Cytoskeletal ß-tubulin and cysteine cathepsin L deregulation by SARS-CoV-2 spike protein interaction with the neuronal model cell line SH-SY5Y.

SARS-CoV-2 mainly infects the respiratory tract but can also target other organs, including the central nervous system. While it was recently shown that cells of the blood-brain-barrier are permissive to SARS-CoV-2 infection in vitro, it remains debated whether neurons can be infected. In this study, we demonstrate that vesicular stomatitis virus particles pseudotyped with the spike protein of SARS-CoV-2 variants WT, Alpha, Delta and Omicron enter the neuronal model cell line SH-SY5Y. Cell biological analyses of the pseudo-virus treated cultures showed marked alterations in microtubules of SH-SY5Y cells. Because the changes in ß-tubulin occurred in most cells, but only few were infected, we further asked whether interaction of the cells with spike protein might be sufficient to cause molecular and structural changes. For this, SH-SY5Y cells were incubated with trimeric spike proteins for time intervals of up to 24 h. CellProfiler™-based image analyses revealed changes in the intensities of microtubule staining in spike protein-incubated cells. Furthermore, expression of the spike protein-processing protease cathepsin L was found to be up-regulated by wild type, Alpha and Delta spike protein pseudotypes and cathepsin L was found to be secreted from spike protein-treated cells. We conclude that the mere interaction of the SARS-CoV-2 with neuronal cells can affect cellular architecture and proteolytic capacities. The molecular mechanisms underlying SARS-CoV-2 spike protein induced cytoskeletal changes in neuronal cells remain elusive and require future studies.

Cathepsin V regulates cell cycle progression and histone stability in the nucleus of breast cancer cells.

Introduction: We previously identified that Cathepsin V (CTSV) expression is associated with poor prognosis in ER+ breast cancer, particularly within the Luminal A subtype. Examination of the molecular role of the protease within Luminal A tumours, revealed that CTSV promotes tumour cell invasion and proliferation, in addition to degradation of the luminal transcription factor, GATA3, via the proteasome. Methods: Cell line models expressing CTSV shRNA or transfected to overexpress CTSV were used to examine the impact of CTSV on cell proliferation by MTT assay and flow cytometry. Western blotting analysis was used to identify the impact of CTSV on histone and chaperone protein expression. Cell fractionation and confocal microscopy was used to illustrate the presence of CTSV in the nuclear compartment. Results: In this work we have identified that CTSV has an impact on breast cancer cell proliferation, with CTSV depleted cells exhibiting delayed progression through the G2/M phase of the cell cycle. Further investigation has revealed that CTSV can control nuclear expression levels of histones H3 and H4 via regulating protein expression of their chaperone sNASP. We have discovered that CTSV is localised to the nuclear compartment in breast tumour cells, mediated by a bipartite nuclear localisation signal (NLS) within the CTSV sequence and that nuclear CTSV is required for cell cycle progression and histone stability in breast tumour cells. Discussion: Collectively these findings support the hypothesis that targeting CTSV may have utility as a novel therapeutic target in ER+ breast cancer by impairing cell cycle progression via manipulating histone stabilisation.

Investigations on Primary Cilia of Nthy-ori 3-1 Cells upon Cysteine Cathepsin Inhibition or Thyrotropin Stimulation.

In the thyroid gland, cysteine cathepsins are secreted upon thyrotropin stimulation for thyroglobulin processing, and they are present at the primary cilia of thyroid epithelial cells. Treatment with protease inhibitors resulted in the loss of cilia from rodent thyrocytes and caused redistribution of the thyroid co-regulating G protein-coupled receptor Taar1 to the endoplasmic reticulum. These findings suggest that ciliary cysteine cathepsins are important to maintain sensory and signaling properties for the proper regulation and homeostasis of thyroid follicles. Therefore, it is important to better understand how cilia structure and frequencies are maintained in human thyroid epithelial cells. Hence, we aimed to investigate the potential role of cysteine cathepsins for the maintenance of primary cilia in the normal human Nthy-ori 3-1 thyroid cell line. This was approached by determining cilia lengths and frequencies in cysteine peptidase inhibition conditions in Nthy-ori 3-1 cell cultures. Cilia lengths were shortened upon 5 h of cysteine peptidase inhibition with cell-impermeable E64. Likewise, cilia lengths and frequencies were decreased upon additional overnight treatment with the cysteine peptidase-targeting, activity-based probe DCG-04. The results suggest that cysteine cathepsin activity is required for the maintenance of the cellular protrusions not only in rodents, but also in human thyrocytes. Hence, thyrotropin stimulation was used to simulate physiological conditions that eventually lead to cathepsin-mediated thyroglobulin proteolysis, which is initiated in the thyroid follicle lumen. Immunoblotting revealed that thyrotropin stimulation conditions result in the secretion of little procathepsin L and some pro- and mature cathepsin S but no cathepsin B from the human Nthy-ori 3-1 cells. Unexpectedly, however, 24 h incubation periods with thyrotropin shortened the cilia although higher amounts of cysteine cathepsins were present in the conditioned media. These data point to the necessity of further studies to delineate which of the cysteine cathepsins plays the most prominent role in cilia shortening and/or elongation. Collectively, the results of our study provide corroboration for the hypothesis of thyroid autoregulation by local mechanisms that our group previously proposed.

Metallacarborane Cluster Anions of the Cobalt Bisdicarbollide-Type as Chaotropic Carriers for Transmembrane and Intracellular Delivery of Cationic Peptides.

Cobalt bisdicarbollides (COSANs) are inorganic boron-based anions that have been previously reported to permeate by themselves through lipid bilayer membranes, a propensity that is related to their superchaotropic character. We now introduce their use as selective and efficient molecular carriers of otherwise impermeable hydrophilic oligopeptides through both artificial and cellular membranes, without causing membrane lysis or poration at low micromolar carrier concentrations. COSANs transport not only arginine-rich but also lysine-rich peptides, whereas low-molecular-weight analytes such as amino acids as well as neutral and anionic cargos (phalloidin and BSA) are not transported. In addition to the unsubstituted isomers (known as ortho- and meta-COSAN), four derivatives bearing organic substituents or halogen atoms have been evaluated, and all six of them surpass established carriers such as pyrenebutyrate in terms of activity. U-tube experiments and black lipid membrane conductance measurements establish that the transport across model membranes is mediated by a molecular carrier mechanism. Transport experiments in living cells showed that a fluorescent peptide cargo, FITC-Arg8, is delivered into the cytosol.

Lack of L-type amino acid transporter 2 in murine thyroid tissue induces autophagy.

Proteolytic cleavage of thyroglobulin (Tg) for thyroid hormone (TH) liberation is followed by TH release from thyroid follicles into the circulation, enabled by TH transporters. The existence of a functional link between Tg-processing cathepsin proteases and TH transporters has been shown to be independent of the hypothalamus-pituitary-thyroid axis. Thus, lack of cathepsin K, combined with genetic defects in the TH transporters Mct8 and Mct10, that is the Ctsk-/-/Mct8-/y/Mct10-/- genotype, results in persistent Tg proteolysis due to autophagy induction. Because amino acid transport by L-type amino acid transporter 2 (Lat2) has been described to regulate autophagy, we asked whether Lat2 availability is affected in Ctsk-/-/Mct8-/y/Mct10-/- thyroid glands. Our data revealed that while mRNA amounts and subcellular localization of Lat2 remained unaltered in thyroid tissue of Ctsk-/-/Mct8-/y/Mct10-/- mice in comparison to WT controls, the Lat2 protein amounts were significantly reduced. These data suggest a direct link between Lat2 function and autophagy induction in Ctsk-/-/Mct8-/y/Mct10-/- mice. Indeed, thyroid tissue of Lat2-/- mice showed enhanced endo-lysosomal cathepsin activities, increased autophagosome formation, and enhanced autophagic flux. Collectively, these results suggest a mechanistic link between insufficient Lat2 protein function and autophagy induction in the thyroid gland of male mice.

Age-dependent response to T4 overtreatment and recovery on systemic and organ level.

Thyroid hormone (TH) metabolism and cellular TH action are influenced by ageing. To investigate the response to thyroxine (T4) overtreatment, a kinetic study was conducted in young and aged mice with chronic hyperthyroidism and hormone withdrawal. Five and 22 months old male mice were treated with T4 or PBS over 5 weeks, followed by observation for up to 12 days. Serial analysis was performed for thyroid function parameters, transcript levels of TH target genes, deiodinase type 1 (DIO1) activity as well as serum lipids at 12, 24, 72, 144, 216, and 288 h after cessation of T4 administration. Higher FT3 concentrations and higher renal DIO1 activities were noted in aged mice 12 h after T4 withdrawal and marked thyroid-stimulating hormone elevation was found in aged mice after 12 days compared to respective controls. A biphasic expression pattern occurred for TH target genes in all organs and a hypothyroid organ state was observed at the end of the study, despite normalization of TH serum concentrations after 72 h. In line with this, mirror-image kinetics were detected for serum cholesterol and triglycerides in aged and young mice. Recovery from TH overtreatment in mice involves short- and medium-term adaption of TH metabolism on systemic and organ levels. Increased renal DIO1 activity may contribute to higher T3 concentrations and prolonged thyrotoxicosis followed by hypothyroidism in an aged-mouse organism. Translation of these findings in the clinical setting seems warranted and may lead to better management of hyperthyroidism and prevention of T4 overtreatment in aged patients.

Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells.

Trace amine-associated receptor 1 (rodent Taar1/human TAAR1) is a G protein-coupled receptor that is mainly recognized for its functions in neuromodulation. Previous in vitro studies suggested that Taar1 may signal from intracellular compartments. However, we have shown Taar1 to localize apically and on ciliary extensions in rodent thyrocytes, suggesting that at least in the thyroid, Taar1 may signal from the cilia at the apical plasma membrane domain of thyrocytes in situ, where it is exposed to the content of the follicle lumen containing putative Taar1 ligands. This study was designed to explore mouse Taar1 (mTaar1) trafficking, heterologously expressed in human and rat thyroid cell lines in order to establish an in vitro system in which Taar1 signaling from the cell surface can be studied in future. The results showed that chimeric mTaar1-EGFP traffics to the apical cell surface and localizes particularly to spherical structures of polarized thyroid cells, procilia, and primary cilia upon serum-starvation. Moreover, mTaar1-EGFP appears to form high molecular mass forms, possibly homodimers and tetramers, in stably expressing human thyroid cell lines. However, only monomeric mTaar1-EGFP was cell surface biotinylated in polarized human thyrocytes. In polarized rat thyrocytes, mTaar1-EGFP is retained in the endoplasmic reticulum, while cilia were reached by mTaar1-EGFP transiently co-expressed in combination with an HA-tagged construct of the related mTaar5. We conclude that Taar1 trafficking to cilia depends on their integrity. The results further suggest that an in vitro cell model was established that recapitulates Taar1 trafficking in thyrocytes in situ, in principle, and will enable studying Taar1 signaling in future, thus extending our general understanding of its potential significance for thyroid autoregulation.

The Amino Acid Transporter Mct10/Tat1 Is Important to Maintain the TSH Receptor at Its Canonical Basolateral Localization and Assures Regular Turnover of Thyroid Follicle Cells in Male Mice.

Cathepsin K-mediated thyroglobulin proteolysis contributes to thyroid hormone (TH) liberation, while TH transporters like Mct8 and Mct10 ensure TH release from thyroid follicles into the blood circulation. Thus, thyroid stimulating hormone (TSH) released upon TH demand binds to TSH receptors of thyrocytes, where it triggers Gαq-mediated short-term effects like cathepsin-mediated thyroglobulin utilization, and Gαs-mediated long-term signaling responses like thyroglobulin biosynthesis and thyrocyte proliferation. As reported recently, mice lacking Mct8 and Mct10 on a cathepsin K-deficient background exhibit excessive thyroglobulin proteolysis hinting towards altered TSH receptor signaling. Indeed, a combination of canonical basolateral and non-canonical vesicular TSH receptor localization was observed in Ctsk-/-/Mct8-/y/Mct10-/- mice, which implies prolonged Gαs-mediated signaling since endo-lysosomal down-regulation of the TSH receptor was not detected. Inspection of single knockout genotypes revealed that the TSH receptor localizes basolaterally in Ctsk-/- and Mct8-/y mice, whereas its localization is restricted to vesicles in Mct10-/- thyrocytes. The additional lack of cathepsin K reverses this effect, because Ctsk-/-/Mct10-/- mice display TSH receptors basolaterally, thereby indicating that cathepsin K and Mct10 contribute to TSH receptor homeostasis by maintaining its canonical localization in thyrocytes. Moreover, Mct10-/- mice displayed reduced numbers of dead thyrocytes, while their thyroid gland morphology was comparable to wild-type controls. In contrast, Mct8-/y, Mct8-/y/Mct10-/-, and Ctsk-/-/Mct8-/y/Mct10-/- mice showed enlarged thyroid follicles and increased cell death, indicating that Mct8 deficiency results in altered thyroid morphology. We conclude that vesicular TSH receptor localization does not result in different thyroid tissue architecture; however, Mct10 deficiency possibly modulates TSH receptor signaling for regulating thyrocyte survival.

