Inhibition of TRPM8 function by prostacyclin receptor agonists requires coupling to Gq/11 proteins.

BACKGROUND AND PURPOSE: The TRPM8 ion channel is involved in innocuous cold sensing and has a potent anti-inflammatory action. Its activation by lower temperature or chemical agonists such as menthol and icilin induces analgesic effects, reversing hypersensitivity and reducing chronic pain. On the other hand, prostacyclin (PGI2) enhances pain and inflammation by activating the IP receptors. Due to the critical roles of TRPM8 and IP receptors in the regulation of inflammatory pain, and considering their overlapping expression pattern, we analysed the functional interaction between human TRPM8 and IP receptors. EXPERIMENTAL APPROACH: We transiently expressed human TRPM8 channels and IP receptors in HEK293T cells and carried out intracellular calcium and cAMP measurements. Additionally, we cultured neurons from the dorsal root ganglia (DRGs) of mice and determined the increase in intracellular calcium triggered by the TRPM8 agonist, icilin, in the presence of the IP receptor agonist cicaprost, the IP receptor antagonist Cay10441, and the Gq/11 inhibitor YM254890. KEY RESULTS: Activation of IP receptors by selective agonists (cicaprost, beraprost, and iloprost) inhibited TRPM8 channel function, independently of the Gs-cAMP pathway. The potent inhibition of TRPM8 channels by IP receptor agonists involved Gq/11 coupling. These effects were also observed in neurons isolated from murine DRGs. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate an unusual signalling pathway of IP receptors by coupling to Gq/11 proteins to inhibit TRPM8 channel function. This pathway may contribute to a better understanding of the role of TRPM8 channels and IP receptors in regulating pain and inflammation.

EDEM1 regulates the insulin mRNA level by inhibiting the endoplasmic reticulum stress-induced IRE1/JNK/c-Jun pathway.

Pancreatic beta cells produce and secrete insulin as a response to rises in blood glucose. Despite the advances in understanding glucose-regulated insulin transcription and translation the mechanisms triggering the synthesis of new insulin molecules are still incompletely described. In this report, we identify EDEM1 as a new modulator of insulin synthesis and secretion. In the presence of EDEM1, INS-1E cells secrete significantly more insulin upon glucose stimulation compared to control cells. We found that overexpression of EDEM1 inhibited the IRE1/JNK/c-Jun pathway, leading to an increase in the insulin mRNA level. Similarly, EDEM1 transduced human islets secreted significantly more insulin upon stimulation. Furthermore, EDEM1 improved insulin secretion restoring normoglycemia and glucose tolerance in diabetic rats. We propose EDEM1 as a regulator of the UPR via IRE1/XBP1s and IRE1/JNK/c-Jun signaling cascades and insulin transcription in pancreatic ß-cells, supporting EDEM1 as a potential target for the treatment of diabetes.

Methionine oxidation selectively enhances T cell reactivity against a melanoma antigen.

The impact of the peptide amino acids side-chain modifications on the immunological recognition has been scarcely explored. We investigate here the effect of methionine oxidation on the antigenicity of the melanoma immunodominant peptide 369-YMDGTMSQV-377 (YMD). Using CD8+ T cell activation assays, we found that the antigenicity of the sulfoxide form is higher when compared to the YMD peptide. This is consistent with free energy computations performed on HLA-A∗02:01/YMD/TCR complex showing that this is lowered upon oxidation, paired with a steep increase in order at atomic level. Oxidized YMD forms were identified at the melanoma cell surface by LC-MS/MS analysis. These results demonstrate that methionine oxidation in the antigenic peptides may generate altered peptide ligands with increased antigenicity, and that this oxidation may occur in vivo, opening up the possibility that high-affinity CD8+ T cells might be naturally primed in the course of melanoma progression, as a result of immunosurveillance.

Targeting EDEM protects against ER stress and improves development and survival in C. elegans.

EDEM-1, EDEM-2 and EDEM-3 are key players for the quality control of newly synthesized proteins in the endoplasmic reticulum (ER) by accelerating disposal and degradation of misfolded proteins through ER Associated Degradation (ERAD). Although many previous studies reported the role of individual ERAD components especially in cell-based systems, still little is known about the consequences of ERAD dysfunction under physiological and ER stress conditions in the context of a multicellular organism. Here we report the first individual and combined characterization and functional interplay of EDEM proteins in Caenorhabditis elegans using single, double, and triple mutant combinations. We found that EDEM-2 has a major role in the clearance of misfolded proteins from ER under physiological conditions, whereas EDEM-1 and EDEM-3 roles become prominent under acute ER stress. In contrast to SEL-1 loss, the loss of EDEMs in an intact organism induces only a modest ER stress under physiological conditions. In addition, chronic impairment of EDEM functioning attenuated both XBP-1 activation and up-regulation of the stress chaperone GRP78/BiP, in response to acute ER stress. We also show that pre-conditioning to EDEM loss in acute ER stress restores ER homeostasis and promotes survival by activating ER hormesis. We propose a novel role for EDEM in fine-tuning the ER stress responsiveness that affects ER homeostasis and survival.

New panel of biomarkers to discriminate between amelanotic and melanotic metastatic melanoma.

Melanoma is a form of skin cancer that can rapidly invade distant organs. A distinctive feature of melanomas is their pigmentation status, as melanin is present in most skin melanomas, whilst many metastatic tumors could become amelanotic. Besides the obvious malfunction of the key genes of the melanin pathway, the amelanotic tumors could bear a characteristic molecular signature accounting for their aggressivity. Using mass spectrometry-based proteomics we report here a distinctive panel of biomarkers for amelanotic aggressive melanoma that differ from the less invasive pigmented cells. The developed method allows the label-free quantification of proteins identified by LC-MS/MS analysis. We found a set of proteins comprising AHNAK, MYOF, ANXA1, CAPN2, ASPH, EPHA2, THBS1, TGM2, ACTN4 along with proteins involved in cell adhesion/migration (integrins, PLEC, FSCN1, FN1) that are highly expressed in amelanotic melanoma. Accompanying the down regulation of pigmentation specific proteins such as tyrosinase and TYRP1, these biomarkers are highly specific for a type of highly invasive melanoma. Interestingly, the LC-MS/MS proteomics analysis in hypoxia revealed that the abundance of this specific set of proteins found in normoxia was rather unaltered in these conditions. These biomarkers could therefore predict a metastatic behaviour for the amelanotic cells in the early stages of the tumor development and thus serve in melanoma prognostic. Applying this algorithm to related databases including melanoma samples published by independent laboratories/public databases we confirm the specificity of the newly found signatures. Overall, we begin to unravel the molecular alterations in the amelanotic melanoma and how basic proteomics offers insights into how to assess the clinical, pathological and misdiagnosis differences between the main subtypes of melanoma.

Affinity Proteomics and Deglycoproteomics Uncover Novel EDEM2 Endogenous Substrates and an Integrative ERAD Network.

Various pathologies result from disruptions to or stress of endoplasmic reticulum (ER) homeostasis, such as Parkinson's disease and most neurodegenerative illnesses, diabetes, pulmonary fibrosis, viral infections, and cancers. A critical process in maintaining ER homeostasis is the selection of misfolded proteins by the ER quality-control system for destruction via ER-associated degradation (ERAD). One key protein proposed to act during the first steps of misfolded glycoprotein degradation is the ER degradation-enhancing α-mannosidase-like protein 2 (EDEM2). Therefore, characterization of the EDEM2-associated proteome is of great interest. We took advantage of using melanoma cells overexpressing EDEM2 as a cancer model system, to start documenting at the deglycoproteome level (N-glycosites identification) the emerging link between ER homeostasis and cancer progression. The dataset created for identifying the EDEM2 glyco clients carrying high mannose/hybrid N-glycans provides a comprehensive N-glycosite analysis mapping over 1000 N-glycosites on more than 600 melanoma glycoproteins. To identify EDEM2-associated proteins, we used affinity proteomics and proteome-wide analysis of sucrose density fractionation in an integrative workflow. Using intensity and spectral count-based quantification, we identify seven new EDEM2 partners, all of which are involved in ER quality-control system and ERAD. Moreover, we defined novel endogenous candidates for EDEM2-dependent ERAD by combining deglycoproteomics, stable isotope labeling with amino acids in cell culture-based proteomics, and biochemical methods. These included tumor antigens and several ER-transiting endogenous melanoma proteins, including integrin alpha-1 and protocadherin 2, the expression of which was negatively correlated with that of EDEM2. Tumor antigens are key in the antigen presentation process, whereas integrin alpha-1 and protocadherin 2 are involved in melanoma metastasis and invasion. EDEM2 could therefore have a regulatory role in melanoma through the modulation of degradation and trafficking in these glycoproteins. The data presented herein suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously suggested.

