BackgroundCathepsin K is a cysteine peptidase known for its importance in osteoclast-mediated bone resorption. Inhibitors of cathepsin K are in clinical trials for treatment of osteoporosis. However, side effects of first generation inhibitors included altered levels of related cathepsins in peripheral organs and in the central nervous system (CNS). Cathepsin K has been recently detected in brain parenchyma and it has been linked to neurobehavioral disorders such as schizophrenia. Thus, the study of the functions that cathepsin K fulfils in the brain becomes highly relevant.ResultsCathepsin K messenger RNA was detectable in all brain regions of wild type (WT) mice. At the protein level, cathepsin K was detected by immunofluorescence microscopy in vesicles of neuronal and non-neuronal cells throughout the mouse brain. The hippocampus of WT mice exhibited the highest levels of cathepsin K activity in fluorogenic assays, while the cortex, striatum, and cerebellum revealed significantly lower enzymatic activities. At the molecular level, the proteolytic network of cysteine cathepsins was disrupted in the brain of cathepsin K-deficient (Ctsk-/-) animals. Specifically, cathepsin B and L protein and activity levels were altered, whereas cathepsin D remained largely unaffected. Cystatin C, an endogenous inhibitor of cysteine cathepsins, was elevated in the striatum and hippocampus, pointing to regional differences in the tissue response to Ctsk ablation. Decreased levels of astrocytic glial fibrillary acidic protein, fewer and less ramified profiles of astrocyte processes, differentially altered levels of oligodendrocytic cyclic nucleotide phosphodiesterase, as well as alterations in the patterning of neuronal cell layers were observed in the hippocampus of Ctsk-/- mice. A number of molecular and cellular changes were detected in other brain regions, including the cortex, striatum/mesencephalon, and cerebellum. Moreover, an overall induction of the dopaminergic system was found in Ctsk-/- animals which exhibited reduced anxiety levels as well as short- and long-term memory impairments in behavioral assessments.ConclusionWe conclude that deletion of the Ctsk gene can lead to deregulation of related proteases, resulting in a wide range of molecular and cellular changes in the CNS with severe consequences for tissue homeostasis. We propose that cathepsin K activity has an important impact on the development and maintenance of the CNS in mice.

Proteases play essential roles in protein degradation, protein processing, and extracellular matrix remodeling in all cell types and tissues. They are also involved in protein turnover for maintenance of homeostasis and protein activation or inactivation for cell signaling. Proteases range in function and specificity, with some performing distinct substrate cleavages, while others accomplish proteolysis of a wide range of substrates. As such, different cell types use specialized molecular mechanisms to regulate the localization of proteases and their function within the compartments to which they are destined. Here, we focus on the cysteine family of cathepsin proteases and legumain, which act predominately within the endo-lysosomal pathway. In particular, recent knowledge on cysteine cathepsins and their primary regulator legumain is scrutinized in terms of their trafficking to endo-lysosomal compartments and other less recognized cellular locations. We further explore the mechanisms that regulate these processes and point to pathological cases which arise from detours taken by these proteases. Moreover, the emerging biological roles of specific forms and variants of cysteine cathepsins and legumain are discussed. These may be decisive, pathogenic, or even deadly when localizing to unusual cellular compartments in their enzymatically active form, because they may exert unexpected effects by alternative substrate cleavage. Hence, we propose future perspectives for addressing the actions of cysteine cathepsins and legumain as well as their specific forms and variants. The increasing knowledge in non-canonical aspects of cysteine cathepsin- and legumain-mediated proteolysis may prove valuable for developing new strategies to utilize these versatile proteases in therapeutic approaches.

