Daniela Sarnataro

Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins

An essential but insufficient step for apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in epithelial cells is their association with detergent-resistant microdomains (DRMs) or rafts. In this paper, we show that in MDCK cells both apical and basolateral GPI-APs associate with DRMs during their biosynthesis. However, only apical and not basolateral GPI-APs are able to oligomerize into high molecular weight complexes. Protein oligomerization begins in the medial Golgi, concomitantly with DRM association, and is dependent on protein–protein interactions. Impairment of oligomerization leads to protein missorting. We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs. Two alternative apical sorting models are presented.

The Cannabinoid CB1 Receptor Antagonist Rimonabant (SR141716) Inhibits Human Breast Cancer Cell Proliferation through a Lipid Raft-Mediated Mechanism

The endocannabinoid system has been shown to modulate key cell-signaling pathways involved in cancer cell growth. In this study, we show that cannabinoid receptor type 1 (CB1) antagonist Rimonabant (SR141716) inhibited human breast cancer cell proliferation, being more effective in highly invasive metastatic MDA-MB-231 cells than in less-invasive T47D and MCF-7 cells. The SR141716 antiproliferative effect was not accompanied by apoptosis or necrosis and was characterized by a G1/S-phase cell cycle arrest, decreased expression of cyclin D and E, and increased levels of cyclin-dependent kinase inhibitor p27KIP1. We have also shown that SR141716 exerted a significant antiproliferative action, in vivo, by reducing the volume of xenograft tumors induced by MDA-MB-231 injection in mice. On the other hand, at the concentration range in which we observed the antiproliferative effect in tumor cells, we did not observe evidence of any genotoxic effect on normal cells. Our data also indicate that the SR141716 antiproliferative effect requires lipid raft/caveolae integrity to occur. Indeed, we found that CB1 receptor (CB1R) is completely displaced from lipid rafts in SR141716-treated MDA-MB-231 cells, and cholesterol depletion by methyl-β-cyclodextrin strongly prevented SR141716-mediated antiproliferative effect. Taken together, our results suggest that SR141716 inhibits human breast cancer cell growth via a CB1R lipid raft/caveolae-mediated mechanism.

PrPC is sorted to the basolateral membrane of epithelial cells independently of its association with rafts.

PrPC is a glycosylphosphatidylinositol‐anchored protein expressed in neurons as well as in the cells of several peripheral tissues. Although the normal function of PrPC remains unknown, a conformational isoform called PrPSc (scrapie) has been proposed to be the infectious agent of transmissible spongiform encephalopathies in animals and humans. Where and how the PrPC to PrPSc conversion occurs in the cells is not yet known. Therefore, dissecting the intracellular trafficking of the wild‐type prion protein, as well as of the scrapie isoform, can be of major relevance to the pathogenesis of the diseases. In this report we have analyzed the exocytic pathway of transfected mouse PrPC in thyroid and kidney polarized epithelial cells. In contrast to the majority of glycosylphosphatidylinositol‐anchored proteins, we found that PrPC is localized mainly on the basolateral domain of the plasma membrane of both cell lines. This is reminiscent of the predominant somatodendritic localization found in neurons. However, similarly to apical glycosylphosphatidylinositol‐proteins, PrPC associates with detergent‐resistant microdomains, which have been suggested to have a role in apical sorting of glycosylphosphatidylinositol‐proteins, as well as in the conversion process of PrPC to PrPSc. In order to discriminate whether detergent‐resistant microdomains have a direct role in PrPSc conversion, or whether they are involved in the transport of the protein to the site of its conversion, we have examined the effect of disruption of detergent‐resistant microdomain association on PrPC intracellular traffic. Consistent with the unusual basolateral localization of this glycosylphosphatidylinositol‐linked protein, our data exclude a classical role for detergent‐resistant microdomains in the post‐trans‐Golgi network sorting and transport of PrPC to the plasma membrane.