EDEM3 Domains Cooperate to Perform Its Overall Cell Functioning.

EDEM3 recognizes and directs misfolded proteins to the ER-associated protein degradation (ERAD) process. EDEM3 was predicted to act as lectin or as a mannosidase because of its homology with the GH47 catalytic domain of the Man1B1, but the contribution of the other regions remained unresolved. Here, we dissect the molecular determinants governing EDEM3 function and its cellular interactions. LC/MS analysis indicates very few stable ER interactors, suggesting EDEM3 availability for transient substrate interactions. Sequence analysis reveals that EDEM3 consists of four consecutive modules defined as GH47, intermediate (IMD), protease-associated (PA), and intrinsically disordered (IDD) domain. Using an EDEM3 knock-out cell line, we expressed EDEM3 and domain deletion mutants to address EDEM3 function. We find that the mannosidase domain provides substrate binding even in the absence of mannose trimming and requires the IMD domain for folding. The PA and IDD domains deletions do not impair the trimming, but specifically modulate the turnover of two misfolded proteins, NHK and the soluble tyrosinase mutant. Hence, we demonstrate that EDEM3 provides a unique ERAD timing to misfolded glycoproteins, not only by its mannose trimming activity, but also by the positive and negative feedback modulated by the protease-associated and intrinsically disordered domain, respectively.

EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein-protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.

Inhibition of N-glycan processing modulates the network of EDEM3 interactors.

We present here data on EDEM3 network of ER resident interactors and the changes induced upon this network by perturbing the early ER N-glycan processing with mannosidase and glucosidase inhibitors. By coupling immunoprecipitation with mass spectrometry we identified EDEM3 interactors and assigned statistical significance to those most abundant ER-residents that might form functional complexes with EDEM3. We further show that this ER interaction network changes in both content and abundance upon treatment with kifunensine (kif) and N-butyldeoxynojirimycin (NB-DNJ) which suggests that when interfering with the N-glycan processing pathway, the functional complexes involving EDEM3 adapt to maintain the cellular homeostasis. In order to increase the scope of EDEM3 network contenders, the set of MS identified species was further supplemented with putative interactors derived from in silico simulations performed with STRING. Finally, the most interesting candidates to this network were further validated by immunoprecipitation coupled with Western Blotting, which strengthened the confidence in the inferred interactions. The data corroborated herein suggest that besides ER residents, EDEM3 interacts also with proteins involved in the ERAD cargo recognition and targeting to degradation translocation into the cytosol, including UBA1 and UBA2 ubiquitinating enzymes. In addition, the results indicate that this network of EDEM3 interactors is highly sensitive to interfering with early ER N-glycan processing.

Novel function of the endoplasmic reticulum degradation-enhancing α-mannosidase-like proteins in the human hepatitis B virus life cycle, mediated by the middle envelope protein.

Cells replicating the human hepatitis B virus (HBV) express high levels of degradation-enhancing α-mannosidase-like proteins (EDEMs), a family of proteins involved in the endoplasmic reticulum associated degradation, one of the pathways activated during the unfolded protein response. Owing to their α-1,2 mannosidase activity, the EDEM1-3 proteins are able to process the N-linked glycans of misfolded or incompletely folded proteins, providing the recognition signal for their subsequent degradation. The HBV small (S), medium (M), and large (L) surface proteins bear an N-linked glycosylation site in the common S domain that is partially occupied in all proteins. The M protein contains an additional site in its preS2 domain, which is always functional. Here, we report that these oligosaccharides are processed by EDEMs, more efficiently by EDEM3, which induces degradation of L and S proteins, accompanied by a reduction of subviral particles production. In striking contrast, M not only is spared from degradation but its trafficking is also accelerated leading to an improved secretion. This unusual behavior of the M protein requires strictly the mannose trimming of the preS2 N-linked glycan. Furthermore, we show that HBV secretion is significantly inhibited under strong endoplasmic reticulum stress conditions when M expression is prevented by mutagenesis of the viral genome. These observations unfold unique properties of the M protein in the HBV life cycle during unfolded protein response and point to alternative mechanisms employed by EDEMs to alleviate this stress in case of necessity by promoting glycoprotein trafficking rather than degradation.

Epitope located N-glycans impair the MHC-I epitope generation and presentation.

