Imaging of protease functions – current guide to spotting cysteine cathepsins in classical and novel scenes of action in mammalian epithelial cells and tissues

The human genome encodes some hundreds of proteases. Many of these are well studied and understood with respect to their biochemistry, molecular mechanisms of proteolytic cleavage, expression patterns, molecular structure, substrate preferences and regulatory mechanisms, including their endogenous inhibitors. Moreover, precise determination of protease localisation within subcellular compartments, peri- and extracellular spaces has been extremely useful in elucidating biological functions of peptidases. This can be achieved by refined methodology as will be demonstrated herein for the cysteine cathepsins. Besides localisation, it is now feasible to study in situ enzymatic activity at the various levels of subcellular compartments, cells, tissues, and even whole organisms including mouse

In vitro studies on the mechanisms of inhibited thyroidal iodide uptake and destructive thyroiditis as adverse effects of amiodarone and interferon-gamma

The clinical use of the potent anti-arrhythmic drug amiodarone is limited due to adverse effects e.g. in the thyroid comprising both hypo- and hyperthyroidism. Amiodarone-induced thyroid dysfunction can occur in subjects with normal thyroid glands but also in those with an underlying thyroid abnormality resulting in an aggravation of the condition. Hashimoto´s thyroiditis and the presence of autoantibodies are common risk factors for developing amiodarone-induced hypothyroidism (AIH), it is though not known whether the thyroid effects of amiodarone can be modulated by the present immunologic activity or vice versa. In amiodarone-induced thyrotoxicosis (AIT), the thyroid iodide uptake is low thereby excluding 131I as a treatment of choice. This is suggestively caused by the cytotoxic effect of the drug resulting in a destructive thyroiditis with leakage of colloid content including preformed thyroid hormones and radioiodide. The thyroid epithelial integrity can also be impaired by proinflammatory cytokines. The aim of this thesis was to analyze the effect of amiodarone on the transepithelial transport of iodide and the effect of the major proinflammatory cytokine interferon-gamma on the thyroid epithelial barrier function. The maintenance of epithelial integrity is a prerequisite for the polarized thyroid phenotype that in turn enables the directed transport and lumenal concentration of iodide. Primary cultures of pig thyrocytes were seeded on filter in a bicameral chamber system thereby allowing the in vivo epithelial characteristics to be maintained and also to enable the simultaneous but separate monitoring of the transport of the tracer 125I- at the basolateral and apical plasma membranes. In the presence of amiodarone, the apical iodide permeability was reduced, an effect that was long-lasting and independent of cAMP and the iodine-content of the drug. Prior to the loss of epithelial barrier function induced by high doses of amiodarone, endocytosis from the apical domain was reduced and vacuoles of varying sizes were present in the cytoplasm. There was also a change in mitochondrial structure and function; a fission-like mitochondrial pattern and a biphasic effect on the membrane potential (hyperpolarization followed by depolarization), all being early pro-apoptotic signs. Interferon-gamma was shown to impair the epithelial barrier function in primary cultures of human thyrocytes, an event that was concomitant with the down-regulation and changed localization of the tight junction protein claudin-1. In conclusion, we suggest that the failure of the amiodarone-treated glands to concentrate radioiodide can be caused by a reduced apical iodide permeability and that the cytotoxic effect of amiodarone eventually causing apoptosis and loss of epithelial integrity can be a mechanism for the follicular destruction seen in AIT. The loss of epithelial barrier function by interferon-gamma might be a mechanism for exposure of hidden autoantigens to the immune system

Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditions

Background: Cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. Nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. Although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. Therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and pH values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells. Results: The proteolytic potencies of cysteine cathepsins B, K, L and S towards thyroglobulin were analyzed under conditions simulating oxidizing versus reducing environments with neutral to acidic pH values. Thyroglobulin, the precursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteine cathepsins. Thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances, starts in the extracellular follicle lumen before being continued within endo-lysosomes. Our study shows that all cathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these are reportedly non-favorable for cysteine proteases. All analyzed cathepsins generated distinct fragments of thyroglobulin at extracellular versus endo-lysosomal conditions as demonstrated by SDS-PAGE followed by immunoblotting or N-terminal sequencing. Moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin S at extracellular conditions, while cathepsins B, K and L worked most efficiently in this respect at endo-lysosomal conditions. Conclusion: The results revealed distinct cleavage patterns at all conditions analyzed, indicating compartment-specific processing of thyroglobulin by cysteine cathepsins. In particular, proteolytic activity of cathepsin S towards the substrate thyroglobulin can now be understood as instrumental for extracellular thyroid hormone liberation. Our study emphasizes that the proteolytic functions of cysteine cathepsins in the thyroid are not restricted to endo-lysosomes but include pivotal roles in extracellular substrate utilization. We conclude that understanding of the interplay and fine adjustment of protease networks in vivo is better approachable by simulating physiological conditions in protease activity assays.Dentistry, Faculty ofOral Biological and Medical Sciences (OBMS), Department ofNon UBCReviewedFacult

Nuclear cysteine cathepsin variants in thyroid carcinoma cells

The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins