Physical and Functional Interaction of CARMA1 and CARMA3 with Iκ Kinase γ-NFκB Essential Modulator

CARMA proteins are scaffold molecules that contain a caspase recruitment domain and a membrane-associated guanylate kinase-like domain. CARMA1 plays a critical role in mediating activation of the NFkappaB transcription factor following antigen receptor stimulation of both B and T lymphocytes. However, the biochemical mechanism by which CARMA1 regulates activation of NFkappaB remains to be determined. Here we have shown that CARMA1 and CARMA3 physically associate with Ikappa kinase gamma/NFkappaB essential modulator (IkappaKgamma-NEMO) in lymphoid and non-lymphoid cells. CARMA1 participates to an inducible large molecular complex that contains IkappaKgamma/NEMO, Bcl10, and IkappaKalpha/beta kinases. Expression of the NEMO-binding region of CARMA3 exerts a dominant negative effect on Bcl10-mediated activation of NFkappaB. Thus, our results provide direct evidence for physical and functional interaction between CARMA and the IkappaK complex and offer a biochemical framework to understand the molecular activities controlled by CARMA-1, -2, and -3 and Bcl10

Defective Thyroglobulin: Cell Biology of Disease

The primary functional units of the thyroid gland are follicles of various sizes comprised of a monolayer of epithelial cells (thyrocytes) surrounding an apical extracellular cavity known as the follicle lumen. In the normal thyroid gland, the follicle lumen is filled with secreted protein (referred to as colloid), comprised nearly exclusively of thyroglobulin with a half-life ranging from days to weeks. At the cellular boundary of the follicle lumen, secreted thyroglobulin becomes iodinated, resulting from the coordinated activities of enzymes localized to the thyrocyte apical plasma membrane. Thyroglobulin appearance in evolution is essentially synchronous with the appearance of the follicular architecture of the vertebrate thyroid gland. Thyroglobulin is the most highly expressed thyroid gene and represents the most abundantly expressed thyroid protein. Wildtype thyroglobulin protein is a large and complex glycoprotein that folds in the endoplasmic reticulum, leading to homodimerization and export via the classical secretory pathway to the follicle lumen. However, of the hundreds of human thyroglobulin genetic variants, most exhibit increased susceptibility to misfolding with defective export from the endoplasmic reticulum, triggering hypothyroidism as well as thyroidal endoplasmic reticulum stress. The human disease of hypothyroidism with defective thyroglobulin (either homozygous, or compound heterozygous) can be experimentally modeled in thyrocyte cell culture, or in whole animals, such as mice that are readily amenable to genetic manipulation. From a combination of approaches, it can be demonstrated that in the setting of thyroglobulin misfolding, thyrocytes under chronic continuous ER stress exhibit increased susceptibility to cell death, with interesting cell biological and pathophysiological consequences

Increased hexosamine biosynthetic pathway flux alters cell-cell adhesion in INS-1E cells and murine islets

Purpose In type 2 Diabetes, beta-cell failure is caused by loss of cell mass, mostly by apoptosis, but also by simple dysfunction (dedifferentiation, decline of glucose-stimulated insulin secretion). Apoptosis and dysfunction are caused, at least in part, by glucotoxicity, in which increased flux of glucose in the hexosamine biosynthetic pathway plays a role. In this study, we sought to clarify whether increased hexosamine biosynthetic pathway flux affects another important aspect of beta-cell physiology, that is beta-cell-beta-cell homotypic interactions. Methods We used INS-1E cells and murine islets. The expression and cellular distribution of E-cadherin and beta-catenin was evaluated by immunofluorescence, immunohistochemistry and western blot. Cell-cell adhesion was examined by the hanging-drop aggregation assay, islet architecture by isolation and microscopic observation. Results E-cadherin expression was not changed by increased hexosamine biosynthetic pathway flux, however, there was a decrease of cell surface, and an increase in intracellular E-cadherin. Moreover, intracellular E-cadherin delocalized, at least in part, from the Golgi complex to the endoplasmic reticulum. Beta-catenin was found to parallel the E-cadherin redistribution, showing a dislocation from the plasmamembrane to the cytosol. These changes had as a phenotypic consequence a decreased ability of INS-1E to aggregate. Finally, in ex vivo experiments, glucosamine was able to alter islet structure and to decrease surface abundandance of E-cadherin and beta-catenin. Conclusion Increased hexosamine biosynthetic pathway flux alters E-cadherin cellular localization both in INS-1E cells and murine islets and affects cell-cell adhesion and islet morphology. These changes are likely caused by alterations of E-cadherin function, highlighting a new potential target to counteract the consequences of glucotoxicity on beta-cells

