Nuclear Cathepsin Research Articles

Cathepsin X deficiency alters the processing and localisation of cathepsin L and impairs cleavage of a nuclear cathepsin L substrate.

Proteases function within sophisticated networks. Altering the activity of one protease can have sweeping effects on other proteases, leading to changes in their activity, structure, specificity, localisation, stability, and expression. Using a suite of chemical tools, we investigated the impact of cathepsin X, a lysosomal cysteine protease, on the activity and expression of other cysteine proteases and their inhibitors in dendritic cells. Among all proteases examined, cathepsin X gene deletion specifically altered cathepsin L levels; pro-cathepsin L and its single chain accumulated while the two-chain form was unchanged. This effect was recapitulated by chemical inhibition of cathepsin X, suggesting a dependence on its catalytic activity. We demonstrated that accumulation of pro- and single chain cathepsin L was not due to a lack of direct cleavage by cathepsin X or altered glycosylation, secretion, or mRNA expression but may result from changes in lysosomal oxidative stress or pH. In the absence of active cathepsin X, nuclear cathepsin L and cleavage of the known nuclear cathepsin L substrate, Lamin B1, were diminished. Thus, cathepsin X activity selectively regulates cathepsin L, which has the potential to impact the degree of cathepsin Lproteolysis, the nature of substrates that it cleaves, and the location of cleavage.

Cystatin B deficiency results in sustained histone H3 tail cleavage in postnatal mouse brain mediated by increased chromatin-associated cathepsin L activity.

Cystatin B (CSTB) is a cysteine cathepsin inhibitor whose biallelic loss-of-function mutations in human result in defects in brain development and in neurodegeneration. The physiological function of CSTB is largely unknown, and the mechanisms underlying the human brain diseases remain poorly understood. We previously showed that CSTB modulates the proteolysis of the N-terminal tail of histone H3 (H3cs1) during in vitro neurogenesis. Here we investigated the significance of this mechanism in postnatal mouse brain. Spatiotemporal analysis of H3cs1 intensity showed that while H3cs1 in wild-type (wt) mice was found at varying levels during the first postnatal month, it was virtually absent in adult brain. We further showed that the high level of H3cs1 coincides with chromatin association of de novo synthesized cathepsin L suggesting a role for nuclear cathepsin L in brain development and maturation. On the contrary, the brains of Cstb -/- mice showed sustained H3cs1 proteolysis to adulthood with increased chromatin-associated cathepsin L activity, implying that CSTB regulates chromatin-associated cathepsin L activity in the postnatal mouse brain. As H3 tail proteolysis has been linked to cellular senescence in vitro, we explored the presence of several cellular senescence markers in the maturing Cstb -/- cerebellum, where we see increased levels of H3cs1. While several markers showed alterations in Cstb -/- mice, the results remained inconclusive regarding the association of deficient CSTB function with H3cs1-induced senescence. Together, we identify a molecular role for CSTB in brain with implications for brain development and disease.

Trafficking of Full-Length and N-Terminally Truncated Cathepsin B in Human Colorectal Carcinoma Cells

Cathepsin B is an endo-lysosomal cysteine protease. However, its increased expression and altered localization to the extracellular space, to mitochondria, or to the nucleus has been linked to tumor progression. In the present study, we show enhanced levels of cathepsin B in adenocarcinoma tissue in comparison to adjacent normal colon. Additionally, cathepsin B was observed in the nuclear compartment of mucosal cells in adenocarcinoma tissue samples and in the nuclei of the colorectal carcinoma cell line HCT116. Accordingly, a distinct 40-kDa form of cathepsin B was detected in HCT116 cells, which is proposed to represent a specific form lacking the signal peptide and parts of the propeptide. Trafficking studies with an EGFP-tagged N-terminally truncated form, mimicking the 40-kDa form, demonstrated accumulation in aggresome-like inclusion bodies, while EGFP-tagged full-length cathepsin B revealed regular sorting to endo-lysosomes. We conclude that the identity of nuclear cathepsin B in colorectal adenocarcinoma (in situ) and in carcinoma cells (in vitro) cannot be attributed to either full-length or 40-kDa N-terminally truncated cathepsin B forms. Hence, future studies are needed to demonstrate which form/s of cathepsin B may be sorted to the nuclei of colorectal carcinoma cells, and whether redundant regulation of related cathepsin expression occurs.

