NPC1L1 and SR-BI are involved in intestinal cholesterol absorption from small-size lipid donors.

In the human intestinal content after a meal, cholesterol is dispersed in a complex mixture of emulsified droplets, vesicles, mixed micelles and precipitated material. The aim of this study was to determine the contribution of the main intestinal cholesterol transporters (NPC1L1, SR-BI) to the absorption processes, using different cholesterol-solubilizing donors. Cholesterol donors prepared with different taurocholate concentrations were added to an apical medium of differentiated TC7/Caco-2 cells. As the taurocholate concentrations increased, cholesterol donor size decreased (from 712 to 7 nm in diameter), which enhanced cholesterol absorption in a dose-dependent manner (38-fold). Two transport processes were observed: (1) absorption from large donors exhibited low-capacity transport with no noticeable transporter contribution; (2) efficient cholesterol absorption occurs from small lipid donors (

A plasminogen-like protease in thyroid rough microsomes degrades thyroperoxidase and thyroglobulin.

Proteasome activity takes place in the cytosolic compartment and acts to degrade several proteins translated and unfolded. In transfected CHO cells expressing thyroid peroxidase (TPO), just-translated TPO undergoes proteasome activity, and then a second proteolytic system degrades more mature forms of TPO. A plasminogen-like (Pl-like) protease is found in microsomal liver membranes and in the thyroid. In the thyroid, this Pl-like protease is localized in the follicular lumen and efficiently degrades thyroglobulin (Tg) in vitro. Here we checked for the presence, in purified endoplasmic reticulum (ER) membranes of transfected CHO and in rough microsomes purified from thyroid tissue, of a second proteolytic system, different from the proteasome, and active against the two major proteins of the thyroid gland, TPO and Tg. We first confirmed that this proteolytic system was able to degrade folded endogenous TPO. We showed also that externally added TPO (folded form) was degraded by opened vesicles of ER in the same system. For thyroid tissue, we showed that added TPO, as well as purified Tg, was degraded by some unknown membrane-associated protease(s) in human and porcine thyroid rough microsomes, whereas BSA and IgG were not. These results indicated that major thyroid glycoproteins are preferential substrates of such protease(s). Immunoblot and zymography experiments identified the unknown membrane-associated protease in rough microsomes from thyroid tissues as being a Pl-like protease. These results highly suggest that this system acts as a nonproteasomal degradation enzyme at the ER level, and we hypothesize that it contributes in regulating the level of major thyroid glycoproteins.

The plasminogen-like molecule apically secreted by epithelial thyroid cells is sulfated.

Plasminogen (Pl), a circulating protease synthesized in the liver, is also present in several tissues. In the thyroid gland a Pl-like protease was found in the apical lumen where it is involved, through its proteolytic activity, in luminal degradation of thyroglobulin (Tg). Here, we showed for the first time that the Pl-like protease apically secreted by epithelial thyroid cells is sulfated, both on tyrosine residue(s) and on oligosaccharide side chains. The Pl molecule is composed of a large N-terminal moiety made of five distinct Kringle domains (K1-K5) separated by small peptidic fragments, and of a C-terminal domain with serine protease activity. Using a software tool able to predict tyrosine sulfation sites in protein sequences we localized the potential tyrosine sulfation sites of Pl. Then, we became aware that, whatever the species considered, at least three of the four potential tyrosine sulfation sites of Pl were located on Kringle sites, and more precisely, for K1, on the highly conserved binding domain of K1. We determined with the same software tool which potential sulfation sites were the most likely to be really sulfated. We hypothesize that the sulfation of these sites modulates the binding properties of Pl.

A plasminogen-like protein, present in the apical extracellular environment of thyroid epithelial cells, degrades thyroglobulin in vitro.

The prothyroid hormone, thyroglobulin (Tg), is stored at high concentrations in the thyroid follicular lumen as a soluble 19S homo-dimer and as heavier soluble (27S and 37S) and insoluble (Tgm) forms. Follicular degradation of Tg may contribute to maintaining Tg concentrations compatible with follicle integrity. Here, we report on the presence of a plasminogen-like protein in the follicular lumen of normal human thyroids and its synthesis and apical secretion by cultured epithelial thyroid cells. Since all the main luminal forms of Tg are cleaved by this plasminogen-like protein, we suggest that it contributes to Tg degradation in the follicular lumen.

Association of the thyrotropin receptor with calnexin, calreticulin and BiP. Efects on the maturation of the receptor.

The thyrotropin receptor (TSHR) is a member of the G protein-coupled receptor superfamily. It has by now been clearly established that the maturation of the glycoproteins synthesized in the endoplasmic reticulum involves interactions with molecular chaperones, which promote the folding and assembly of the glycoproteins. In this study, we investigated whether calnexin (CNX), calreticulin (CRT) and BiP, three of the main molecular chaperones present in the endoplasmic reticulum, interact with the TSHR and what effects these interactions might have on the folding of the receptor. In the first set of experiments, we observed that in a K562 cell line expressing TSHR, about 50% of the receptor synthesized was degraded by the proteasome after ubiquitination. In order to determine whether TSHR interact with CNX, CRT and BiP, coimmunoprecipitation experiments were performed. TSHR was found to be associated with all three molecular chaperones. To study the role of the interactions between CNX and CRT and the TSHR, we used castanospermine, a glucosidase I and II inhibitor that blocks the interactions between these chaperones and glycoproteins. In K562 cells expressing the TSHR, these drugs led to a faster degradation of the receptor, which indicates that these interactions contribute to stabilizing the receptor after its synthesis. The overexpression of calnexin and calreticulin in these cells stabilizes the receptor during the first hour after its synthesis, whereas the degradation of TSHR increased in a cell line overexpressing BiP and the quantity of TSHR able to acquire complex type oligosaccharides decreased. These results show that calnexin, calreticulin and BiP all interact with TSHR and that the choice made between these two chaperone systems is crucial because each of them has distinct effects on the folding and stability of this receptor at the endoplasmic reticulum level.