patologia del metabolismo lipidico

Struttura e metabolismo delle lipoproteine plasmatiche, Enzimi e trasportatori chiave del metabolismo delle lipoproteine, Recettori delle lipoproteine, Metabolismo delle lipoproteine contenenti apoB, Biogenesi delle HDL e trasporto inverso del colesterolo, Principali malattie del metabolismo lipidico

Thyroglobulin from Molecular and Cellular Biology to Clinical Endocrinology

Thyroglobulin (Tg) is a vertebrate secretory protein synthesized in the thyrocyte endoplasmic reticulum (ER) where it acquires N-linked glycosylation and conformational maturation (including formation of many disulfide bonds), leading to homodimerization. Its primary functions include iodide storage and thyroid hormonogenesis. Tg consists largely of repeating domains, and many tyrosyl residues in these domains become iodinated to form monoiodo- and diiodotyrosine, whereas only a small portion of Tg structure is dedicated to hormone formation. Interestingly, evolutionary ancestors, dependent upon thyroid hormone for development, synthesize thyroid hormones without the complete Tg protein architecture. Nevertheless, in all vertebrates, Tg follows a strict pattern of region I, II-III, and the Cholinesterase-Like (ChEL) domain. In vertebrates, Tg first undergoes intracellular transport through the secretory pathway, which requires the assistance of thyrocyte ER chaperones and oxidoreductases, as well as coordination of distinct regions of Tg, to achieve a native conformation. Curiously, regions II-III and ChEL behave as fully independent folding units that could function as successful secretory proteins by themselves. However, the large Tg region I (bearing the primary thyroxine-forming site) is incompetent by itself for intracellular transport, requiring the downstream regions II-III and ChEL to complete its folding. A combination of nonsense mutations, frameshift mutations, splice site mutations, and missense mutations in Tg occur spontaneously to cause congenital hypothyroidism and thyroidal ER stress. These Tg mutants are unable to achieve a native conformation within the ER, interfering with the efficiency of Tg maturation and export to the thyroid follicle lumen for iodide storage and hormonogenesis

The cholinesterase-like domain of thyroglobulin functions as an intramolecular chaperone.

Thyroid hormonogenesis requires secretion of thyroglobulin, a protein comprising Cys-rich regions I, II, and III (referred to collectively as region I-II-III) followed by a cholinesterase-like (ChEL) domain. Secretion of mature thyroglobulin requires extensive folding and glycosylation in the ER. Multiple reports have linked mutations in the ChEL domain to congenital hypothyroidism in humans and rodents; these mutations block thyroglobulin from exiting the ER and induce ER stress. We report that, in a cell-based system, mutations in the ChEL domain impaired folding of thyroglobulin region I-II-III. Truncated thyroglobulin devoid of the ChEL domain was incompetent for cellular export; however, a recombinant ChEL protein ("secretory ChEL") was secreted efficiently. Coexpression of secretory ChEL with truncated thyroglobulin increased intracellular folding, promoted oxidative maturation, and facilitated secretion of region I-II-III, indicating that the ChEL domain may function as an intramolecular chaperone. Additionally, we found that the I-II-III peptide was cosecreted and physically associated with secretory ChEL. A functional ChEL domain engineered to be retained intracellularly triggered oxidative maturation of I-II-III but coretained I-II-III, indicating that the ChEL domain may also function as a molecular escort. These insights into the role of the ChEL domain may represent potential therapeutic targets in the treatment of congenital hypothyroidism