Role of Kallikrein 7 in Body Weight and Fat Mass Regulation.

Increased plasma and adipose tissue protease activity is observed in patients with type 2 diabetes and obesity. It has been proposed that specific proteases contribute to the link between obesity, adipose tissue inflammation and metabolic diseases. We have recently shown that ablation of the serine protease kallikrein-related peptidase 7 (Klk7) specifically in adipose tissue preserves systemic insulin sensitivity and protects mice from obesity-related AT inflammation. Here, we investigated whether whole body Klk7 knockout (Klk7-/-) mice develop a phenotype distinct from that caused by reduced Klk7 expression in adipose tissue. Compared to littermate controls, Klk7-/- mice gain less body weight and fat mass both under chow and high fat diet (HFD) feeding, are hyper-responsive to exogenous insulin and exhibit preserved adipose tissue function due to adipocyte hyperplasia and lower inflammation. Klk7-/- mice exhibit increased adipose tissue thermogenesis, which is not related to altered thyroid function. These data strengthen our recently proposed role of Klk7 in the regulation of body weight, energy metabolism, and obesity-associated adipose tissue dysfunction. The protective effects of Klk7 deficiency in obesity are likely linked to a significant limitation of adipocyte hypertrophy. In conclusion, our data indicate potential application of specific KLK7 inhibitors to regulate KLK7 activity in the development of obesity and counteract obesity-associated inflammation and metabolic diseases.

The Thyroid Hormone Transporter Mct8 Restricts Cathepsin-Mediated Thyroglobulin Processing in Male Mice through Thyroid Auto-Regulatory Mechanisms That Encompass Autophagy.

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.

Mice Hypomorphic for Keap1, a Negative Regulator of the Nrf2 Antioxidant Response, Show Age-Dependent Diffuse Goiter with Elevated Thyrotropin Levels.

Background: Familial nontoxic multinodular goiter (MNG) is a rare disease. One of the associated genes is Kelch-like ECH-associated protein 1 (KEAP1), which encodes the main inhibitor of nuclear factor erythroid 2-related transcription factor 2 (Nrf2), a central mediator of antioxidant responses. The association of KEAP1 with familial MNG is based on only two loss-of-function mutations identified in two families, only one of which included proper phenotyping and adequate demonstration of co-segregation of the phenotype and the mutation. There is no experimental evidence from model organisms to support that decreased Keap1 levels can, indeed, cause goiter. This study used mice hypomorphic for Keap1 to test whether decreased Keap1 expression can cause goiter, and to characterize the activation status of Nrf2 in their thyroid. Methods: C57BL/6J Keap1flox/flox (Keap1 knock-down [Keap1KD]) mice were studied at 3 and 12 months of age. Plasma and thyroid glands were harvested for evaluation of thyroid function tests and for gene and protein expression by real-time polymerase chain reaction and immunoblotting, respectively. Results: Keap1KD mice showed diffuse goiter that began to develop in early adult life and became highly prominent and penetrant with age. The goiter was characterized by a markedly increased size of thyroid follicles, most notably of the colloid compartment, and by absence of thyroid nodules or hyperplasia. Keap1KD mice also showed decreased T4 levels in early adult life that were eventually well compensated over time by increased thyrotropin (TSH) levels. Nrf2 was activated in the thyroid of Keap1KD mice. Despite a known stimulatory effect of Nrf2 on thyroglobulin (Tg) gene transcription and Tg protein abundance, the expression levels were decreased in the thyroid of Keap1KD mice. No clear patterns were observed in the expression profiles of other thyroid hormone synthesis-specific factors, with the exception of Tg-processing and Tg-degrading cathepsins, including an increase in mature forms of cathepsins D, L, and S. Conclusions: Keap1KD mice develop age-dependent diffuse goiter with elevated TSH levels. The precise mechanism accounting for the thyroidal phenotype remains to be elucidated, but it may involve enhanced Tg solubilization and excessive lysosomal Tg degradation.

Procathepsin V Is Secreted in a TSH Regulated Manner from Human Thyroid Epithelial Cells and Is Accessible to an Activity-Based Probe.

