Production of an antigenic peptide by insulin-degrading enzyme.

Most antigenic peptides presented by major histocompatibility complex (MHC) class I molecules are produced by the proteasome. Here we show that a proteasome-independent peptide derived from the human tumor protein MAGE-A3 is produced directly by insulin-degrading enzyme (IDE), a cytosolic metallopeptidase. Cytotoxic T lymphocyte recognition of tumor cells was reduced after metallopeptidase inhibition or IDE silencing. Separate inhibition of the metallopeptidase and the proteasome impaired degradation of MAGE-A3 proteins, and simultaneous inhibition of both further stabilized MAGE-A3 proteins. These results suggest that MAGE-A3 proteins are degraded along two parallel pathways that involve either the proteasome or IDE and produce different sets of antigenic peptides presented by MHC class I molecules.

Thyrotropin activates guanosine 5'-diphosphate/guanosine 5'-triphosphate exchange on the rate-limiting endocytic catalyst, Rab5a, in human thyrocytes in vivo and in vitro.

CONTEXT: We have previously reported that the TSH receptor/cAMP cascade enhances the coordinate expression of the rate-limiting endocytic catalysts, Rab5a and Rab7, which respectively promote thyroglobulin (Tg) internalization and transfer to lysosomes, thereby accelerating thyroid hormone secretion. OBJECTIVE: We address whether TSH further controls Rab5a activity by promoting its GTP-bound state. DESIGN: We compared Rab5a activation in seven pairs of hyperactive and corresponding quiescent thyroid tissues; TSH effect was reproduced on polarized cultures of normal human thyrocytes. PATIENTS: We studied seven euthyroid patients bearing hyperactive autonomous adenomas; normal thyroid tissue for culture. MAIN OUTCOME MEASUREMENTS: Rab5a GDP/GTP exchange factor activity [Rab5a-guanine nucleotide exchange factor (GEF)], expression of Rabex-5 (a Rab5a-GEF), and function of thyrocytes in vitro were the main outcome measures. RESULTS: In autonomous adenomas, constitutive activation increased both total activity and sedimentability (membrane recruitment) of Rab5a-GEF, compared with perinodular tissues. Increased Rab5a-GEF activity correlated with increased expression of Rabex-5 and Rab5a, as well as with Tg store depletion. In polarized human thyrocyte monolayers, TSH did not affect total Rab5a-GEF activity after 2 h but promoted its membrane recruitment; after 4 d, TSH increased both Rab5a-GEF activity and Rabex-5 expression and recruitment onto membranes where Rabex-5 coimmunoprecipitated with Rabaptin-5 and Rab5a. Sedimentable Rab5a-GEF perfectly correlated with apical endocytosis and lysosomal transfer of 125I-Tg, and with basolateral secretion of 125I-derived hormones. CONCLUSION: This study provides the first clinical and experimental evidence that regulation of the activity of a rate-limiting endocytic catalyst finely tunes a tightly controlled cellular function that ultimately governs whole body metabolism.

Infection by Mycoplasma hyorhinis strongly enhances uptake of antisense oligonucleotides: a reassessment of receptor-mediated endocytosis in the HepG2 cell line.

This paper shows that the approximately 66 kDa band, previously isolated from the HepG2 cell line as an oligonucleotide (ON) plasma membrane 'receptor', is induced by Mycoplasma infection. Moreover, this band has been identified as the invariant membrane protein of Mycoplasma hyorhinis, p70, based on ribosomal DNA sequencing combined with ON ligand blotting after p70 immunoprecipitation by a monoclonal antibody. Whereas antibiotic treatment of infected HepG2 cells strongly decreased ON capture, as measured by a biochemical assay, conversely, deliberate infection of HeLa cells with M.hyorhinis dramatically promoted ON uptake but did not affect receptor-mediated endocytosis of transferrin. This was confirmed by confocal microscopy of infected HepG2 cells, which also showed an indistinguishable labelling pattern after exposure of living cells to fluorescent ON and after p70 immunolabelling in permeabilised fixed cells. We propose that ON binds to p70 on M.hyorhinis attached at the cell surface, after which the complex is internalised by 'piggy-back' endocytosis.

Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2 cell line: evidence for non-conventional intracellular trafficking.

Having identified an oligonucleotide (ON) receptor in the HepG2 cell line, we have re-examined here the kinetics of ON uptake, subcellular distribution and intracellular localisation in these cells, at concentrations relevant for the study of a receptor-dependent process. Kinetic parameters of ON endocytosis were comparable with those of the receptor-mediated endocytosis tracer, transferrin (uptake equilibrium, saturation with concentration, specific competition and rapid efflux) and were clearly distinct from those of fluid-phase endocytosis. By analytical subcellular fractionation, particulate ON showed a bimodal distribution after 2 h of uptake, with a low-density peak superimposed on the distribution of endosomes, and a high-density peak overlapping lysosomes. After an overnight chase, only the high-density peak remained, but it could be dissociated from lysosomes, based on its refractoriness to displacement upon chloroquine-induced swelling. After 2 h of uptake at 300 nM ON-Alexa, a punctate pattern was resolved, by confocal microscopy, from those of transferrin, of a fluid-phase tracer, and of vital staining of lysosomes by LysoTracker. At 3 microM ON-Alexa, its pattern largely overlapped with the fluid-phase tracer and LysoTracker. Taken together, these data suggest that ON may be internalised at low concentrations by receptor-mediated endocytosis into unique endosomes, then to dense structures that are distinct from lysosomes. The nature of these two compartments and their significance for ON effect deserve further investigation.

Regulation of matrix metalloproteinase (MMP) activity by the low-density lipoprotein receptor-related protein (LRP). A new function for an "old friend".