Increase of [Ca2+]i via activation of ATP receptors in PC-Cl3 rat thyroid cell line

In PC-Cl3 rat thyroid cell line, ATP and UTP provoked a transient increase in [Ca2+](i), followed by a lower sustained phase. Removal of extracellular Ca2+ reduced the initial transient response and completely abolished the plateau phase. Thapsigargin (TG) caused a rapid rise in [Ca2+](i) and subsequent addition of ATP was without effect. The transitory activation of [Ca2+](i) was dose-dependently attenuated in cells pretreated with the specific inhibitor of phospholipase C (PLC), U73122. These data suggest that the ATP-stimulated increment of [Ca2+](i) required InsP(3) Formation and binding to its specific receptors in Ca2+ stores. Desensitisation was demonstrated with respect to the calcium response to ATP and UTP in Fura 2-loaded cells. Further studies were performed to investigate whether the effect of ATP on Ca2+ entry into PC-Cl3 cells was via L-type voltage-dependent Ca2+ channels (L-VDCC) and/or by the capacitative pathway. Nifedipine decreased ATP-induced increase on [Ca2+](i). Addition of 2 mM Ca2+ induced a [Ca2+](i) rise after pretreatment of the cells with TG or with 100 muM ATP in Ca2+-free medium, These data indicate that Ca2+ entry into PC-Cl3 stimulated with ATP occurs through both an L-VDCC and through a capacitative pathway. Using buffers with differing Na+ concentrations, we found that the effects of ATP were dependent of extracellular Na+, suggesting that a Na+/Ca2+ exchange mechanism is also operative. These data suggest the existence, in PC-Cl3 cell line, of a P2Y purinergic receptor able to increase the [Ca2+](i), via PLC activation, Ca2+ store depletion, capacitative Ca2+ entry and L-VDCC activation

ER stress impairs MHC Class I surface expression and increases susceptibility of thyroid cells to NK-mediated cytotoxicity.

We recently reported that, in thyroid cells, ER stress triggered by thapsigargin or tunicamycin, two well known ER stressing agents, induced dedifferentiation and loss of the epithelial phenotype in rat thyroid cells. In this study we sought to evaluate if, in thyroid cells, ER stress could affect MHC class I expression and the possible implications of this effect in the alteration of function of natural killer cells, suggesting a role in thyroid pathology. In both, a human line of fetal thyroid cells (TAD-2 cells) and primary cultures of human thyroid cells, thapsigargin and tunicamicin triggered ER stress evaluated by BiP mRNA levels and XBP-1 splicing. In both cell types, TAD-2 cell line and primary cultures, major histocompatibility complex class I (MHC-I) plasmamembrane expression was significantly reduced by ER stress. This effect was accompanied by signs of natural killer activation. Thus, natural killer cells dramatically increased IFN-γ production and markedly increased their cytotoxicity against thyroid cells. Together, these data indicate that ER stress induces a decrease of MHC class I surface expression in thyroid cells, resulting in reduced natural killer-cell self-tolerance

Prediction of the secondary structure of the carboxy-terminal third of rat thyroglobulin.

A secondary structure prediction has been made using the available primary sequence data of the proposed carboxy-terminal of rat thyroglobulin. The model predicts 22% alfa-helix, 28% beta-structure and 17% beta turns. Out of the 8 possible carbohydrate acceptor-sites (Asn-x-Ser/Thr), 3 (residues 136, 368, 782) are associated with peptide sequences which favour the formation of beta-turn or loop-structures and are located in high hydrophilic regions. The entire sequence is predicted to be made up of two domains: one of them is highly structured, contains the hormonogenic sites, a cluster of tyrosines and at least one carbohydrate acceptor site