PMC:539297 / 11900-15419 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/539297","sourcedb":"PMC","sourceid":"539297","source_url":"http://www.ncbi.nlm.nih.gov/pmc/539297","text":"Homozygous CbCln3Δex7/8 cells and Cln3Δex7/8 knock-in mice process cathepsin D abnormally\nWe next investigated the basis for subunit c accumulation, testing the hypothesis that cathepsin D is abnormal since it is required for ATP synthase subunit c degradation in the lysosome [13]. We first tested cathepsin D transport and processing in homozygous CbCln3Δex7/8 cells and Cln3Δex7/8 mice using anti-cathepsin D antibody that recognizes unprocessed and processed cathepsin D isoforms. Immunostaining of wild-type and homozygous CbCln3Δex7/8 cells revealed a perinuclear and punctate vesicular cathepsin D distribution, consistent with its transport and processing through the secretory pathway and delivery to the lysosome (Fig. 5a). However, in homozygous CbCln3Δex7/8 cells, cathepsin D distribution was less vesicular and more perinuclear-clustered than in wild-type cells. Immunoblots of homozygous CbCln3Δex7/8 cell and Cln3Δex7/8 tissue extracts also showed altered relative levels of cathepsin D isoforms (Fig. 5b). Cathepsin D isoforms, identified by relative molecular weights, represent the ~45 kDa precursor, the ~43 kDa intermediate single chain form of the enzyme, and the 31 kDa heavy chain of the double-chain form of the mature enzyme [14]. In homozygous CbCln3Δex7/8 cell and Cln3Δex7/8 tissue extracts, the precursor and heavy chains were reduced, and the single chain was slightly elevated compared to wild-type extracts. The cellular growth media did not contain altered levels of cathepsin D, indicating enzyme secretion was not affected. Heterozygous Cln3Δex7/8 mice and CbCln3Δex7/8 cells were indistinguishable from wild-type, as expected for a recessive disease phenotype (data not shown).\nFigure 5 Cathepsin D localization and processing in wild-type and homozygous CbCln3Δex7/8 cells a. Immunostaining of wild-type and homozygous CbCln3Δex7/8 precursor cells with anti-cathepsin D antibody, recognizing unprocessed and processed forms of cathepsin D protein is shown. CbCln3+/+ cells (left panel) exhibited a perinuclear and cytoplasmic punctate signal. Cathepsin D signal in homozygous CbCln3Δex7/8 cells (right panel) was more often perinuclear, with less cytoplasmic punctate signal, compared to wild-type CbCln3+/+ cells. 40 × magnification. b. α-Cathepsin D-probed immunoblots of total wild-type versus homozygous Cln3Δex7/8 knock-in tissue or CbCln3Δex7/8 cellular extracts are shown. The ~45 kDa cathepsin D band, representing precursor, was the predominant band in wild-type (wt) tissue and cellular extracts, with lower levels of mature enzyme (single chain, ~43 kDa, and heavy chain, ~31 kDa). Conversely, homozygous Cln3Δex7/8 and CbCln3Δex7/8 mutant (m) extracts exhibited reduced levels of precursor and heavy chain of the double-chain form of the enzyme, with elevated levels of single-chain mature enzyme. The impact of the altered cathepsin D processing on enzymatic activity was next tested to determine if altered enzymatic activity accounts for inefficient subunit c turnover. In a fluorogenic in vitro assay, cathepsin D activity in total cellular extracts was not significantly altered in homozygous CbCln3Δex7/8 cells (376 ± 89 RFU/μg total protein), versus wild-type cells (324 ± 58 RFU/μg total protein), although a consistent trend towards increased enzymatic activity in mutant cells was observed. Thus, cathepsin D transport and processing are disrupted in homozygous CbCln3Δex7/8 cells in a manner such that enzymatic activity appears to be relatively 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