The significance of cysteine cathepsins for the liberation of thyroid hormones from the precursor thyroglobulin was previously shown by in vivo and in vitro studies. Cathepsin L is most important for thyroglobulin processing in mice. The present study aims at specifying the possible contribution of its closest relative, cysteine cathepsin L2/V, to thyroid function. Immunofluorescence analysis on normal human thyroid tissue revealed its predominant localization at the apical plasma membrane of thyrocytes and within the follicle lumen, indicating the secretion of cathepsin V and extracellular tasks rather than its acting within endo-lysosomes. To explore the trafficking pathways of cathepsin V in more detail, a chimeric protein consisting of human cathepsin V tagged with green fluorescent protein (GFP) was stably expressed in the Nthy-ori 3-1 thyroid epithelial cell line. Colocalization studies with compartment-specific markers and analyses of post-translational modifications revealed that the chimeric protein was sorted into the lumen of the endoplasmic reticulum and subsequently transported to the Golgi apparatus, while being N-glycosylated. Immunoblotting showed that the chimeric protein reached endo-lysosomes and it became secreted from the transduced cells. Astonishingly, thyroid stimulating hormone (TSH)-induced secretion of GFP-tagged cathepsin V occurred as the proform, suggesting that TSH upregulates its transport to the plasma membrane before it reaches endo-lysosomes for maturation. The proform of cathepsin V was found to be reactive with the activity-based probe DCG-04, suggesting that it possesses catalytic activity. We propose that TSH-stimulated secretion of procathepsin V is the default pathway in the thyroid to enable its contribution to thyroglobulin processing by extracellular means.

Significance of nuclear cathepsin V in normal thyroid epithelial and carcinoma cells.

Altered expression and/or localization of cysteine cathepsins is believed to involve in thyroid diseases including cancer. Here, we examined the localization of cathepsins B and V in human thyroid tissue sections of different pathological conditions by immunolabeling and morphometry. Cathepsin B was mostly found within endo-lysosomes as expected. In contrast, cathepsin V was detected within nuclei, predominantly in cells of cold nodules, follicular and papillary thyroid carcinoma tissue, while it was less often detected in this unusual localization in hot nodules and goiter tissue. To understand the significance of nuclear cathepsin V in thyroid cells, this study aimed to establish a cellular model of stable nuclear cathepsin V expression. As representative of a specific form lacking the signal peptide and part of the propeptide, N-terminally truncated cathepsin V fused to eGFP recapitulated the nuclear localization of endogenous cathepsin V throughout the cell cycle in Nthy-ori 3-1 cells. Interestingly, the N-terminally truncated cathepsin V-eGFP was more abundant in the nuclei during S phase. These findings suggested a possible contribution of nuclear cathepsin V forms to cell cycle progression. Indeed, we found that N-terminally truncated cathepsin V-eGFP expressing cells were more proliferative than those expressing full-length cathepsin V-eGFP or wild type controls. We conclude that a specific molecular form of cathepsin V localizes to the nucleus of thyroid epithelial and carcinoma cells, where it might involve in deregulated pathways leading to hyperproliferation. These findings highlight the necessity to better understand cathepsin trafficking in health and disease. In particular, cell type specificity of mislocalization of cysteine cathepsins, which otherwise act in a functionally redundant manner, seems to be important to understand their non-canonical roles in cell cycle progression.

Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)-A CleavEx Based Analysis.

Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix, modulating the pericellular environment in physiology and in pathologies. The interconnection between these families remains elusive. To assess the cross-activation of these families, we developed a peptide, fusion protein-based exposition system (Cleavage of exposed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences. Initial assessment identified ten MMP activation domain sequences which were validated by Edman degradation. The analysis revealed that membrane-type MMPs (MT-MMPs) are targeted by KLK14 for activation. Correspondingly, proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT-MMPs was confirmed by Edman degradation. The effectiveness of proMMP activation was analyzed by gelatin zymography, confirming the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation. As both, KLKs and MT-MMPs, are implicated in cancer, their cross-activation may constitute an important factor in tumor progression and metastasis.

Auto-Regulation of the Thyroid Gland Beyond Classical Pathways.

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.

Function of Cathepsin K in the Central Nervous System of Male Mice is Independent of Its Role in the Thyroid Gland.

