Cell Biology
The innate immune response is the first line of defense against viral infection. Host pattern recognition receptors (PRRs) detect pathogen-associated molecular patterns (PAMPs) and trigger several signaling pathways following viral infections. One such pathway, the cGAS-STING pathway, detects cytosolic DNA to induce the production and secretion of type I interferons.
The cGAS-STING pathway is activated by the presence of cytosolic DNA. cGAS (cGMP-AMP synthase), a cytosolic DNA detector, binds double-stranded DNA, dimerizes, and catalyzes the production of the second messenger cyclic GMP-AMP (cGAMP) from GTP and ATP. STING, an ER adaptor protein, binds to cGAMP and becomes activated through dimerization, which results in its trafficking from the ER to the Golgi. At the Golgi, STING recruits the kinase TBK1 and the transcription factor IRF3. TBK1 phosphorylates STING, itself, and IRF3. Phosphorylation activates IRF3, causing its translocation to the nucleus, where it induces the production of type I interferons.
To counteract the host immune response, human cytomegalovirus (HCMV) encodes several immunoevasin proteins. HCMV glycoproteins such as US6 inhibit antigen presentation by blocking peptide transport via the transporter associated with antigen processing (TAP). The region spanning amino acids 89–108 of US6 was identified as responsible for TAP inhibition.
Our studies have identified a novel interaction and function of US6. For the first time, we show that US6 interacts with the host p24 proteins TMED2 and TMED10. US6 inhibits the cytosolic DNA-triggered cGAS-STING pathway and reduces the production of IFNβ1. We have discovered a correlation between the binding of US6 to TMED2 and TMED10 and its ability to inhibit the production of type I interferons. Using sequential mutants, we show that US6 possesses two distinct and separable regions responsible for its functions. Microscopy reveals that US6 delays STING trafficking from the ER to the Golgi.
While altered thyroid phenotypes can arise due to cathepsin deficiencies, functional compensation via the upregulation of cathepsin L, despite normal serum thyroid stimulating hormone (TSH) levels, in cathepsin K-deficient (Ctsk-/-) mice is unexpected. Furthermore, these mice show normal serum thyroid hormone (TH) status by increased monocarboxylate transporter Mct8-mediated TH export. Thus, alternative thyroid regulatory mechanisms that are independent of the canonical hypothalamus-pituitary-thyroid axis might exist, making Ctsk-/- mice a suitable model to study “thyroid auto-regulation”. The first part of the present study elucidates the possible roles of Mct8 and Mct10 in non-canonical regulation of cathepsin-mediated thyroglobulin (Tg) proteolysis. We assessed the thyroid phenotypes in combined cathepsin K and TH transporter deficiency, i.e., in Ctsk-/-/Mct10-/-, Ctsk-/-/Mct8-/y, and Ctsk-/-/Mct8-/y/Mct10-/- mice. Results revealed that induced lysosomal biogenesis due to autophagy, possibly triggered by thyrotoxicity, causes persistent Tg proteolysis in a counterintuitive manner in Mct8-deficient genotypes. The second part of the study examines the factors triggering autophagy in the Ctsk-/-/Mct8-/y/Mct10-/- murine model. Since Lat2 regulates autophagy in kidney and pancreas, we propose that Lat2 regulates autophagy in thyrocytes as well. Indeed, Lat2 protein amounts were significantly decreased in autophagy-induced Ctsk-/-/Mct8-/y/Mct10-/- mice. Furthermore, Lat2-/- thyroid glands showed autophagy induction and lysosomal biogenesis. The third part of this study explores whether altered TSH receptor localization can lead to differences in thyroid gland architecture. Results revealed that Mct10 deficiency results in vesicular TSH receptors as opposed to canonical basolateral TSH receptors in controls. Although altered TSH receptor localization does not result in gross alterations in thyroid gland morphology, thyrocyte survival is possibly regulated by Mct10.