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The thyroid gland is both a thyroid hormone (TH) generating as well as a TH responsive organ. It is hence crucial that cathepsin-mediated proteolytic cleavage of the precursor thyroglobulin is regulated and integrated with the subsequent export of TH into the blood circulation, which is enabled by TH transporters such as monocarboxylate transporters Mct8 and Mct10. Previously, we showed that cathepsin K-deficient mice exhibit the phenomenon of functional compensation through cathepsin L upregulation, which is independent of the canonical hypothalamus-pituitary-thyroid axis, thus, due to auto-regulation. Since these animals also feature enhanced Mct8 expression, we aimed to understand if TH transporters are part of the thyroid auto-regulatory mechanisms. Therefore, we analyzed phenotypic differences in thyroid function arising from combined cathepsin K and TH transporter deficiencies, i.e., in Ctsk-/-/Mct10-/-, Ctsk-/-/Mct8-/y, and Ctsk-/-/Mct8-/y/Mct10-/-. Despite the impaired TH export, thyroglobulin degradation was enhanced in the mice lacking Mct8, particularly in the triple-deficient genotype, due to increased cathepsin amounts and enhanced cysteine peptidase activities, leading to ongoing thyroglobulin proteolysis for TH liberation, eventually causing self-thyrotoxic thyroid states. The increased cathepsin amounts were a consequence of autophagy-mediated lysosomal biogenesis that is possibly triggered due to the stress accompanying intrathyroidal TH accumulation, in particular in the Ctsk-/-/Mct8-/y/Mct10-/- animals. Collectively, our data points to the notion that the absence of cathepsin K and Mct8 leads to excessive thyroglobulin degradation and TH liberation in a non-classical pathway of thyroid auto-regulation.

This mini-review asks how self-regulation of the thyroid gland is realized at the cellular and molecular levels by canonical and non-canonical means. Canonical pathways of thyroid regulation comprise thyroid stimulating hormone-triggered receptor signaling. As part of non-canonical regulation, we hypothesized an interplay between protease-mediated thyroglobulin processing and thyroid hormone release into the circulation by means of thyroid hormone transporters like Mct8. We proposed a sensing mechanism by different thyroid hormone transporters, present in specific subcellular locations of thyroid epithelial cells, selectively monitoring individual steps of thyroglobulin processing, and thus, the cellular thyroid hormone status. Indeed, we found that proteases and thyroid hormone transporters are functionally inter-connected, however, in a counter-intuitive manner fostering self-thyrotoxicity in particular in Mct8- and/or Mct10-deficient mice. Furthermore, the possible role of the G protein-coupled receptor Taar1 is discussed, because we detected Taar1 at cilia of the apical plasma membrane of thyrocytes in vitro and in situ. Eventually, through pheno-typing Taar1-deficient mice, we identified a co-regulatory role of Taar1 and the thyroid stimulating hormone receptors. Recently, we showed that inhibition of thyroglobulin-processing enzymes results in disappearance of cilia from the apical pole of thyrocytes, while Taar1 is re-located to the endoplasmic reticulum. This pathway features a connection between thyrotropin-stimulated secretion of proteases into the thyroid follicle lumen and substrate-mediated self-assisted control of initially peri-cellular thyroglobulin processing, before its reinternalization by endocytosis, followed by extensive endo-lysosomal liberation of thyroid hormones, which are then released from thyroid follicles by means of thyroid hormone transporters.

Background: The trace amine-associated receptor 1 (Taar1) is one member of the Taar family of G-protein-coupled receptors (GPCR) accepting various biogenic amines as ligands. It has been proposed that Taar1 mediates rapid, membrane-initiated effects of thyronamines, the endogenous decarboxylated and deiodinated relatives of the classical thyroid hormones T₄ and T₃. Objectives: Although the physiological actions of thyronamines in general and 3-iodothyronamine (T₁AM) in particular are incompletely understood, studies published to date suggest that synthetic T₁AM-activated Taar1 signaling antagonizes thyromimetic effects exerted by T₃. However, the location of Taar1 is currently unknown. Methods: To fill this gap in our knowledge we employed immunofluorescence microscopy and a polyclonal antibody to detect Taar1 protein expression in thyroid tissue from Fisher rats, wild-type and taar1-deficient mice, and in the polarized FRT cells. Results: With this approach we found that Taar1 is expressed in the membranes of subcellular compartments of the secretory pathway and on the apical plasma membrane of FRT cells. Three-dimensional analyses further revealed Taar1 immunoreactivity in cilial extensions of postconfluent FRT cell cultures that had formed follicle-like structures. Conclusions: The results suggest Taar1 transport along the secretory pathway and its accumulation in the primary cilium of thyrocytes. These findings are of significance considering the increasing interest in the role of cilia in harboring functional GPCR. We hypothesize that thyronamines can reach and activate Taar1 in thyroid follicular epithelia by acting from within the thyroid follicle lumen, their potential site of synthesis, as part of a nonclassical mechanism of thyroid autoregulation.

