we have compared the gene expression profiles in normal and tumor prostate tissues by serial analysis of gene expression (16.Waghray A. Schober M. Feroze F. Yao F. Virgin J. Chen Y.Q. Cancer Res. 2001; 61: 4283-4286PubMed Google Scholar). Cathepsin H was one of the genes that were overexpressed in tumor samples. In this study, we further characterize the expression pattern of cathepsin H in human prostate tumor cells. All cell lines used in this study were obtained from the American Type Culture Collection. DU-145, PC-3, and LNCaP prostate tumor cells were maintained in RPMI 1640 medium (Invitrogen) containing 5% fetal bovine serum. Human renal 293 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) with 5% fetal bovine serum. Paraffin-embedded prostate tissue sections were obtained from Harper Hospital, which is affiliated with Wayne State University. Paraffin-embedded prostate tissues were deparaffinized and stained as described previously (16.Waghray A. Schober M. Feroze F. Yao F. Virgin J. Chen Y.Q. Cancer Res. 2001; 61: 4283-4286PubMed Google Scholar). One μg of total RNA was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen) and oligo(dT). Cathepsin H cDNA was PCR-amplified using 5′-GACGCTCTGGGCCGCCACCTC-3′ and 5′-GCGGCTGCCACGGCTCACACC-3′ primers for 26 cycles at 94 °C for 30 s, 62 °C for 30 s, and 68 °C for 90 s. Nested PCR reaction was performed using 5′-ATTAGAATTCATGTGGGCCACGCTGCCGCT-3′ and 5′-GATAGCCTCGAGCACCAGAGGGATGGGGTAGG- 3′ primers targeted to the open reading frame of cathepsin H and tagged with the appropriate restriction enzyme sites for 30 cycles with the same PCR profile as described above. The cathepsin H open reading frame was cloned and sequenced. reverse transcription-PCR prostatic intraepithelial neoplasia yellow fluorescence protein reverse transcription-PCR prostatic intraepithelial neoplasia yellow fluorescence protein Briefly, 1 μg of total RNA from DU-145, PC-3, and LNCaP cells was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen). One μl of this reaction mixture was PCR-amplified using primers flanking the deleted region (see Fig. 2B for primer sequences). Genomic DNA was isolated from DU-145, PC-3, and LNCaP cells using DNAzol (Invitrogen), and 1 μl of 1:100 diluted genomic DNA was PCR-amplified using primers flanking the deleted region including the intron region (see Fig. 2B for primer sequences). PCR for both mRNA and genomic DNA was carried out in a 25-μl reaction for 30 cycles at 94 °C for 30 s, 64 °C (RT-PCR)/70 °C (genomic PCR) for 30 s, and 72 °C for 30 s. The PCR products were run on 8% polyacrylamide gels. PC-3 and 293 cells were transiently transfected using LipofectAMINE Plus reagent (Invitrogen) with pdEYFP-N1 vector, pdEYFP-N1-CTSH, or pdEYFP-N1-CTSHΔ10–21. The yellow fluorescent protein tagging vector (pdEYFP-N1) was kindly provided by Drs. R. Pepperkok and S. Wiemann (17.Simpson J.C. Wellenreuther R. Poustka A. Pepperkok R. Wiemann S. EMBO Rep. 2000; 1: 287-292Crossref PubMed Scopus (295) Google Scholar). Cells were used for different assays as described. PC-3 and 293 cells grown on 100-mm dishes were transiently transfected as mentioned above. After 48 h, the cells were viewed under a fluorescence microscope with 485 nm excitation and 520 nm emissions for the distribution of the cathepsin H-EYFP fusion protein. Western blotting was performed as described previously (18.Chen Y.Q. Cipriano S.C. Arenkiel J.M. Miller F.R. Cancer Res. 1995; 55: 4536-4539PubMed Google Scholar). A fixed amount of protein was loaded onto a 12% SDS-PAGE gel, transferred to a nitrocellulose membrane, and detected with polyclonal anti-cathepsin H antibodies (Athens Research Technology and Santa Cruz Biotechnology) or anti-green fluorescence protein antibody (CLONTECH). Purified cathepsin H (Athens Research Technology) was incubated with a fixed concentration of substrate with or without inhibitors, and its activity was measured as described previously (19.Barrett A.J. Kirschke H. Methods Enzymol. 1981; 80,: 535-561Crossref PubMed Scopus (1722) Google Scholar, 20.Keppler D. Sloane B.F. Enzyme Protein. 1996; 49: 94-105Crossref PubMed Scopus (75) Google Scholar). The different substrates used were Pyr-Arg-Thr-Lys-Arg-AMC (Peptide Institute, Inc., Lexington, KY) and Z-Phe-Arg-AMC, Z-Arg-Arg-AMC, and L-Arg-AMC (Bachem, King of Prussia, PA). Inhibitors used were CA-074, E-64 (Bachem), and Ser(O-Bzl)-CHN2, a cathepsin H-selective inhibitor kindly donated by Dr. Elliott Shaw (The Friedrich Miescher Institute, Basel, Switzerland) (21.Angliker H. Wikstrom P. Kirschke H. Shaw E. Biochem. J. 1989; 262: 63-68Crossref PubMed Scopus (29) Google Scholar). Cathepsin H enzymatic activity in different cells was determined using the cathepsin H-selective synthetic substrate L-Arg-AMC in the presence of puromycin. Cell lysates from a fixed number of DU-145, PC-3, and LNCaP cells and from 293 cells transfected with CTSH (the full-length form of cathepsin H) or CTSHΔ10–21 (a truncated form of cathepsin H with a 12-amino acid deletion in its signal peptide region) were used. Twenty μl (200,000 cells) of cell lysates were preincubated in a 30-μl assay buffer (0.1 m sodium phosphate, 4 mm EDTA, 10 mml-cysteine, 0.05 mm puromycin, and 0.05% Triton X-100, pH 6.8) at room temperature for 15 min. Cathepsin H activity was measured in a 96-well microfluor plate (Dynax) by adding 50 μl of 250 μmsubstrate to the preincubated mixture. The absorbance was read every minute for a period of 30 min at excitation/emission 355/460 nm using a Spectra Fluoroskan Plus (Tecan). For measurement of pericellular (cell surface and secreted) cathepsin H activity, transiently transfected cells were detached using Versene and resuspended in pericellular assay buffer (Hank's balanced salt solution lacking sodium bicarbonate and containing 0.6 mmCaCl2, 0.6 mm MgCl2, 2 mml-cysteine, 0.05 mm puromycin, and 25 mm Pipes, pH 7.0) at a concentration of 1 × 106 cells/ml. The enzymatic reaction for secreted cathepsin H was run as described above by adding 150 μl (150,000 cells) cell suspension/well onto 96-well plates followed by the addition of 150 μl of 250 μm substrate to the assay buffer (22.Hulkower K.I. Butler C.C. Linebaugh B.E. Klaus J.L. Keppler D. Giranda V.L. Sloane B.F. Eur. J. Biochem. 2000; 267: 4165-4170Crossref PubMed Scopus (45) Google Scholar). Transiently transfected 293 cells were washed with phosphate-buffered saline and collected by scraping into homogenization solution (0.25 m sucrose and 1 mm EDTA, pH 7.4). The cells were spun down at 1,000 rpm for 10 min at 4 °C, resuspended in 1.5 ml of homogenization solution, and lysed by repeated passage (40 times) through a 27.5-gauge needle. The homogenates were fractionated by differential centrifugation. Nuclei were collected after centrifugation at 800 × gfor 10 min at 4 °C, and the pellet was resuspended in 100 μl of homogenization buffer. The postnuclear supernatant was centrifuged at 25,000 × g for 20 min to collect mitochondria and lysosomes and then centrifuged at 100,000 × g for 30 min to obtain microsomes. The postmicrosomal supernatant represented the soluble cytosolic proteins (23.Mehtani S. Gong Q. Panella J. Subbiah S. Peffley D.M. Frankfater A. J. Biol. Chem. 1998; 273: 13236-13244Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The concentration of protein in each fraction was determined. Previous serial analysis of gene expression analysis indicated an increased expression of cathepsin H in prostate tumor tissue (16.Waghray A. Schober M. Feroze F. Yao F. Virgin J. Chen Y.Q. Cancer Res. 2001; 61: 4283-4286PubMed Google Scholar). The serial analysis of gene expression result was confirmed by immunohistochemistry. Strong staining for cathepsin H was seen in high-grade PIN and adenocarcinoma, in contrast to the weak staining seen in normal prostate glands (Fig. 1). Over 40 glands with focal or diffuse high-grade PIN were examined. All of them exhibited uniform, strong immunostaining. This was particularly evident in glands where high-grade PIN was focal (Fig. 1B). All low-grade (Fig. 1C), moderate (Fig. 1D), and high-grade (Fig. 1E) carcinomas examined showed higher levels of cathepsin H normal Cathepsin H protein expression was to the cells were for cathepsin H. from were and