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en: Pro-cathepsin D prevents aberrant protein aggregation dependent on endoplasmic reticulum protein CLN6.

en: We previously expressed a chimeric protein in which the small heat-shock protein αB-crystallin (αBC) is fused at its N-terminus to the C-terminus of the first transmembrane segment of the endoplasmic reticulum (ER) protein mitsugumin 23 and confirmed its localization to the ER. Moreover, overexpression of this N-terminally modified αBC was shown to prevent the aggregation of the coexpressed R120G αBC variant, which is highly aggregation-prone and associated with the hereditary myopathy αB-crystallinopathy. To uncover a molecular mechanism by which the ER-anchored αBC negatively regulates the protein aggregation, we isolated proteins that bind to the ER-anchored αBC and identified the lysosomal protease cathepsin D (CTSD) as one such interacting protein. Proteolytically active CTSD is produced by multi-step processing of pro-cathepsin D (proCTSD), which is initially synthesized in the ER and delivered to lysosomes. When overexpressed, CTSD itself prevented the coexpressed R120G αBC variant from aggregating. This anti-aggregate activity was also elicited upon overexpression of the W383C CTSD variant, which is predominantly sequestered in the ER and consequently remains unprocessed, suggesting that proCTSD, rather than mature CTSD, serves to suppress the aggregation of the R120G αBC variant. Meanwhile, overexpression of the A58V CTSD variant, which is identical to wild-type CTSD except for the Ala58Val substitution within the pro-peptide, did not suppress the protein aggregation, indicating that the integrity of the pro-peptide is required for proCTSD to exert its anti-aggregate activity. Based on our previous finding that overexpression of the ER transmembrane protein CLN6 (ceroid-lipofuscinosis, neuronal 6), identified as an interacting protein of the ER-anchored αBC, prevents the R120G αBC variant from aggregating, the CLN6-proCTSD coupling was hypothesized to underpin the functionality of proCTSD within the ER. Indeed, CTSD, when overexpressed in CLN6-depleted cells, was unable to exert its anti-aggregate activity, supporting our view. Collectively, we show here that proCTSD prevents the protein aggregation through the functional association with CLN6 in the microenvironment surrounding the ER membrane, shedding light on a novel aspect of proCTSD and its potential involvement in CTSD-related disorders characterized by the accumulation of aberrant protein aggregates.

en: Molecular Genetics and Metabolism

ja: Molecular Genetics and Metabolism


en: CLN6's luminal tail-mediated functional interference between CLN6 mutants as a novel pathomechanism for the neuronal ceroid lipofuscinoses.

en: CLN6 (Ceroid Lipofuscinosis, Neuronal, 6) is a 311-amino acid protein spanning the endoplasmic reticulum membrane. Mutations in CLN6 are linked to CLN6 disease, a hereditary neurodegenerative disorder categorized into the neuronal ceroid lipofuscinoses. CLN6 disease is an autosomal recessive disorder and individuals affected with this disease have two identical (homozygous) or two distinct (compound heterozygous) CLN6 mutant alleles. Little has been known about CLN6's physiological roles and the disease mechanism. We recently found that CLN6 prevents protein aggregate formation, pointing to impaired CLN6's anti-aggregate activity as a cause for the disease. To comprehensively understand the pathomechanism, overall anti-aggregate activity derived from two different CLN6 mutants needs to be investigated, considering patients compound heterozygous for CLN6 alleles. We focused on mutant combinations involving the S132CfsX18 (132fsX) prematurely terminated protein, produced from the most frequent mutation in CLN6. The 132fsX mutant nullified anti-aggregate activity of the P299L CLN6 missense mutant but not of wild-type CLN6. Wild-type CLN6's resistance to the 132fsX mutant was abolished by replacement of amino acids 297-301, including Pro297 and Pro299, with five alanine residues. Given that removal of CLN6's C-terminal fifteen amino acids 297-311 (luminal tail) did not affect the resistance, we suggested that CLN6's luminal tail, when unleashed from Pro297/299-mediated conformational constraints, is improperly positioned by the 132fsX mutant, thereby blocking the induction of anti- aggregate activity. We here reveal a novel mechanism for dissipating CLN6 mutants' residual functions, providing an explanation for the compound heterozygosity-driven pathogenesis.

en: Biomedical Research

ja: Biomedical Research