As cell biologists and specialized in proteases, our group has made significant contributions to the understanding of the role of cathepsins, in the maintenance of thyroid function. In this study, we examined the significance of cathepsin V in thyroid physiology and carcinogenesis. Immunofluorescence analysis of non-cancerous and cancerous human thyroid tissue showed the localization of cathepsin V at the apical plasma membrane domain of thyrocytes and within the follicle lumen of normal thyroid tissue, whereas cathepsin V was found within nuclei in cells of follicular and papillary thyroid carcinoma tissue. These results prompted us to explore which molecular form of cathepsin V can be secreted and/or sorted to the nuclear compartment of thyrocytes. To this end, we created two cell lines stably expressing full-length (hCV-eGFP) and N-terminally truncated cathepsin V (h56NCV-eGFP), both tagged with eGFP at their C-termini, which are referred to as Nthyori-CV and Nthyori-NCV, respectively. We found that hCV-eGFP chimeric protein follows the canonical transport pathway of cathepsins from the rough endoplasmic reticulum (ER) lumen through the Golgi apparatus to endo-lysosomes. In addition, our results revealed that Nthyori-CV cells secrete the proform of the hCV-eGFP chimeric protein in a TSH-regulated pattern into the extracellular space. On the other hand, h56NCV-eGFP chimeric protein which lacks the ER lumen-targeting signal peptide and part of the propeptide escaped the secretory pathway, which explains its unusual presence in the cytosol and accumulation in the nuclei of Nthyori-NCV cells. In particular, it was more abundant in the nuclei during S phase, suggesting that nuclear cathepsin V might be involved in the regulation of cell cycle progression of thyrocytes. Thus, we investigated the effect of h56NCV-eGFP expression on the proliferation rate of thyrocytes. We found that Nthyori-NCV cells are more proliferative than both Nthyori-CV and wild-type controls.
NKG2D is a crucial Natural Killer (NK) cell activating receptor, and the murine cytomegalovirus (MCMV) employs multiple immunoevasins in order to avoid NKG2D-mediated activation. One of the MCMV immunoevasins, gp40 (m152), downregulates the cell surface NKG2D ligand, RAE-1 gamma, thus limiting NK cell activation. My study establishes the molecular mechanism by which gp40 retains RAE-1 gamma in the secretory pathway. Using flow cytometry and pulse chase analysis, I demonstrate that gp40 retains RAE-1 gamma in the early secretory pathway, and that this effect depends on the binding of gp40 to a host protein, TMED10, a member of the p24 protein family. I also show that the TMED10-based retention mechanism can be saturated, and that gp40 has a backup mechanism as it masks RAE-1 gamma on the cell surface, blocking the interaction with the NKG2D receptor and thus NK cell activation.
Planar surfaces with geometric protein patterns have been developed for various applications in biotechnology, such as orienting cells, arranging membrane proteins, or studying protein-ligand binding in massively parallel approaches. Geometric shapes and dimensions of protein patterns vary depending on the application. For the study of proteins in living cells, protein patterns in the micrometer range are commonly used; these are called protein micropatterns. Diverse lithography techniques have been used and were further developed to fabricate even complicated protein micropatterns on various surfaces. One of the first techniques to immobilize proteins in geometric patterns on glass surfaces was microcontact printing, which is a rather simple stamping approach that immobilizes proteins by physical absorption onto surfaces.
MHC class I are peptide receptors that present the cell´s proteome at the cell surface to T cells. They thus play an essential role in the adaptive immune response against cells that are infected by viruses, bacteria, or that carry tumorigenic mutations. We have adapted the technology of protein micropatterns to the field of MHC class I and have used microcontact printing to immobilize anti-MHC class I antibodies on glass surfaces to develop an MHC class I capture assay. The development of this assay consisted of optimization and trial experiments; they finally established a robust assay that can be used to specifically capture MHC class I in living cells.
In the field of MHC class I antigen presentation, we have identified two applications for the capture assay. First, a novel peptide binding assay was developed that allows for the monitoring of specific peptide binding to captured MHC class I in living cells. Further development of the assay led to the finding that the use of conformation-specific antibodies allows for differential capture of different structural forms of MHC class I. Excitingly, this enables the investigation of conformation-sp
The thyroid gland is an endocrine organ responsible for producing thyroid hormones, which are essential for normal growth and development, and for maintaining functional homeostasis of different organ systems. The thyroid is known to be classically regulated by the hypothalamic-pituitary-thyroid (HPT) axis which, in short, involves hypothalamic release of thyrotropin-releasing hormone (TRH) in response to low levels of thyroid hormones in the circulation. Through positive feedback, the TRH signals to the pituitary to release thyroid stimulating hormone (TSH), which, in turn, binds and activates thyrocytes to release thyroid hormones. Replenishment of thyroid hormones in circulation then serves as a negative feedback to quench TRH and TSH release. Growing evidence, including the non-genomic effect of thyroid hormones (T3 and T4), the selective thyroid hormone transporters that regulate cellular thyroid hormone uptake and release (including in the thyrocytes), as well as the discovery of endogenous thyroid hormone derivatives, the thyronamines, and their physiological effects, collectively challenge the classical view on thyroid regulation. The implications of that is that more complex regulatory mechanisms might exist, that are capable modulating thyroid function, also at the level of the thyroid follicle itself – the functional unit of the thyroid gland. Such pathways may be independent of the HPT axis, and are collectively referred to as “thyroid auto-regulation”.
