Nuclear Localization of Cystatin B, the Cathepsin Inhibitor Implicated in Myoclonus Epilepsy (EPM1)

Riccio, Massimo; Giaimo, Rossella Di; Pianetti, Simona; Palmieri, Pier Paolo; Melli, Marialuisa; Santi, Spartaco

doi:10.1006/excr.2000.5085

Cited by 91 publications

(75 citation statements)

References 28 publications

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“…2B). The comparison between the fluorescence and transmission images showed that the ectopic expression of GFP-tagged CSTB is located in both the nucleus and the cytoplasm of both cells analyzed, which is consistent with a previous report (26). The green fluorescence perfectly overlapped with the CSTB immunoreactivity.…”

Section: Cstb2 Gene Expression In Hccsupporting

confidence: 90%

“…CSTB inhibits lysosomal cathepsin, and the intracellular distribution of CSTB is dependent on the differentiation status of the cells (32). Indeed, CSTB is found mainly in the nucleus of proliferating cells or in both the nucleus and the cytoplasm of differentiated cells (26). However, these results suggest that endogenous CSTB is localized in the cytoplasm, whereas ectopic CSTB is localized in both the nucleus and cytoplasm of Hep3B or 293T cells.…”

Section: Discussionmentioning

confidence: 93%

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Identification of Cystatin B as a Potential Serum Marker in Hepatocellular Carcinoma

Lee¹,

Yu²,

Park³

et al. 2008

Clinical Cancer Research

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Purpose: The poor survival rate of hepatocellular carcinoma (HCC) is in part due to the inability to diagnose patients at an early stage. Therefore, the aim of this study was to search for candidate serum marker for HCC and to test their ability to distinguish a HCC from benign liver disease. Experimental Design: Genome-wide analysis by a microarray in 40 HCC patients was done between HCC and paired nontumor liver tissues. Expression of cystatin B (CSTB) was examined by mRNA expression analysis and immunohistochemistry.The serum CSTB levels were measured using a sandwich ELISA method in four groups, including normal healthy subjects (group 1, n = 52) and patients with noncirrhotic chronic hepatitis (group 2, n = 53), cirrhosis (group 3, n = 43), and HCC (group 4, n = 62). Results: Microarray and statistical analyses identified 248 genes that were expressed differently between HCC and nontumor liver tissues. One of them, CSTB, was expressed preferentially in the HCCs compared with the nontumor tissues, 36 of 45 specimens (80%) by Northern blot and semiquantitative reverse transcription-PCR analyses. The serum CSTB level was much higher in HCC patients than in those with nonmalignant chronic liver disease (groups 2 and 3; P < 0.0001).The receiver operating characteristic curve indicated 5.34 ng/mL to be the optimal value for CSTB, and the sensitivity and specificity for this CSTB value were 85.5% (95% confidence interval, 74.2-93.1%) and 53.1% (95% confidence interval, 42.7-63.4%), respectively, in distinguishing between patients with HCC and those with nonmalignant chronic liver disease. Conclusion: CSTB is specifically overexpressed in most HCCs and is also elevated in the serum of a large proportion of HCC patients. CSTB or the combination of CSTB and a-fetoprotein may be a useful marker for diagnosing patients with HCC with a high sensitivity.

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Section: Cstb2 Gene Expression In Hccsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 93%

Identification of Cystatin B as a Potential Serum Marker in Hepatocellular Carcinoma

Lee¹,

Yu²,

Park³

et al. 2008

Clinical Cancer Research

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“…This is in agreement with previous findings in which CSTB was found to reside in the nucleus in proliferating neuronal cells but distributed to the cytoplasm in differentiated human neuroblastoma and rat primary cerebellar granule cells. 28 Since findings of the distribution of CSTB are contradictory in other neuronal cell types, 29 further systematic evaluation is necessary to determine the role of CSTB during cell differentiation.…”

Section: Discussionmentioning

confidence: 99%

Loss of lysosomal association of cystatin B proteins representing progressive myoclonus epilepsy, EPM1, mutations

et al. 2004

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Loss-of-function mutations in the cystatin B (CSTB), a cysteine protease inhibitor, gene underlie progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1), characterized by myoclonic and tonic -clonic seizures, ataxia and a progressive course. A minisatellite repeat expansion in the promoter region of the CSTB gene is the most common mutation in EPM1 patients and leads to reduced mRNA levels. Seven other mutations altering the structure of CSTB, or predicting altered splicing, have been described. Using a novel monoclonal CSTB antibody and organelle-specific markers in human primary myoblasts, we show here that endogenous CSTB localizes not only to the nucleus and cytoplasm but also associates with lysosomes. Upon differentiation to myotubes, CSTB becomes excluded from the nucleus and lysosomes, suggesting that the subcellular distribution of CSTB is dependent on the differentiation status of the cell. Four patient mutations altering the CSTB polypeptide were transiently expressed in BHK-21 cells. The p.Lys73fsX2-truncated mutant protein shows diffuse cytoplasmic and nuclear distribution, whereas p.Arg68X is rapidly degraded. Two missense mutations, the previously described p.Gly4Arg affecting the highly conserved glycine, critical for cathepsin binding, and a novel mutation, p.Gln71Pro, fail to associate with lysosomes. These data imply an important lysosome-associated physiological function for CSTB and suggest that loss of this association contributes to the molecular pathogenesis of EPM1.