Matrix metalloproteinases (MMPs) are essential contributors to a microenvironment that promotes tumour progression. During the two last decades, inhibition of MMPs has become the focus of considerable interest for cancer therapy, and numerous synthetic metalloproteinase inhibitors have been developed by the pharmaceutical industry. However, clinical trials have shown disappointing efficacy or unexpected toxicity and new targets are thus eagerly awaited. The identification of endocytic clearance of several MMPs by the low-density lipoprotein receptor-related protein (LRP) might provide insight into novel strategies for controlling MMP level during malignant processes. This review attempts to summarize recent aspects on the cellular and molecular basis of LRP-mediated endocytic disposal of MMPs.

Differential subcellular membrane recruitment of Src may specify its downstream signalling.

Most Src family members are diacylated and constitutively associate with membrane "lipid rafts" that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at "rafts" remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to "lipid rafts"; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 degrees C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. "lipid rafts". By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ( approximately 70%) cholesterol extraction with methyl-beta-cyclodextrin (MbetaCD) did not abolish "rafts" floatation, but strongly decreased Src association with floating "rafts" and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to MbetaCD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at "non-raft" domains on endosomes, then via PI3-kinase-Akt on a distinct set of "rafts" at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.

Constitutive diffuse activation of phosphoinositide 3-kinase at the plasma membrane by v-Src suppresses the chemotactic response to PDGF by abrogating the polarity of PDGF receptor signalling.

Cancer cells depend on chemotaxis for invasion and frequently overexpress and/or activate Src. We previously reported that v-Src accelerates motility by promoting phosphoinositide 3-kinase (PI3-K) signalling but abrogates chemotaxis. We here addressed the mechanism of the loss of chemotactic response to platelet-derived growth factor (PDGF) gradients in fibroblasts harbouring a thermosensitive v-Src kinase. At non-permissive temperature, PDGF receptor (PDGFR) signalling, assessed by phosphoY(751)-specific antibodies (a docking site for PI3-K), was not detected without PDGF and showed a concentration-dependent PDGF response. Both immunolabeling of PI3-K (p110) and live cell imaging of its product (phosphatidylinositol 3,4,5 tris-phosphate) showed PI3-K recruitment and activation at lamellipodia polarized towards a PDGF gradient. Centrosomes and PDGFR- and Src-bearing endosomes were also oriented towards this gradient. Upon v-Src thermoactivation, (i) Y(751) phosphorylation was moderately induced without PDGF and synergistically increased with PDGF; (ii) PI3-K was recruited and activated all along the plasma membrane without PDGF and did not polarize in response to a PDGF gradient; and (iii) polarization of centrosomes and of PDGFR-bearing endosomes were also abrogated. Thus, PDGF can further increase PDGFR auto-phosphorylation despite strong Src kinase activity, but diffuse downstream activation of PI3-K by Src abrogates cell polarization and chemotaxis: "signalling requires silence".

Lactacystin decreases amyloid-beta peptide production by inhibiting beta-secretase activity.

The human amyloid precursor protein (APP) is processed by the nonamyloidogenic and the amyloidogenic catabolic pathways. The sequential cleavage of APP by the beta- and gamma-secretase activities, known as the amyloidogenic processing of APP, leads to the formation of the amyloid-beta peptide (Abeta). Abeta is the main constituent of the amyloid core of senile plaques, a typical hallmark of Alzheimer's disease. In addition to secretases, other cellular proteolytic activities, like the proteasome, might participate in the metabolism of APP. We investigated the consequence of proteasome inhibition on the amyloidogenic processing of human APP. CHO cells and primary cultures of rat cortical neurons expressing human APP or a protein corresponding to its beta-cleaved C-terminal fragment (C99) were treated with lactacystin, an irreversible inhibitor of the chymotrypsin-like activity of the proteasome. Lactacystin significantly decreased the level of Abeta produced from APP in both cellular models, whereas the production of Abeta from C99 was not affected. Lactacystin did not inhibit gamma-secretase activity but was found to inhibit the beta-cleavage of APP, leading to a proportional decrease in Abeta production. Although lactacystin did not inhibit the catalytic activity of recombinant BACE1, a decrease in neuronal beta-secretase activity was measured after treatment with lactacystin.

Src triggers circular ruffling and macropinocytosis at the apical surface of polarized MDCK cells.

We addressed the role of Src on cortical actin dynamics and polarized endocytosis in MDCK cells harboring a thermosensitive v-src mutant. Shifting monolayers established at 40 degrees C (non-permissive temperature) to 34 degrees C (permissive temperature) rapidly reactivated v-Src kinase, but tight junctions and cell polarity resisted for >6 h. At this interval, activated v-src was recruited on apical vesicles, induced cortactin-associated apical circular ruffles productive of macropinosomes, thereby accelerating apical pinocytosis by approximately fivefold. Ruffling and macropinosome formation were selectively abrogated by inhibitors of actin polymerization, phosphoinositide 3-kinase, phospholipase C, and phospholipase D, which all returned apical pinocytosis to the level observed at 40 degrees C, underscoring the distinct control of apical micropinocytosis and macropinocytosis. Src promoted microtubule-dependent fusion of macropinosomes to the apical recycling endosome (ARE), causing its strong vacuolation. However, preservation of tubulation and apical polarity indicated that its function was not affected. The ARE was labeled for v-src, Rab11, and rabankyrin-5 but not early endosome antigen 1, thus distinguishing two separate Rab5-dependent apical pathways. The mechanisms of Src-induced apical ruffling and macropinocytosis could shed light on the triggered apical enteroinvasive pathogens entry and on the apical differentiation of osteoclasts.