Correction for Burton et al., “Targeting the Nuclear Cathepsin L CCAAT Displacement Protein/Cut Homeobox Transcription Factor-Epithelial Mesenchymal Transition Pathway in Prostate and Breast Cancer Cells with the Z-FY-CHO Inhibitor”

This article refers to:Targeting the Nuclear Cathepsin L CCAAT Displacement Protein/Cut Homeobox Transcription Factor-Epithelial Mesenchymal Transition Pathway in Prostate and Breast Cancer Cells with the Z-FY-CHO Inhibitor

Studying the Genomic Role of Cathepsin-D in ER+ Breast Cancer

Estrogen receptor alpha (ERα), a transcription factor implicated in induction of cell growth in breast cancer, is a therapeutic target that is expressed in >70% of breast tumors. The transcriptional activity of ERα is controlled by ligands and increased through its interaction with co-activators such as the p160/SRC and p300/CBP families. In an attempt to identify the ligand-specific protein complexes involved in transcriptional regulation by ERα, BioID and TurboID screens were performed in two ER+ breast cancer cell lines, T-47D and ZR-75-1. Surprisingly, Cathepsin-D (Cath-D), a lysosomal aspartyl endoproteinase that is an ER target gene, was identified in these screens. Cath-D expression is associated with a poor prognosis and increased metastasis rate in breast cancer irrespective of its catalytic activities {Glondu, 2001 #119}[i]. Cath-D is localized in part to the nucleus where it interacts with TRPS1, a repressor of GATA-mediated transcription and modulator of ERα signaling {Bach, 2015 #117}[ii]. Co-silencing Cath-D and TRPS1 suppressed cell proliferation and inhibited growth under soft agar, suggesting that they cooperate to drive tumorigenesis {Bach, 2015 #117}[ii]. We hypothesized that Cath-D plays genomic as well as non-genomic roles in breast tumor aggressiveness and may alter ERα-mediated transcription. The nuclear localization of Cath-D was confirmed by immunofluorescence using different commercialized antibodies and observed in western blots of chromatin-bound fractions in three different ERα+ breast cancer cell lines, T-47D, ZR-75 and MCF-7. Specificity of the antibodies was confirmed using siRNA-mediated suppression of Cath-D. Moreover, Cath-D was also identified in proximity to TurboID-ERα by LC-MS after chromatin fractionation. The proximity of ERα and Cath-D both in the cytoplasm and nucleus was confirmed by proximity Ligation Assay (PLA) in three ER+ cell lines. Co-immunoprecipitation assays indicated physical interaction of Cath-D with ERα in T-47D cell extracts. Further, Cath-D was detected by ChIP-qPCR on estrogen response elements (EREs) of two ERα target genes, TFF1 and GREB1 in T-47D and ZR-75 cells. These results suggest that Cath-D can interact with ERα on DNA and play genomic roles in ER+ breast cancer cells. [i] Glondu, M., et al. (2001). “A mutated cathepsin-D devoid of its catalytic activity stimulates the growth of cancer cells.” Oncogene20(47): 6920-6929. [ii] Bach, A. S., et al. (2015). “Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells.” Oncotarget6(29): 28084-28103.

Significance of nuclear cathepsin V in normal thyroid epithelial and carcinoma cells

Altered expression and/or localization of cysteine cathepsins is believed to involve in thyroid diseases including cancer. Here, we examined the localization of cathepsins B and V in human thyroid tissue sections of different pathological conditions by immunolabeling and morphometry. Cathepsin B was mostly found within endo-lysosomes as expected. In contrast, cathepsin V was detected within nuclei, predominantly in cells of cold nodules, follicular and papillary thyroid carcinoma tissue, while it was less often detected in this unusual localization in hot nodules and goiter tissue. To understand the significance of nuclear cathepsin V in thyroid cells, this study aimed to establish a cellular model of stable nuclear cathepsin V expression. As representative of a specific form lacking the signal peptide and part of the propeptide, N-terminally truncated cathepsin V fused to eGFP recapitulated the nuclear localization of endogenous cathepsin V throughout the cell cycle in Nthy-ori 3-1 cells. Interestingly, the N-terminally truncated cathepsin V-eGFP was more abundant in the nuclei during S phase. These findings suggested a possible contribution of nuclear cathepsin V forms to cell cycle progression. Indeed, we found that N-terminally truncated cathepsin V-eGFP expressing cells were more proliferative than those expressing full-length cathepsin V-eGFP or wild type controls. We conclude that a specific molecular form of cathepsin V localizes to the nucleus of thyroid epithelial and carcinoma cells, where it might involve in deregulated pathways leading to hyperproliferation. These findings highlight the necessity to better understand cathepsin trafficking in health and disease. In particular, cell type specificity of mislocalization of cysteine cathepsins, which otherwise act in a functionally redundant manner, seems to be important to understand their non-canonical roles in cell cycle progression.