The degradation process of the antigens specific to MHC-I presentation depends mainly on the proteasomal proteases in the cytosol. However, since many antigens are glycoproteins, including tumor antigens or viruses envelope proteins, their glycosylation status could also affect their processing and presentation. Here, we investigate the processing of tyrosinase, a multiple glycosylated tumor antigen overexpressed in human malignant melanoma. By LC-MS/MS analysis of human tyrosinase expressed in a melanoma cell, we show that all seven sites of tyrosinase are at least partially N-glycosylated. Using human CD8+ T-cell clones specific for the tyrosinase epitope YMDGTMSQV (369-377), including an N-glycosylation site, we found that transfectants of single and triple N-glycosylation mutants are recognized by specific T cells. Importantly, single, triple, and the aglycosylated tyrosinase mutants lacking the epitope located N-glycosylation site (N371D) were able to trigger higher CD8+ T-cell activation. The LC/MS analysis showed significant increase of the amount of YMDGTMSQV peptide resulted from accelerated oligomerization and degradation of aglycosylated mutants. The generation of the antigenic peptide by the antigen processing machinery is therefore largely independent of tyrosinase N-glycosylation. However, while distal N-glycans had no effect on the epitope generation, the mutants lacking the N371 glycan generated the antigenic peptide more efficiently. We conclude that epitope located N-glycans limit the ability of human tyrosinase to provide HLA-A2-restricted antigen for recognition by specific CD8+ T cells.

Combinatorial MAPLE gradient thin film assemblies signalling to human osteoblasts.

There is increased interest in smart bioactive materials to control tissue regeneration for the engineering of cell instructive scaffolds. We introduced combinatorial matrix-assisted pulsed laser evaporation (C-MAPLE) as a new method for the fabrication of organic thin films with a compositional gradient. Synchronized C-MAPLE of levan and oxidized levan was employed to assemble a two-compound biopolymer film structure. The gradient of the film composition was validated by fluorescence microscopy. In this study, we investigated the cell response induced by the compositional gradient using imaging of early osteoblast attachment and analysis of signalling phosphoprotein expression. Cells attached along the gradient in direct proportion to oxidized levan concentration. During this process distinct areas of the binary gradient have been shown to modulate the osteoblasts' extracellular signal-regulated kinase signalling with different propensity. The proposed fabrication method results in the preparation of a new bioactive material, which could control the cell signalling response. This approach can be extended to screen new bioactive interfaces for tissue regeneration.

Expression and subcellular localization of RAGE in melanoma cells.

The receptor for advanced glycation end products (RAGE) is involved in multiple stages of tumor development and malignization. To gain further knowledge on the RAGE role in tumor progression, we investigated the receptor expression profile and its subcellular localization in melanoma cells at different stages of malignancy. We found that RAGE clustered at membrane ruffles and leading edges, and at sites of cell-to-cell contact in primary melanoma cells (e.g., MelJuSo), in contrast with a more dispersed localization in metastatic cells (e.g., SK-Mel28). RAGE silencing by RNAi selectively inhibited migration of MelJuSo cells, whilst having no influence on SK-Mel28 cell migration, in a "wound healing" assay. Western blot detection of RAGE showed a more complex RAGE oligomerization in MelJuSo cells compared to melanocytes and SK-Mel28 cells. By competing the binding of antibodies with recombinant soluble RAGE, an oligomeric form running at approximately 200 kDa was detected, as it was the monomeric RAGE of 55-60 kDa. SDS-PAGE electrophoresis under reducing versus nonreducing conditions indicated that the oligomer of about 200 kDa is formed by disulfide bonds, but other interactions are likely to be important for RAGE multimerization in melanoma cells. Immunofluorescence microscopy revealed that treatment with two cholesterol-chelating drugs, nystatin and filipin, significantly affected RAGE localization in MelJuSo cells. SK-Mel28 cells showed a reduced RAGE glycosylation and association with cholesterol-rich membranes and also a considerable downregulation of the soluble forms. Our results indicate that RAGE isoform expression and subcellular localization could be important determinants for the regulation of its function in tumor progression.

Value of dopachrome tautomerase detection in the assessment of melanocytic tumors.