Trace amine-associated receptor 1 (Taar1) was identified in 2001 as a receptor related to the serotonin receptor family. Taar1 was found to be expressed in thyroid and pancreas. Therefore, we designed this study in which Taar1 expression and function in the thyroid gland and pancreas of mice were analysed.
The sub-cellular localization of a receptor like Taar1 determines its availability for putative ligands. Therefore, sub-cellular localization of Taar1 was analysed in thyroid tissue. Taar1 localization was confined to primary cilia. It is also noteworthy that sub-cellular localization of the Tg-processing protease, cathepsin L, was confined to primary cilia of thyrocytes. Thus, we were interested if an interaction of Taar1 with cathepsins takes place in the thyroid, and whether this affects the sub-cellular localization of Taar1. To this aim, sub-cellular localization of Taar1 was investigated in FRT cells treated with cathepsin inhibitors. Experiments revealed that Taar1 localization is altered upon inhibition of cathepsin activity.
Furthermore, we determined the phenotype of the thyroid gland of taar1-/- mice in comparison to WT controls. Serum thyroid hormone concentrations were slightly decreased upon Taar1 deficiency, which was correlated with decreased expression levels and proteolytic activities of cathepsins in the thyroid gland of taar1-/- mice vs WT controls. Thereby, taar1-/- mice were characterized as mildly hypothyroid, revealing the importance of Taar1 for regulation of thyroid gland functions.
Taar1 was shown previously to be also expressed in β cells of the pancreas. Thus, we aimed to investigate blood glucose concentrations upon Taar1 deficiency. However, the data showed that blood glucose levels were not affected by Taar1 deficiency. Collectively, this study revealed that Taar1 is important for various aspects of endocrine regulation in mice.
Supported by Deutsche Forschungsgemeinschaft in the framework of SPP 1629, BR1308/11-1 and 11-2.
Introduction: Thyroid hormones (TH) are important for almost all organs in the body. TH transport into target cells is crucial for their biological action. The thyroid gland, as the TH producing organ, is also a target of its hormones. Therefore regulation of the processing of the TH precursor molecule thyroglobulin and transport of TH across the plasma membrane of thyroid epithelial cells is vital for thyroid development and function.
Objectives and Methods: We hypothesized that the thyroid gland must exhibit TH sensing mechanisms to ensure extra- and intra-thyroidal TH homeostasis and therefore investigated the relationship between thyroglobulin processing enzymes and TH transporters. Thereby, the auto-regulation of the mouse thyroid gland in general and in a TSH-independent manner was addressed. Hence, mouse models were bred combining both, TH transporter- and cysteine cathepsin-deficiency, and a computer-based thyroid phenotyping tool was developed to quantitatively analyse thyroid morphology and functional states. TH transporter expression and localization patterns were analysed in cysteine cathepsin-deficient animals as models of impaired thyroglobulin processing, and in reverse, the protein levels of cathepsins in TH transporter-deficient mice with altered serum TH levels were addressed.
Results: The established diagnostic tool offers a non-biased and reproducible thyroid phenotyping system to basic thyroid research. The functional assessment of the angio-follicular unit by our phenotyping tool reveals how the structure-function-relationship contributes to the compensation of functional losses in the mouse thyroid gland. Additionally, thyroid gland tissue of Mct8- and Mct10- double-deficient mice revealed both, increased cathepsin L expression levels and increased thyroglobulin solubilisation and degradation. Interestingly, animals deficient in both, cathepsin K and Mct8, exhibited apical Lat2 signal, whereas the knock-out of Mct10 led to decreased Lat2 levels in thyrocytes.
Conclusion: These results indicate that a possible sensing mechanism might involve TH transporter Lat2 as `transceptor´ in a localisation-dependent signalling pathway linking sensing capabilities and lysosomal function in thyrocytes, the latter being disrupted in Mct8- and Mct10- double-deficient mice leading to increased TH liberation potential despite the already toxic intra-thyroidal TH levels. In summary, TSH-independent regulative measures of the thyroid gland were described and point towards a more complex thyroid regulation than described before.