Lost or Forgotten: The nuclear cathepsin protein isoforms in cancer.

While research into the role of cathepsins has been progressing at an exponential pace over the years, research into their respective isoform proteins has been less frenetic. In view of the functional and biological potential of such protein isoforms in model systems for cancer during their initial discovery, much later they have offered a new direction in the field of cathepsin basic and applied research. Consequently, the analysis of such isoforms has laid strong foundations in revealing other important regulatory aspects of the cathepsin proteins in general. In this review article, we address these key aspects of cathepsin isoform proteins, with particular emphasis on how they have shaped what is now known in the context of nuclear cathepsin localization and what potential these hold as nuclear-based therapeutic targets in cancer.

Targeting the Nuclear Cathepsin L CCAAT Displacement Protein/Cut Homeobox Transcription Factor-Epithelial Mesenchymal Transition Pathway in Prostate and Breast Cancer Cells with the Z-FY-CHO Inhibitor.

The epithelial mesenchymal transition (EMT) promotes tumor migration and invasion by downregulating epithelial markers such as E-cadherin and upregulating mesenchymal markers such as vimentin. Cathepsin L (Cat L) is a cysteine protease that can proteolytically activate CCAAT displacement protein/cut homeobox transcription factor (CUX1). We hypothesized that nuclear Cat L may promote EMT via CUX1 and that this could be antagonized with the Cat L-specific inhibitor Z-FY-CHO. Mesenchymal prostate (ARCaP-M and ARCaP-E overexpressing Snail) and breast (MDA-MB-468, MDA-MB-231, and MCF-7 overexpressing Snail) cancer cells expressed lower E-cadherin activity, higher Snail, vimentin, and Cat L activity, and a p110/p90 active CUX1 form, compared to epithelial prostate (ARCaP-E and ARCaP-Neo) and breast (MCF-7 and MCF-7 Neo) cancer cells. There was increased binding of CUX1 to Snail and the E-cadherin promoter in mesenchymal cells compared to epithelial prostate and breast cells. Treatment of mesenchymal cells with the Cat L inhibitor Z-FY-CHO led to nuclear-to-cytoplasmic relocalization of Cat L, decreased binding of CUX1 to Snail and the E-cadherin promoter, reversed EMT, and decreased cell migration/invasion. Overall, our novel data suggest that a positive feedback loop between Snail-nuclear Cat L-CUX1 drives EMT, which can be antagonized by Z-FY-CHO. Therefore, Z-FY-CHO may be an important therapeutic tool to antagonize EMT and cancer progression.