Dopachrome tautomerase (DCT) and tyrosinase (Tyr) are melanogenic enzymes and structurally related melanosomal proteins. The present study investigates DCT expression comparatively with Tyr, the most tested melanoma biomarker, aiming to evaluate DCT potential in the assessment of melanocytic tumors and gain insights into the molecular and pathological characterization of DCT-phenotype in tumor progression. DCT and Tyr are simultaneously analyzed in melanoma cell lines by semiquantitative RT-PCR, western blot, and N-glycan analysis, and in cell populations of melanocytic tumors by immunohistofluorescence using a novel anti-hDCT antibody against an extended sequence within DCT luminal domain. DCT, unlike Tyr, is fully processed along the secretory pathway in both pigmented and amelanotic melanoma cells. In 53 nevi and 116 primary malignant melanomas, 81% and 52%, respectively, are DCT+/Tyr+, showing that DCT is a stable antigen, retained by most tumors and partially expressed in Tyr-negative cell populations. The DCT/Tyr disjunction is a process correlated with melanocyte neoplastic transformation and malignant progression. A tumor architecture--DCT-phenotype-containing DCT+/Tyr- cell populations selected into the innermost dermis from double-positive cells is detected in 35% of DCT+/Tyr+ specimens. The DCT-phenotype is associated with enhanced neurotization in benign nevi and with ulceration in thin malignant melanomas. The intradermal DCT+/Tyr- clones in superficial melanomas acquire the expression and specific subcellular distribution of unfavorable prognostic markers. DCT assessment shows specific antigen patterns with potential significance in the outcome of melanocytic lesions, connecting DCT, a mediator of a melanoma stress-resistant pathway, and an antiapoptotic molecule to DCT- phenotypes that are possibly more stable and stress resistant.

Characterization of functional transient receptor potential melastatin 8 channels in human pancreatic ductal adenocarcinoma cells.

OBJECTIVE: Recently, the transient receptor potential melastatin 8 (TRPM8) channel has emerged as a putative biomarker for pancreatic ductal adenocarcinoma (PDA). This study aimed to evaluate the expression of TRPM8 and its modulation by specific agonists and antagonists in PDA cells. METHODS: We examined the protein expression of TRPM8 in 3 different PDA cell lines and compared it with a nontumoral epithelial cell line of human pancreatic origin using Western blotting and immunocytochemical analysis. To assess the function of TRPM8 channels, we measured the TRPM8 currents in whole-cell mode of the patch clamp technique. To explore the putative involvement of TRPM8 in cell migration, we investigated the motility of PDA cells using the scratch-wound assay. RESULTS: Pancreatic ductal adenocarcinoma cells express functional plasma membrane TRPM8 channels, which are responsive after exposure to agonists (menthol and icilin) and antagonists N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride salt. The silencing of TRPM8 expression by small interfering RNA augments the migration of PDA cells. Conversely, the activated form of TRPM8 inhibits PDA cell motility. CONCLUSIONS: An unglycosylated TRPM8 protein is expressed and is functional in the membrane of PDA cells. Transient receptor potential melastatin 8 inhibits the migration of PDA cells, suggesting a putative role as a biomarker or target for this channel for PDA therapy.

PLK1 is a binding partner and a negative regulator of FOXO3 tumor suppressor.

FOXO family members (FOXOs: FOXO1, FOXO3, FOXO4 and FOXO6) are important transcription factors and tumor suppressors controlling cell homeostasis and cell fate. They are characterized by an extraordinary functional diversity, being involved in regulation of cell cycle, proliferation, apoptosis, DNA damage response, oxidative detoxification, cell differentiation and stem cell maintenance, cell metabolism, angiogenesis, cardiac and other organ's development, aging, and other critical cellular processes. FOXOs are tightly regulated by reversible phosphorylation, ubiquitination, acetylation and methylation. Interestingly, the known kinases phosphorylate only a small percentage of the known or predicted FOXOs phosphorylation sites, suggesting that additional kinases that phosphorylate and control FOXOs activity exist. In order to identify novel regulators of FOXO3, we have employed a proteomics screening strategy. Using HeLa cancer cell line and a Tandem Affinity Purification followed by Mass Spectrometry analysis, we identified several proteins as binding partners of FOXO3. Noteworthy, Polo Like Kinase 1 (PLK1) proto-oncogene was one of the identified FOXO3 binding partners. PLK1 plays a critical role during cell cycle (G2-M transition and all phases of mitosis) and in maintenance of genomic stability. Our experimental results presented in this manuscript demonstrate that FOXO3 and PLK1 exist in a molecular complex through most of the phases of the cell cycle, with a higher occurrence in the G2-M cell cycle phases. PLK1 induces translocation of FOXO3 from the nucleus to the cytoplasm and suppresses FOXO3 activity, measured by the decrease in the pro-apoptotic Bim protein levels and in the cell cycle inhibitor protein p27. Furthermore, PLK1 can directly phosphorylate FOXO3 in an in vitro kinase assay. These results present the discovery of PLK1 proto-oncogene as a binding partner and a negative regulator of FOXO3 tumor suppressor.