Plasma membrane and lysosomal localization of CB1 cannabinoid receptor are dependent on lipid rafts and regulated by anandamide in human breast cancer cells

In this report we show, by confocal analysis of indirect immunofluorescence, that the type‐1 cannabinoid receptor (CB1R), which belongs to the family of G‐protein‐coupled receptors, is expressed on the plasma membrane in human breast cancer MDA‐MB‐231 cells. However, a substantial proportion of the receptor is present in lysosomes. We found that CB1R is associated with cholesterol‐ and sphyngolipid‐enriched membrane domains (rafts). Cholesterol depletion by methyl‐β‐cyclodextrin (MCD) treatment strongly reduces the flotation of the protein on the raft‐fractions (DRM) of sucrose density gradients suggesting that CB1 raft‐association is cholesterol dependent. Interestingly binding of the agonist, anandamide (AEA) also impairs DRM‐association of the receptor suggesting that the membrane distribution of the receptor is dependent on rafts and is possibly regulated by the agonist binding. Indeed MCD completely blocked the clustering of CB1R at the plasma membrane. On the contrary the lysosomal localization of CB1R was impaired by this treatment only after AEA binding.

Oligomerization Is a Specific Requirement for Apical Sorting of Glycosyl‐Phosphatidylinositol‐Anchored Proteins but Not for Non‐Raft‐Associated Apical Proteins

Protein apical sorting in polarized epithelial cells is mediated by two different mechanisms, raft dependent and raft independent. In Madin–Darby canine kidney (MDCK) cells, an essential step for apical sorting of glycosyl‐phosphatidylinositol (GPI)‐anchored proteins (GPI‐APs) is their coalescence into high‐molecular‐weight (HMW) oligomers. Here we show that this mechanism is also functional in Fischer rat thyroid cells, which possess a different sorting phenotype compared with MDCK cells. We demonstrate that, as in MDCK cells, both apical and basolateral GPI‐APs associate with detergent‐resistant microdomains, but that only the apical proteins are able to oligomerize into HMW complexes during their passage through the medial Golgi. We also show that oligomerization is a specific requirement for apical sorting of GPI‐APs and is not used by transmembrane, non‐raft‐associated apical proteins.

TRAP1 and the proteasome regulatory particle TBP7/Rpt3 interact in the endoplasmic reticulum and control cellular ubiquitination of specific mitochondrial proteins

Tumor necrosis factor receptor-associated protein-1 (TRAP1) is a mitochondrial (MITO) antiapoptotic heat-shock protein. The information available on the TRAP1 pathway describes just a few well-characterized functions of this protein in mitochondria. However, our groups use of mass-spectrometric analysis identified TBP7, an AAA-ATPase of the 19S proteasomal subunit, as a putative TRAP1-interacting protein. Surprisingly, TRAP1 and TBP7 colocalize in the endoplasmic reticulum (ER), as demonstrated by biochemical and confocal/electron microscopic analyses, and interact directly, as confirmed by fluorescence resonance energy transfer analysis. This is the first demonstration of TRAP1s presence in this cellular compartment. TRAP1 silencing by short-hairpin RNAs, in cells exposed to thapsigargin-induced ER stress, correlates with upregulation of BiP/Grp78, thus suggesting a role of TRAP1 in the refolding of damaged proteins and in ER stress protection. Consistently, TRAP1 and/or TBP7 interference enhanced stress-induced cell death and increased intracellular protein ubiquitination. These experiments led us to hypothesize an involvement of TRAP1 in protein quality control for mistargeted/misfolded mitochondria-destined proteins, through interaction with the regulatory proteasome protein TBP7. Remarkably, expression of specific MITO proteins decreased upon TRAP1 interference as a consequence of increased ubiquitination. The proposed TRAP1 network has an impact in vivo, as it is conserved in human colorectal cancers, is controlled by ER-localized TRAP1 interacting with TBP7 and provides a novel model of the ER–mitochondria crosstalk.

α-Adducin mutations increase Na/K pump activity in renal cells by affecting constitutive endocytosis: implications for tubular Na reabsorption

Genetic variation in alpha-adducin cytoskeletal protein is implicated in the polymerization and bundling of actin and alteration of the Na/K pump, resulting in abnormal renal sodium transport and hypertension in Milan hypertensive rats and humans. To investigate the molecular involvement of alpha-adducin in controlling Na/K pump activity, wild-type or mutated rat and human alpha-adducin forms were, respectively, transfected into several renal cell lines. Through multiple experimental approaches (microscopy, enzymatic assays, coimmunoprecipitation), we showed that rat and human mutated forms increased Na/K pump activity and the number of pump units; moreover, both variants coimmunoprecipitate with Na/K pump. The increased Na/K pump activity was not due to changes in its basolateral localization, but to an alteration of Na/K pump residential time on the plasma membrane. Indeed, both rat and human mutated variants reduced constitutive Na/K pump endocytosis and similarly affected transferrin receptor trafficking and fluid-phase endocytosis. In fact, alpha-adducin was detected in clathrin-coated vesicles and coimmunoprecipitated with clathrin. These results indicate that adducin, besides its modulatory effects on actin cytoskeleton dynamics, might play a direct role in clathrin-dependent endocytosis. The constitutive reduction of the Na/K pump endocytic rate induced by mutated adducin variants may be relevant in Na-dependent hypertension.

Detergent-resistant membrane domains but not the proteasome are involved in the misfolding of a PrP mutant retained in the endoplasmic reticulum.

Inherited prion diseases are neurodegenerative pathologies related to genetic mutations in the prion protein (PrP) gene, which favour the conversion of PrPC into a conformationally altered pathogenic form, PrPSc. The molecular basis of PrPC/PrPSc conversion, the intracellular compartment where it occurs and how this process leads to neurological dysfunction are not yet known. We have studied the intracellular synthesis, degradation and localization of a PrP mutant associated with a genetic form of Creutzfeldt-Jakob disease (CJD), PrPT182A, in transfected FRT cells. PrPT182A is retained in the endoplasmic reticulum (ER), is mainly associated with detergent-resistant microdomains (DRMs) and is partially resistant to proteinase K digestion. Although an untranslocated form of this mutant is polyubiquitylated and undergoes ER-associated degradation, the proteasome is not responsible for the degradation of its misfolded form, suggesting that it does not have a role in the pathogenesis of inherited diseases. On the contrary, impairment of PrPT182A association with DRMs by cholesterol depletion leads to its accumulation in the ER and substantially increases its misfolding. These data support the previous hypothesis that DRMs are important for the correct folding of PrP and suggest that they might have a protective role in pathological scrapie-like conversion of PrP mutants.

Adiponectin affects lung epithelial A549 cell viability counteracting TNFa and IL-1ß toxicity through AdipoR1

Adiponectin (Acrp30) exerts protective functions on metabolic and cellular processes as energy metabolism, cell proliferation and differentiation by two widely expressed receptors, AdipoR1 and AdipoR2. To date, the biological role of Acrp30 in lung has not been completely assessed but altered levels of Acrp30 and modulated expression of both AdipoRs have been related to establishment and progression of chronic obstructive pulmonary disease (COPD) and lung cancer. Here, we investigated the effects of Acrp30 on A549, a human alveolar epithelial cell line, showing how, in a time and dose-dependent manner, it decreases cell viability and increases apoptosis through ERK1/2 and AKT. Furthermore, we examined the effects of Acrp30 on A549 cells exposed to TNFα and/or IL-1ß, two potent lung inflammatory cytokines. We showed that Acrp30, in dose- and time-dependent manner, reduces cytotoxic effects of TNFα and/or IL-1ß improving cell viability and decreasing apoptosis. In addition, Acrp30 inhibits NF-κB nuclear trans-activation and induces the expression of the anti-inflammatory IL-10 cytokine without modifying that of pro-inflammatory IL-6, IL-8, and MCP-1 molecules via ERK1/2 and AKT. Finally, specifically silencing AdipoR1 or AdipoR2, we observed that NF-κB inhibition is mainly mediated by AdipoR1. Taken together, our data provides novel evidence for a direct effect of Acrp30 on the proliferation and inflammation status of A549 cells strongly supporting the hypothesis for a protective role of Acrp30 in lung. Further studies are needed to fully elucidate the Acrp30 lung effects in vivo but our results confirm this adipokine as a promising therapeutic target in lung diseases.

Lipid Rafts and Clathrin Cooperate in the Internalization of PrPC in Epithelial FRT Cells

Background The cellular prion protein (PrPC) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrPSc). Although endocytosis appears to be required for this conversion, the mechanism of PrPC internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved. Methodology/Principal Findings We have investigated the mechanism of PrPC endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrPC internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrPC endocytosis. PrPC internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrPC co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrPC can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization. Conclusions/Significance These findings are of particular interest if we consider that the internalization route/s undertaken by PrPC can be crucial for the ability of different prion strains to infect and to replicate in different cell lines.