Abstract 1602: Snail transcription factor regulates nuclear cathepsin L activity

Abstract Triple negative breast cancer (TNBC) are defined as tumors lacking the expression of estrogen receptor-alpha, progesterone receptor and human epidermal growth factor receptor-2, which accounts for approximately 15% of total breast cancer patients, and is more prevalent and lethal among young African, African-American and Latino women patients. The primary cause of breast cancer death is metastasis, which is regulated by factors such as epithelial mesenchymal transition (EMT), a dynamic process that promotes cell motility and decreases cell-cell adhesion. Snail transcription factor promotes EMT by repressing epithelial genes such as E-cadherin, while increasing mesenchymal genes such as vimentin. Cathepsin L (Cat L) cysteine protease promotes cell invasion and nuclear Cat L has recently been associated with poor prognosis in colon and breast cancer. However, the mechanism by which Cat L localizes to the nucleus is unknown. CDP/CUX transcription factor is a substrate for Cat L which when proteolytically cleaved by Cat L from the p200 to the p110 isoform can transcriptionally activate Snail and repress E-cadherin. We have recently published that Snail overexpression increases Cat L activity in prostate cancer cells which can be antagonized by muscadine grape skin extract (MSKE), a natural product rich in anthocyanin. We hypothesize that a feedback loop exists whereby Snail Transcription factor may promote nuclear Cat L expression and activity in TNBC which subsequently cleaves CDP/CUX and further increases Snail transcription, leading to increased EMT; this can be abrogated by Cat L inhibitor or MSKE. We utilized various breast cancer cell lines as well as MCF-7 cells stably overexpressing Snail (MCF-7 Snail) or control (MCF-7 Neo) to evaluate the expression of Snail, Cat L, CDP/CUX and EMT marker expression (E-cadherin, vimentin) by western blot and immunofluorescence. Migration and invasion assays were performed including treatments with Cat L specific inhibitor (Z-FY-CHO) or MSKE. We observed more Snail, Cat L and CDP/CUX cleavage products (p110 and p90 isoforms) expression in MCF-7 Snail cells and TNBC cells (MDA-MB-231, MDA-MB-468) as compared to MCF-7 control cells. Interestingly, immunofluorescent and cell fractionation analyses revealed that Snail overexpression promoted nuclear Cat L and CDP/CUX p110 isoform expression, and EMT (low E-cadherin, high vimentin, migration and invasion) which could be abrogated by Z-FY-CHO or MSKE. We are currently staining for Snail and Cat L in breast cancer tissue of varying races/grades including TNBC, as well as confirming by chromatin immunoprecipitation that the CDP/CUX cleavage products seen in TNBC can bind Snail and/or E-cadherin promoters. In conclusion, our study shows that Snail promotes nuclear localization of Cat L which may promote EMT via CDP/CUX, and that inhibition with Cat L inhibitors or natural products such as MSKE may be a good therapeutic target for TNBC. Citation Format: Liza J. Burton, Valerie Odero-Marah. Snail transcription factor regulates nuclear cathepsin L activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1602.

Abstract B93: Nuclear cathepsin L (cat L) is associated with epithelial mesenchymal transition (emt) in prostate cancer cells

Abstract Prostate cancer (PCa) is the most frequently diagnosed cancer in men, the second leading cause of male cancer deaths in the U.S, and also presents the greatest racial disparity of any cancer in the U.S. The incidence and mortality of PCa is higher in African-American men (AA), compared to other ethnic groups. The Epithelial Mesenchymal Transition (EMT) is a key event in tumor migration and invasion processes whereby epithelial cells lose polarity together with cell-cell contacts associated with loss of epithelial markers such as E-cadherin and then undergo a dramatic remodeling of the cytoskeleton associated with expression of mesenchymal markers such as vimentin, as well as increased production of extracellular matrix (ECM)-degrading proteases. Snail promotes EMT by using its zinc finger domains to repress epithelial-associated target genes such as E-cadherin and occludin. Cathepsin L (Cat L) is a cysteine protease involved in cell invasion and has recently been associated with poor prognosis in colon and breast cancer when Cat L is localized in the nucleus. CDP/CUX can be proteolytically activated by Cat L from p200 inactive form to p110 and p90 active forms, and subsequently repress E-cadherin and activate Snail by binding to their promoter regions. We recently published that Snail could up-regulate Cat L expression and activity in prostate cancer cells. We hypothesized that nuclear Cat L may promote EMT and prostate cancer progression and that antagonizing Cat L activity would lead to Mesenchymal Epithelial Transition (MET). We stained African American and Caucasian human normal and prostate cancer tissue from the Fox Chase Cancer Center for Snail and Cat L to examine expression. We utilized prostate cancer cell lines and EMT models to analyze EMT markers and Cat L expression by western blot analysis, Q-PCR and immunofluorescence. We also examined Cat L activity using gelatin zymography and subsequently treated mesenchymal cells with 1 µM, 5 µM or 20 µM Z-FY-CHO (Cat L specific inhibitor) for 72 hours. We also performed invasion and migration assays. Staining of human prostate patient tissue revealed that Snail and Cat L levels were lower in normal tissue vs cancer tissue and nuclear localization of Cat L appeared in earlier stages of prostate cancer in African American tissue compared to that of Caucasian cancer tissue. Interestingly, analysis of epithelial (ARCaP-E) and mesenchymal (ARCaP-M or ARCaP-E overexpressing Snail) prostate cancer cells revealed that mesenchymal cells displaying higher Snail expression showed increased Cat L expression and activity, in addition to Cat L being localized in the cytoplasm and nucleus, whereas Cat L was predominantly cytoplasmic in epithelial cells with low Snail expression. Additionally, the African American cell lines (E006-AA and E006-AA ht) displayed high Snail and Cat L expression/activity compared to LNCaP cells. Mesenchymal cells expressed higher p110 and p90 active form of CDP/CUX than epithelial cells that expressed the p200 inactive form. Additionally, Snail overexpression increased p110/90 CDP/CUX in the nuclear fraction of ARCaP-E overexpressing Snail cells. Treatment of mesenchymal cells with Cat L inhibitor, Z-FY-CHO resulted in decreased vimentin and Cat L expression, while E-cadherin increased, suggestive of MET. This was associated with decreased cell migration and invasion. We further observed that treatment with Z-FY-CHO changes the localization of Cat L from nuclear to cytoplasmic. Overall, our novel data suggests that Snail may promote nuclear Cat L that may be associated with proteolytic degradation of CDP/CUX and EMT, and the inhibition of Cat L could play a role in reverting EMT and preventing tumor progression. Citation Format: Liza J. Burton, Jodi Dougan, Valerie Odero-Marah. Nuclear cathepsin L (cat L) is associated with epithelial mesenchymal transition (emt) in prostate cancer cells. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B93.