Supported by the Deutsche Forschungsgemeinschaft, in the framework of the SPP 1629 “Thyroid Trans Act” project, BR 1308/11-1.
Cysteine cathepsins are proteases involved in distinct processes essential for cellular homeostasis in a variety of tissues including the gastrointestinal tract. Since cathepsins are part of a complex proteolytic network, any imbalance in their localization and activity can influence the structural and functional integrity of the intestine. In the present study, we demonstrate that absence of cathepsin B in mice results in elevated levels of cathepsin X in a segment-specific manner. Furthermore, we provide evidence for the notion that each part of the gastrointestinal tract is characterized by its distinct proteolytic profile which requires tightly balanced protein transport logistics in intestine epithelial cells.
Moreover, several reports have shown that the so called “endo-lysosomal” enzymes of the gastrointestinal tract often localize to unusual sites such as in the cytosol or in mitochondria, and in the nuclei of both, epithelial and carcinoma cells. Therefore, in our subsequent in vitro study, we investigated the localization and expression of cathepsins B and L in the normal enterocyte cell line IEC6 in comparison with the colon carcinoma cell lines Caco2, HCT116, and SW620 throughout different phases of the cell cycle. Our results revealed the presence of cathepsins B and L in the nucleus of colon carcinoma cells, and demonstrated the abundance of cytosolic cathepsins which were found to be co localized with β tubulin during M phase in the carcinoma cells, while cathepsin L was the only enzyme detectable in the nuclei of normal epithelial cells. Additionally, immunoblotting of subcellular fractions demonstrated a distinct molecular form of cathepsin B in the nuclei of carcinoma cells. Furthermore, the cell cycle analysis demonstrated the necessity of cathepsin L activity for cell cycle progression of HCT116 cells. Next, we compared the trafficking of enhanced green fluorescent protein tagged full length and N terminally truncated cathepsins B and L in colon carcinoma cells, which exhibited a juxtanuclear localization in aggresomes of HCT116 cells, whereas full-length cathepsin B followed the expected transport pathway to endo lysosomes.
Overall, our studies showed the segment-specific profile of cysteine cathepsins in the gastrointestinal tract of mice, and suggested a significant role of cysteine cathepsins during cell cycle progression. The results of this study are important to understand the normal gastrointestinal tract homeostasis, and contribute to shedding light on the onset and progression of colon cancer.
Major Histocompatibility Complex (MHC) class I molecules are cell surface glycoprotein complexes that present endogenous peptides to cytotoxic T cells of the immune system. The trimeric MHC class I molecules (heavy chain, light chain, and peptide) are assembled in the early secretory pathway - the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC), and the cis side of the Golgi apparatus. MHC class I molecules then progress to the medial Golgi and follow the secretory pathway to the cell surface.
The many known human and murine allotypes of MHC class I differ in their rates of cell surface transport. This difference might be caused by allotype-specific protein folding and assembly, transport to ER exit sites (ERES), recruitment into ER-to-Golgi transport carriers, or retrieval processes from the ERGIC or the cis-Golgi to the ER. In this work, I have evaluated the contribution of each of these processes to MHC class I cell surface transport with two murine allotypes as model proteins, H-2Db (slow transport) and H-2Kb (fast transport), using radioactive pulse-chase experiments, in vitro generation of ER-to-Golgi transport carriers, and protein folding assays. From the results, I conclude that both proteins are recruited to ER-to-Golgi transport carriers with the same efficiency, but that a novel pre-ER exit step delays their access to ERES. This step is mediated by proteins of the ER matrix, and it acts on the peptide-bound lumenal domain of MHC class I.
I have also analyzed the ER exit of proteins that participate in MHC class I assembly: the peptide transporter TAP, the chaperone tapasin, and the lectin calreticulin. In my experiments, these proteins leave the ER and may promote MHC class I assembly in post-ER compartments, though only to a minor extent.
My data support a model, in which MHC class I protein complexes are predominantly assembled in the ER. I propose that the cell surface transport rate for MHC class I molecules is determined by the rates of protein folding and unfolding, the affinity of all MHC class I folding intermediates to proteins of the ER matrix, and the proteostasis in the ER and the secretory pathway.
