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Abstract Background The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in prohormone processing initiated in the follicle lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space in thyroid cancer tissue, and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through e.g. extracellular matrix degradation. Methods Transport of cathepsin B in normal thyroid epithelial and carcinoma cells was investigated through immunolocalization of endogenous cathepsin B in combination with probing protease activity. Transport analyses of cathepsin B-eGFP and its active-site mutant counterpart cathepsin B-C29A-eGFP were used to test whether intrinsic sequences of a protease influence its trafficking. Results Our approach employing activity based probes, which distinguish between active and inactive cysteine proteases, demonstrated that both eGFP-tagged normal and active-site mutated cathepsin B chimeras reached the endo-lysosomal compartments of thyroid epithelial cells, thereby ruling out alterations of sorting signals by mutagenesis of the active-site cysteine. Analysis of chimeric protein trafficking further showed that GFP-tagged cathepsin B was transported to the expected compartments, i.e. endoplasmic reticulum, Golgi apparatus and endo-lysosomes of normal and thyroid carcinoma cell lines. However, the active-site mutated cathepsin B chimera was mostly retained in the endoplasmic reticulum and Golgi of KTC-1 and HTh7 cells. Hence the latter, as the least polarized of the three carcinoma cell lines analyzed, exhibited severe transport defects in that it retained chimeras in pre-endolysosomal compartments. Furthermore, secretion of endogenous cathepsin B and of other cysteine peptidases, which occurs at the apical pole of normal thyroid epithelial cells, was most prominent and occurred in a non-directed fashion in thyroid carcinoma cells. Conclusions Transport of endogenous and eGFP-tagged active and inactive cathepsin B in the cultured thyroid carcinoma cells reflected the distribution patterns of this protease in thyroid carcinoma tissue. Hence, our studies showed that sub-cellular localization of proteolysis is a crucial step in regulation of tissue homeostasis. We conclude that any interference with protease trafficking resulting in altered regulation of proteolytic events leads to, or is a consequence of the onset and progression of thyroid cancer.

Spastic paraplegia 35 (SPG35) (OMIM: 612319) or fatty acid hydroxylase-associated neurodegeneration (FAHN) is caused by deficiency of fatty acid 2-hydroxylase (FA2H). This enzyme synthesizes sphingolipids containing 2-hydroxylated fatty acids, which are particularly abundant in myelin. Fa2h-deficient (Fa2h-/-) mice develop symptoms reminiscent of the human disease and therefore serve as animal model of SPG35. In order to understand further the pathogenesis of SPG35, we compared the proteome of purified CNS myelin isolated from wild type and Fa2h-/- mice at different time points of disease progression using tandem mass tag labeling. Data analysis with a focus on myelin membrane proteins revealed a significant increase of the oligodendrocytic myelin paranodal and inner loop protein (Opalin) in Fa2h-/- mice, whereas the concentration of other major myelin proteins was not significantly changed. Western blot analysis revealed an almost 6-fold increase of Opalin in myelin of Fa2h-/- mice aged 21-23 months. A concurrent unaltered Opalin gene expression suggested a decreased turnover of the Opalin protein in Fa2h-/- mice. Supporting this hypothesis, Opalin protein half-life was reduced significantly when expressed in CHO cells synthesizing 2-hydroxylated sulfatide, compared to cells synthesizing only non-hydroxylated sulfatide. Degradation of Opalin was inhibited by inhibitors of lysosomal degradation but unaffected by proteasome inhibitors. Taken together, these results reveal a new function of 2-hydroxylated sphingolipids namely affecting the turnover of a myelin membrane protein. This may play a role in the pathogenesis of SPG35., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)