Nuclear cathepsin L activity is required for cell cycle progression of colorectal carcinoma cells.

Prominent tasks of cysteine cathepsins involve endo-lysosomal proteolysis and turnover of extracellular matrix constituents or plasma membrane proteins for maintenance of intestinal homeostasis. Here we report on enhanced levels and altered subcellular localization of distinct cysteine cathepsins in adenocarcinoma tissue in comparison to adjacent normal colon. Immunofluorescence and immunoblotting investigations revealed the presence of cathepsin L in the nuclear compartment in addition to its expected endo-lysosomal localization in colorectal carcinoma cells. Cathepsin L was represented as the full-length protein in the nuclei of HCT116 cells from which stefin B, a potent cathepsin L inhibitor, was absent. Fluorescence activated cell sorting analyses with synchronized cell cultures revealed deceleration of cell cycle progression of HCT116 cells upon inhibition of cathepsin L activity, while expression of cathepsin L-enhanced green fluorescent protein chimeras accelerated S-phase entry. We conclude that the activity of cathepsin L is high in the nucleus of colorectal carcinoma cells because of lacking stefin B inhibitory activity. Furthermore, we hypothesize that nuclear cathepsin L accelerates cell cycle progression of HCT116 cells thereby supporting the notion that cysteine cathepsins may play significant roles in carcinogenesis due to deregulated trafficking.

Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells.

The lysosomal protease cathepsin D (Cath-D) is overproduced in breast cancer cells (BCC) and supports tumor growth and metastasis formation. Here, we describe the mechanism whereby Cath-D is accumulated in the nucleus of ERα-positive (ER+) BCC. We identified TRPS1 (tricho-rhino-phalangeal-syndrome 1), a repressor of GATA-mediated transcription, and BAT3 (Scythe/BAG6), a nucleo-cytoplasmic shuttling chaperone protein, as new Cath-D-interacting nuclear proteins. Cath-D binds to BAT3 in ER+ BCC and they partially co-localize at the surface of lysosomes and in the nucleus. BAT3 silencing inhibits Cath-D accumulation in the nucleus, indicating that Cath-D nuclear targeting is controlled by BAT3. Fully mature Cath-D also binds to full-length TRPS1 and they co-localize in the nucleus of ER+ BCC where they are associated with chromatin. Using the LexA-VP16 fusion co-activator reporter assay, we then show that Cath-D acts as a transcriptional repressor, independently of its catalytic activity. Moreover, microarray analysis of BCC in which Cath-D and/or TRPS1 expression were silenced indicated that Cath-D enhances TRPS1-mediated repression of several TRPS1-regulated genes implicated in carcinogenesis, including PTHrP, a canonical TRPS1 gene target. In addition, co-silencing of TRPS1 and Cath-D in BCC affects the transcription of cell cycle, proliferation and transformation genes, and impairs cell cycle progression and soft agar colony formation. These findings indicate that Cath-D acts as a nuclear transcriptional cofactor of TRPS1 to regulate ER+ BCC proliferation and transformation in a non-proteolytic manner.