showed Two different forms of cathepsin H the full-length form (CTSH) and a truncated form with deletion of at the signal peptide region (CTSHΔ10–21), were identified by PCR and sequence analysis (Fig. and of the full-length cDNA were to that of cathepsin H reported previously R. Res. 1989; PubMed Scopus Google Scholar). The presence of this truncated form was for the and further confirmed by and of the PCR (Fig. of CTSH to be higher that of CTSHΔ10–21 in tissues as well as DU-145, PC-3, and LNCaP cells (Fig. the deletion was not at a we this deletion occurred at the genomic level in tumor cells. PCR of genomic DNA showed one at the full-length in of the cell lines (Fig. and of the CTSH cDNA sequence with the genomic sequence in the Human data that the cathepsin H gene has of genomic (Fig. All to the Western analysis showed that H (41 kDa) was expressed at a level in DU-145, PC-3, and LNCaP expression of single cathepsin H (28 kDa) was higher in LNCaP cells in DU-145 and PC-3 cells (Fig. In the protein was detected after an of in DU-145 and PC-3 cells (Fig. Therefore, the total amount of cathepsin H protein was higher in LNCaP cells as compared with that in DU-145 and PC-3 cells. the by Western cathepsin H activity was compared in cell lines. the of assay we used human cathepsin H and incubated cathepsin H with different Z-Phe-Arg-AMC, Z-Arg-Arg-AMC, and L-Arg-AMC (19.Barrett A.J. Kirschke H. Methods Enzymol. 1981; 80,: 535-561Crossref PubMed Scopus (1722) Google Scholar). confirmed the of L-Arg-AMC cathepsin H (Fig. we that cathepsin H activity was by the (Fig. This inhibitor was previously to be for cathepsin H B. I. P. Turk V. FEBS Lett. 1996; PubMed Scopus Google Scholar). a cysteine protease was less in cathepsin H of this inhibitor was to cathepsin H activity (Fig. The enzyme was not to the cathepsin inhibitor (Fig. the above assay with cell lysates from LNCaP we that the measured enzyme activity in cell lysates was by but not by (Fig. the of we determined the cathepsin H activity in DU-145, PC-3, and LNCaP cells. The cathepsin H activity in DU-145, PC-3, and LNCaP cells was and 2 (Fig. PC-3 and 293 cells were transfected with CTSH and CTSHΔ10–21 tagged with or transfected with tagging CTSH protein distribution showed a pattern in the (Fig. CTSHΔ10–21 was in the region of the was the in cells (Fig. that CTSHΔ10–21 protein had a different Cells transfected with the vector exhibited diffuse fluorescence in both the and (17.Simpson J.C. Wellenreuther R. Poustka A. Pepperkok R. Wiemann S. EMBO Rep. 2000; 1: 287-292Crossref PubMed Scopus (295) Google Scholar). the transfected 293 cells were fractionated by differential centrifugation. The to and were for the presence of cathepsin proteins (Fig. both forms of cathepsin H were detected in and the level of CTSHΔ10–21 higher in the fraction and in the fraction (Fig. were seen in the fraction (Fig. the to be of cathepsin H, that of the cathepsin H proteins are the deletion of in the signal peptide region the intracellular trafficking of cathepsin H. Western analysis detected cathepsin fusion proteins kDa) in both CTSH and CTSHΔ10–21 transiently transfected 293 cells. The protein in CTSHΔ10–21 was that in to the of (Fig. indicated that cells expressed and at a level. cathepsin H activity is affected to the deletion in the signal peptide we measured the cellular and pericellular (cell surface and secreted) activity in transfected 293 cells. cathepsin H activity was 48 and 1 and pericellular activity was 10 10 and 2 cells in and CTSHΔ10–21 transfected cathepsin H activity was higher in cells compared with cells. In contrast, the pericellular cathepsin H activity was higher in cells in cells (Fig. CTSH expression in an increased cathepsin H activity in cell CTSHΔ10–21 expression to cathepsin H activity (Fig. that the truncated cathepsin H is enzymatically active. In this study, we have the presence of forms of cathepsin H in prostate tissues and cancer cell the full-length form (CTSH) and a truncated form with a deletion of in the signal peptide region characterize different forms of cathepsin H, CTSH and CTSHΔ10–21 cDNA were transfected into PC-3 and 293 cells. and cell data indicated that full-length cathepsin H was associated with and in with V. A. T. T. Kirschke H. H. G. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, the truncated cathepsin H was in the perinuclear the in and reduced in the lysosomal mRNA of CTSHΔ10–21 was expressed at a level that of CTSH in the prostate tissues and tumor cell CTSHΔ10–21 protein to be secreted from transfected cells and was active. Therefore, is that this secreted CTSHΔ10–21 may play a role in tumor are to the role of this truncated form of cathepsin H in prostate cancer progression. form of cathepsin that its signal peptide and of the acid was reported previously L. D. Sloane B.F. 1995; PubMed Scopus (61) Google Scholar). This truncated form of cathepsin was to be associated with and other (23.Mehtani S. Gong Q. Panella J. Subbiah S. Peffley D.M. Frankfater A. J. Biol. Chem. 1998; 273: 13236-13244Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). of CTSHΔ10–21 is to that of the truncated cathepsin B. It is CTSHΔ10–21 is through analysis indicated that the deletion was not at a In genomic PCR data showed deletion at the DNA level. CTSHΔ10–21 may be the result of through an including serial analysis of gene expression have an increased expression of cathepsin H in tumor cells and tissues (9.Gabrijelcic D. Svetic B. Spaic D. Skrk J. Budihna M. Dolenc I. Popovic T. Cotic V. Turk V. Eur. J. Clin. Chem. Clin. Biochem. 1992; 30: 69-74PubMed Google Scholar, J. Stabuc B. Schweiger A. Krasovec M. Cimerman N. Kopitar-Jerala N. Vrhovec I. Clin. Cancer Res. 1997; 3: 1815-1822PubMed Google Scholar, M. Sawaya R. Gokaslan Z.L. Chintala S.K. Rao J.S. Chintala K.S. Cancer Lett. 1996; 104: 121-126Crossref PubMed Scopus (43) Google Scholar, Re E.C. Shuja S. Cai J. Murnane M.J. Br. J. Cancer. 2000; 82: 1317-1326Crossref PubMed Scopus (42) Google Scholar, B. Jung K. Lein M. Turk I. Rudolph B. Hampel G. Schnorr D. Loening S.A. Eur. J. Cancer. 1999; 35: 138-144Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). analysis an increased expression of cathepsin H in intraepithelial and prostate Increased expression of cathepsin H along with cathepsin and A. E. N. Sloane B.F. Shi G.P. H.A. E. A. Int. J. Cancer. 2001; PubMed Scopus Google Scholar) in high-grade PIN that proteins may play an important role in the of prostate cancer progression. Furthermore, higher levels of cathepsin H, the secreted in carcinoma cells may to tumor We found a level of cathepsin H mRNA in LNCaP, DU-145, and PC-3 prostate cancer cells. However, LNCaP cells showed the protein expression and DU-145 and PC-3 cells had but levels of protein expression and activity compared with LNCaP cells. activity were different from those described previously (13.Friedrich B. Jung K. Lein M. Turk I. Rudolph B. Hampel G. Schnorr D. Loening S.A. Eur. J. Cancer. 1999; 35: 138-144Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), in which DU-145 had the It is the is a result of different assay or cell It is that the high level of cathepsin H protein in LNCaP (Fig. cathepsin H activity was higher in LNCaP cells that in and PC-3 cells (Fig. This be by the presence of cathepsin H in activity For has been to be a strong cathepsin H inhibitor B. I. P. Turk V. FEBS Lett. 1996; PubMed Scopus Google Scholar, R. L. Popovic T. Turk V. Eur. J. Biochem. PubMed Scopus Google Scholar). Lysosomal cysteine proteases were to be involved in intracellular protein However, a role for cysteine proteases, such as a role in II extracellular matrix remodeling, and tumor (1.Chapman H.A. Riese R.J. Shi G.P. Annu. Rev. Physiol. 1997; 59: 63-88Crossref PubMed Scopus (671) Google Scholar, V. Turk B. Turk D. EMBO J. 2001; 20: 4629-4633Crossref PubMed Scopus (620) Google B. Turk D. Turk V. Biochim. Biophys. Acta. 2000; 1477: 98-111Crossref PubMed Scopus (676) Google Scholar, J. D.M. Sloane B.F. Biochim. Biophys. Acta. 2000; 1477: PubMed Scopus Google Scholar, S. M. Sloane B.F. Biol. Chem. 1998; Google Scholar, M. K. Sloane B.F. 2000; PubMed Scopus Google Scholar). of form or truncated level of in the function of cysteine proteases in cell