Cathepsin K deficiency in male mice (Ctsk-/-) results in decreased numbers of hippocampal astrocytes and altered neuronal patterning as well as learning and memory deficits. Additionally, cathepsin K carries essential roles in the thyroid gland where it contributes to the liberation of thyroid hormones (TH). Because TH are essential for brain development, in particular for the cerebellum, we investigated whether cathepsin K's function in the thyroid is directly linked to the brain phenotype of Ctsk-/- mice. Serum levels of thyroid stimulating hormone, brain concentrations of free TH, and deiodinase 2 (Dio2) activity in brain parenchyma as well as cerebellar development were comparable in Ctsk-/- and WT animals, suggesting regular thyroid states and TH metabolism. Despite unaltered transcript levels, protein expression of two TH transporters was enhanced in specific brain regions in Ctsk-/- mice, suggesting altered TH supply to these regions. Thyrotropin releasing hormone (Trh) mRNA levels were enhanced threefold in the hippocampus of Ctsk-/- mice. In the striatum of Ctsk-/- mice the mRNA for Dio2 and hairless were approximately 1.3-fold enhanced, while mRNA levels for monocarboxylate transporter 8 and Trh were reduced to 60% and 40%, respectively, pointing to altered striatal physiology. We conclude that the role of cathepsin K in the thyroid gland is not directly associated with its function in the central nervous system (CNS) of mice. Future studies will show whether the brain region-specific alterations in Trh mRNA may eventually result in altered neuroprotection that could explain the neurobehavioral defects of Ctsk-/- mice.

Treatment of rat thyrocytes in vitro with cathepsin B and L inhibitors results in disruption of primary cilia leading to redistribution of the trace amine associated receptor 1 to the endoplasmic reticulum.

Taar1 is a G protein-coupled receptor (GPCR) confined to primary cilia of rodent thyroid epithelial cells. Taar1-deficient mouse thyroid follicles feature luminal accumulation of thyroglobulin suggesting that Taar1 acts as a regulator of extra- and pericellular thyroglobulin processing, which is mediated by cysteine cathepsin proteases present at the apical plasma membrane of rodent thyrocytes. Here, by immunostaining and confocal laser scanning microscopy, we demonstrated co-localization of cathepsin L, but only little cathepsin B, with Taar1 at primary cilia of rat thyrocytes, the FRT cells. Because proteases were shown to affect half-lives of certain receptors, we determined the effect of cathepsin activity inhibition on sub-cellular localization of Taar1 in FRT cells, whereupon Taar1 localization altered such that it was retained in compartments of the secretory pathway. Since the same effect on Taar1 localization was observed in both cathepsin B and L inhibitor-treated cells, the interaction of cathepsin activities and sub-cellular localization of Taar1 was thought to be indirect. Indeed, we observed that cathepsin inhibition resulted in a lack of primary cilia from FRT cells. Next, we proved that primary cilia are a necessity for Taar1 trafficking to reach the plasma membrane of FRT cells, since the disruption of primary cilia by treatment with ß-cyclodextrin resulted in Taar1 retention in compartments of the secretory pathway. Furthermore, in less well-polarized rat thyrocytes, namely in FRTL-5 cells lacking primary cilia, Taar1 was mainly confined to the compartments of the secretory pathway. We conclude that Taar1 localization in polarized thyroid epithelial cells requires the presence of primary cilia, which is dependent on the proteolytic activity of cysteine cathepsins B and L.

Development of Chemical Tools to Monitor Human Kallikrein 13 (KLK13) Activity.

Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH2 demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples.

Aging Alters Phenotypic Traits of Thyroid Dysfunction in Male Mice With Divergent Effects on Complex Systems but Preserved Thyroid Hormone Action in Target Organs.

Clinical manifestation of hyperthyroidism and hypothyroidism vary with age, with an attenuated, oligosymptomatic presentation of thyroid dysfunction (TD) in older patients. We asked, whether in rodents TD phenotypes are influenced by age and whether this involves changes in systemic and/or organ thyroid hormone (TH) signaling. Chronic hyper- or hypothyroidism was induced in male mice at different life stages (5, 12, and 20 months). TH excess resulted in pronounced age-specific body weight changes (increase in youngest and decrease in old mice), neither explained by changes in food intake (similar increase at all ages), nor by thermogenic gene expression in brown adipose tissue (BAT) or TH serum concentrations. Relative increase in body temperature and activity were more pronounced in old compared to young hyperthyroid mice. An attenuated hypothyroid state was found in old mice for locomotor activity and in heart and BAT on functional (less bradycardia) and gene expression level (heart and BAT). In contrast, decrease in body weight was pronounced in old hypothyroid mice. Thus, age has divergent impact on features of TD in mice, whereby effects on highly complex systems, such as energy homeostasis are not proportional to serum TH state, in contrast to organ-specific responses in heart and BAT.