Combination of bortezomib and mitotic inhibitors down-modulate Bcr-Abl and efficiently eliminates tyrosine-kinase inhibitor sensitive and resistant Bcr-Abl-positive leukemic cells.

Emergence of resistance to Tyrosine-Kinase Inhibitors (TKIs), such as imatinib, dasatinib and nilotinib, in Chronic Myelogenous Leukemia (CML) demands new therapeutic strategies. We and others have previously established bortezomib, a selective proteasome inhibitor, as an important potential treatment in CML. Here we show that the combined regimens of bortezomib with mitotic inhibitors, such as the microtubule-stabilizing agent Paclitaxel and the PLK1 inhibitor BI2536, efficiently kill TKIs-resistant and -sensitive Bcr-Abl-positive leukemic cells. Combined treatment activates caspases 8, 9 and 3, which correlate with caspase-induced PARP cleavage. These effects are associated with a marked increase in activation of the stress-related MAP kinases p38MAPK and JNK. Interestingly, combined treatment induces a marked decrease in the total and phosphorylated Bcr-Abl protein levels, and inhibits signaling pathways downstream of Bcr-Abl: downregulation of STAT3 and STAT5 phosphorylation and/or total levels and a decrease in phosphorylation of the Bcr-Abl-associated proteins CrkL and Lyn. Moreover, we found that other mitotic inhibitors (Vincristine and Docetaxel), in combination with bortezomib, also suppress the Bcr-Abl-induced pro-survival signals and result in caspase 3 activation. These results open novel possibilities for the treatment of Bcr-Abl-positive leukemias, especially in the imatinib, dasatinib and nilotinib-resistant CML cases.

AP-3 and Rabip4' coordinately regulate spatial distribution of lysosomes.

The RUN and FYVE domain proteins rabip4 and rabip4' are encoded by RUFY1 and differ in a 108 amino acid N-terminal extension in rabip4'. Their identical C terminus binds rab5 and rab4, but the function of rabip4s is incompletely understood. We here found that silencing RUFY1 gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4' yielded the adaptor protein complex AP-3, of which the hinge region in the ß3 subunit bound directly to the FYVE domain of rabip4'. Rabip4' colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4' in regulating lysosome positioning through an interorganellar pathway.

Tyrosinase degradation is prevented when EDEM1 lacks the intrinsically disordered region.

EDEM1 is a mannosidase-like protein that recruits misfolded glycoproteins from the calnexin/calreticulin folding cycle to downstream endoplasmic reticulum associated degradation (ERAD) pathway. Here, we investigate the role of EDEM1 in the processing of tyrosinase, a tumour antigen overexpressed in melanoma cells. First, we analyzed and modeled EDEM1 major domains. The homology model raised on the crystal structures of human and Saccharomyces cerevisiae ER class I α1,2-mannosidases reveals that the major mannosidase domain located between aminoacids 121-598 fits with high accuracy. We have further identified an N-terminal region located between aminoacids 40-119, predicted to be intrinsically disordered (ID) and susceptible to adopt multiple conformations, hence facilitating protein-protein interactions. To investigate these two domains we have constructed an EDEM1 deletion mutant lacking the ID region and a triple mutant disrupting the glycan-binding domain and analyzed their association with tyrosinase. Tyrosinase is a glycoprotein partly degraded endogenously by ERAD and the ubiquitin proteasomal system. We found that the degradation of wild type and misfolded tyrosinase was enhanced when EDEM1 was overexpressed. Glycosylated and non-glycosylated mutants co-immunoprecipitated with EDEM1 even in the absence of its intact mannosidase-like domain, but not when the ID region was deleted. In contrast, calnexin and SEL 1L associated with the deletion mutant. Our data suggest that the ID region identified in the N-terminal end of EDEM1 is involved in the binding of glycosylated and non-glycosylated misfolded proteins. Accelerating tyrosinase degradation by EDEM1 overexpression may lead to an efficient antigen presentation and enhanced elimination of melanoma cells.