Thyroid hormone (TH) target cells need to adopt mechanisms to maintain sufficient levels of TH to ensure regular functions. This includes thyroid epithelial cells, which generate TH in addition to being TH-responsive. However, the cellular and molecular pathways underlying thyroid auto-regulation are insufficiently understood. In order to investigate whether thyroglobulin processing and TH export are sensed by thyrocytes, we inactivated thyroglobulin-processing cathepsins and TH-exporting monocarboxylate transporters (Mct) in the mouse. The states of thyroglobulin storage and its protease-mediated processing and degradation were related to the levels of TH transporter molecules by immunoblotting and immunofluorescence microscopy. Thyroid epithelial cells of cathepsin-deficient mice showed increased Mct8 protein levels at the basolateral plasma membrane domains when compared to wild type controls. While the protein amounts of the thyroglobulin-degrading cathepsin D remained largely unaffected by Mct8 or Mct10 single-deficiencies, a significant increase in the amounts of the thyroglobulin-processing cathepsins B and L was detectable in particular in Mct8/Mct10 double deficiency. In addition, it was observed that larger endo-lysosomes containing cathepsins B, D, and L were typical for Mct8- and/or Mct10-deficient mouse thyroid epithelial cells. These data support the notion of a crosstalk between TH transporters and thyroglobulin-processing proteases in thyroid epithelial cells. We conclude that a defect in exporting thyroxine from thyroid follicles feeds back positively on its cathepsin-mediated proteolytic liberation from the precursor thyroglobulin, thereby adding to the development of auto-thyrotoxic states in Mct8 and/or Mct10 deficiencies. The data suggest TH sensing molecules within thyrocytes that contribute to thyroid auto-regulation.

The human genome encodes 11 cysteine cathepsins belonging to the papain-like family of cysteine peptidases that are known predominantly as endo-lysosomal enzymes. However, it is now understood that the functions and activities of cysteine cathepsins are not limited to endo-lysosomal compartments, as they are also active in the peri-and extracellular space. The thyroid gland is an endocrine organ where such intra-and extracellular proteolytic activities are required to solubilize the prohormone thyroglobulin from its luminal, covalently cross-linked storage forms for subsequent processing into smaller protein fragments and thyroid hormone liberation. Cathepsin K has been identified as one of the cysteine cathepsins with a crucial role in thyroglobulin processing. However, cathepsin K has mainly been a key focus of attention in the last few years because of its high expression in osteoclasts and due to its essential role as collagenase and elastase important for bone remodelling. Besides its remarkable function as an endopeptidase acting on highmolecular mass, covalently cross-linked extracellular substrates such as type I collagen, elastin or thyroglobulin, cathepsin K is also one of the very few proteolytic enzymes that is able to directly liberate thyroxine from thyroglobulin fragments by exopeptidase action. Thus, thyroid cathepsin K is now accepted as a cysteine peptidase with a vital role in liberation of thyroid hormones, which in turn are essential for homoeostasis by triggering a number of important biological processes, ranging from growth and brain development in young vertebrates to tissue remodelling events during morphogenesis or wound healing, as well as control of metabolic pathways and thermoregulation in adults. This review focuses on thyroid cathepsin K and will discuss how localization and trafficking within thyroid epithelial cells explain its thyroid-specific functions. The effects of targeted cathepsin K gene ablation will be summarized from the perspective of the thyroid gland, and we will propose potential consequences of short-and long-term inhibition of thyroid cathepsin K activity for the main thyroid hormone target tissues, namely bone, cardiovascular and immune systems, intestine, and the central nervous system, in addition to the thyroid gland itself.