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“…As OBP is a nuclear protein while cathepsin B is most commonly a lysosomal protein, it is difficult to imagine how cathepsin B directly cleaves OBP. However, cathepsin B and cathepsin B activity have been identified in the nuclei of multiple cell types (40,41,48,54). Overexpression of OBP is detrimental to viral DNA synthesis and viral plaque formation (2,29,32,52,55).…”

Section: Discussionmentioning

confidence: 99%

Cathepsin B Mediates Cleavage of Herpes Simplex Virus Type 1 Origin Binding Protein (OBP) To Yield OBPC-1, and Cleavage Is Dependent upon Viral DNA Replication

Link

Silva

Schaffer

2007

J Virol

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Although the seven viral proteins required for herpes simplex virus type 1 (HSV-1) DNA replication have been identified, the mechanism by which viral DNA synthesis is regulated is unclear. HSV-1 DNA replication is thought to occur in two stages: origin-dependent DNA replication (stage I) mediated by the origin binding protein (OBP), followed by origin-and OBP-independent DNA replication (stage II). The mechanism that facilitates the switch from stage I to stage II is unknown; however, it must involve the loss of OBP function or OBP itself from the replication initiation complex. Previous studies from this laboratory identified a transcript (UL8.5) and protein (OBPC) that are in frame with and comprise the C terminus of the gene specifying OBP. Because of its DNA binding ability, OBPC has been hypothesized to mediate the switch from stage I to stage II. Here, we identify a second protein (OBPC-2) that is also in frame with the C terminus of OBP but comprises a smaller portion of the protein. We demonstrate that the protein originally identified (OBPC-1) is a cathepsin B-mediated cleavage product of OBP, while OBPC-2 may be the product of the UL8.5 transcript. We further demonstrate that the cleavage of OBP to yield OBPC-1 is dependent upon viral DNA replication. These results suggest that cleavage may be a mechanism by which OBP levels and/or activity are regulated during infection.The 152-kb herpes simplex virus type 1 (HSV-1) genome encodes at least 84 proteins, 7 of which are early genes essential for viral DNA replication in vitro (49). Functionally, the proteins specified by the UL5, UL8, and UL52 genes comprise the helicase/primase complex, which unwinds viral DNA prior to replication. The UL29 gene encodes infected cell protein 8 (ICP8), a single-stranded DNA (ssDNA) binding protein that stabilizes replicating ssDNA. The products of the UL30 and UL42 genes comprise the viral DNA polymerase and polymerase accessory protein, respectively, which together replicate the viral genome. The product of the UL9 gene is the origin binding protein (OBP), the viral DNA replication initiator protein, and the subject of this paper.Initiator proteins are functionally similar in all life forms and are characterized by their ability to bind to specific sequences in origin DNA. This binding results in unwinding of the origin at a site that usually contains an AT-rich sequence. DNA binding and unwinding are followed by the recruitment of the DNA replication machinery. As a means of regulating the DNA replication process, the activities of initiator proteins themselves are highly regulated. For example, the activities of many viral initiator proteins including simian virus 40 large T antigen (44), polyomavirus large T antigen (59), human papilloma virus E1 (27), bovine papillomavirus E1 (65), and the NS1 protein of minute virus of mice (11) are regulated by differential phosphorylation. The activities of some initiator proteins such as the bacteriophage lambda O (60-62) and bovine papillomavirus E1 (28) are also regulated by u...

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Nuclear Localization of Cystatin B, the Cathepsin Inhibitor Implicated in Myoclonus Epilepsy (EPM1)

Cited by 91 publications

References 28 publications

Identification of Cystatin B as a Potential Serum Marker in Hepatocellular Carcinoma

Identification of Cystatin B as a Potential Serum Marker in Hepatocellular Carcinoma

Loss of lysosomal association of cystatin B proteins representing progressive myoclonus epilepsy, EPM1, mutations

Cathepsin B Mediates Cleavage of Herpes Simplex Virus Type 1 Origin Binding Protein (OBP) To Yield OBPC-1, and Cleavage Is Dependent upon Viral DNA Replication

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