Out-of-frame start codons prevent translation of truncated nucleo-cytosolic cathepsin L in vivo

The lysosomal protease cathepsin L has been reported to cleave various functionally important cytosolic or nuclear proteins. To explain nucleo-cytosolic localization of cathepsin L, it has been hypothesized that skipping of the first start codon during translation initiation results in an N-terminally truncated protein lacking the endoplasmic reticulum-import signal. Here we demonstrate that out-of-frame AUGs prevent translation of truncated cathepsin L in cell culture as well as in a new knock-in mouse model. We further evaluate potential roles of nuclear cathepsin L during early embryonic development. Our analysis reveals normal epiblast development of cathepsin L-deficient embryos, but uncovers a pronounced lysosomal storage phenotype in the extra-embryonic tissue of the visceral endoderm. In conclusion, the phenotypes of cathepsin L deficiency can be fully assigned to lack of canonically targeted cathepsin L, while the biogenesis and functionality of nucleo-cytosolic cathepsin L remain elusive.

Can nuclear cathepsin variants promote tumor progression in the thyroid?

Introduction: Cathepsins are proteolytic enzymes that are crucial for thyroid physiology through their processing of the prohormone thyroglobulin followed by thyroid hormone liberation. This processing is initiated in the thyroid follicle lumen and continued in endo-lysosomes. In pathological conditions, such as thyroid carcinoma, cathepsin B is secreted from tumor cells in a non-polarized way, and has been suggested to promote metastasis through degradation of extracellular matrix constituents. Interestingly, nuclear variants of cathepsins have been identified in thyroid carcinoma, with histones being plausible candidates for nuclear substrates. Methods: Here, by overexpressing chimeras of cathepsin variants fused to green fluorescent protein (GFP) in thyroid carcinoma cells, we monitored the subcellular trafficking pathways and destinations of these proteases. Results: We show that variants of cysteine cathepsins B and V are localized in the nucleus of tumor and tumor-associated cells in situ. A variant of cathepsin V was also shown to be associated with microtubules in M-phase cells in vitro. The GFP-tagged cathepsin variants were N-terminally truncated, and therefore lacking the signal peptide, in order to enable a destination different from endo-lysosomes. We show that a 51 amino acid N-terminal truncation of cathepsin B, lacking the signal peptide and part of the propeptide, caused its targeting to the mitochondria. On the other hand, N-terminally truncated cathepsin V at amino acid 56 displayed a nuclear localization. This construct is thus suitable to use for the analysis of the impact of nuclear cathepsin variants on cell functions associated with thyroid malignancy. Conclusion: We conclude that variants of cathepsins are destined to novel locations in thyroid tumor cells and might promote cancer onset and progression through new mechanisms involving e.g. chromatin condensation and transcriptional regulation.

The Protease Degrading Sperm Histones Post-Fertilization in Sea Urchin Eggs Is a Nuclear Cathepsin L That Is Further Required for Embryo Development

Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp) belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.

Localization of nuclear cathepsin L and its association with disease progression and poor outcome in colorectal cancer

Previous in vitro studies have identified a nuclear isoform of Cathepsin L. The aim of this study was to examine if nuclear Cathepsin L exists in vivo and examine its association with clinical, pathological and patient outcome data. Cellular localization (nuclear and cytoplasmic) and expression levels v of Cathespin L in 186 colorectal cancer cases using immunohistochemistry. The molecular weight and activity of nuclear and cytoplasmic Cathepsin L in vivo and in vitro were assessed by Western blotting and ELISA, respectively. Epithelial nuclear staining percentage (p = 0.04) and intensity (p = 0.006) increased with advancing tumor stage, whereas stromal cytoplasmic staining decreased (p = 0.02). Using multivariate statistical analysis, survival was inversely associated with staining intensity in the epithelial cytoplasm (p = 0.01) and stromal nuclei (p = 0.007). In different colorectal cell lines and in vivo tumors, pro- and active Cathepsin L isoforms were present in both the cytoplasm and nuclear samples, with pro-Cathepsin L at 50 kDa and active Cathepsin L at 25 kDa. Purified nuclear and cytoplasmic fractions from cell lines and tumors showed active Cathepsin L activity. The identification of nuclear Cathepsin L may play an important prognostic role in colorectal disease progression and patient outcome. Moreover, these findings suggest that altering active nuclear Cathepsin L may significantly influence disease progression.

Nuclear cathepsin F regulates activation markers in rat hepatic stellate cells.

Activation of hepatic stellate cells during liver fibrosis is a major event facilitating an increase in extracellular matrix deposition. The up-regulation of smooth muscle alpha-actin and collagen type I is indicative of the activation process. The involvement of cysteine cathepsins, a class of lysosomal cysteine proteases, has not been studied in conjunction with the activation process of hepatic stellate cells. Here we report a nuclear cysteine protease activity partially attributed to cathepsin F, which co-localizes with nuclear speckles. This activity can be regulated by treatment with retinol/palmitic acid, known to reduce the hepatic stellate cell activation. The treatment for 48 h leads to a decrease in activity, which is coupled to an increase in cystatin B and C transcripts. Cystatin B knockdown experiments during the same treatment confirm the regulation of the nuclear activity by cystatin B. We demonstrate further that the inhibition of the nuclear activity by E-64d, a cysteine protease inhibitor, results in a differential regulation of smooth muscle alpha-actin and collagen type I transcripts. On the other hand, cathepsin F small interfering RNA transfection leads to a decrease in nuclear activity and a transcriptional down-regulation of both activation markers. These findings indicate a possible link between nuclear cathepsin F activity and the transcriptional regulation of hepatic stellate cell activation markers.

Increased Expression and Activity of Nuclear Cathepsin L in Cancer Cells Suggests a Novel Mechanism of Cell Transformation

It is generally accepted that the role of cathepsin L in cancer involves its activities outside the cells once it has been secreted. However, cathepsin L isoforms that are devoid of a signal peptide were recently shown to be present in the nucleus where they proteolytically process the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor. A role for nuclear cathepsin L in cell proliferation could be inferred from the observation that the CDP/Cux processed isoform can accelerate entry into S phase. Here, we report that in many transformed cells the proteolytic processing of CDP/Cux is augmented and correlates with increased cysteine protease expression and activity in the nucleus. Taking advantage of an antibody that recognizes the prodomain of human cathepsin L, we showed that human cells express short cathepsin L species that do not contain a signal peptide, do not transit through the endoplasmic reticulum, are not glycosylated, and localize to the nucleus. We also showed that transformation by the ras oncogene causes rapid increases both in the production of short nuclear cathepsin L isoforms and in the processing of CDP/Cux. Using a cell-based assay, we showed that a cell-permeable inhibitor of cysteine proteases is able to delay the progression into S phase and the proliferation in soft agar of ras-transformed cells, whereas the non-cell-permeable inhibitor had no effect. Taken together, these results suggest that the role of cathepsin L in cancer might not be limited to its extracellular activities but may also involve its processing function in the nucleus.

Nuclear cathepsin B-like protease cleaves transcription factor YY1 in differentiated cells

Differentiation of pluripotent cells into differentiated cell types involves changes in many aspects of cellular biochemistry. Many of these changes result in alterations of gene expression, which may occur by changing the activity of transcription factors. The cell line NTERA-2 (NT2) can be differentiated into various cell types by incubation with retinoic acid. The differentiated cell type is also permissive for infection with the human herpesvirus cytomegalovirus (CMV). The transcription factor YY1 has been shown to regulate the immediate-early promoter of CMV in a differentiation specific manner by binding to one site at −958 to −950 and to at least two sites in the enhancer. It is demonstrated here that there is a second YY1 site in the modulator between −995 and −987. Levels of YY1 DNA binding activity and protein decrease in NT2 cells as they are differentiated with retinoic acid. This decrease in protein is due to the degradation of YY1 by a cathepsin B-like activity found in nuclear extracts. The cleavage products of YY1 include the intact C-terminal half of the protein, which contains the zinc fingers and the DNA binding activity. This suggests a mechanism that allows expression of the CMV immediate-early promoter in differentiated cells.

Immunohistochemical Studies of Proliferating Cell Nuclear Antigen and Cathepsin D in Transitional Cell Carcinoma of the Urinary Bladder

Immunohistochemical Studies of Proliferating Cell Nuclear Antigen and Cathepsin D in Transitional Cell Carcinoma of the Urinary Bladder