Publications by authors named "Ștefana M Petrescu"

26 Publications

  • Page 1 of 1

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

Mol Cell Proteomics 2021 Jul 29;20:100125. Epub 2021 Jul 29.

Department of Molecular Cell Biology, Institute of Biochemistry, Bucharest, Romania. Electronic address:

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.
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http://dx.doi.org/10.1016/j.mcpro.2021.100125DOI Listing
July 2021

EDEM3 Domains Cooperate to Perform Its Overall Cell Functioning.

Int J Mol Sci 2021 Feb 22;22(4). Epub 2021 Feb 22.

Department of Molecular Cell Biology, Institute of Biochemistry, Splaiul Independentei 296, 060031 Bucharest 17, Romania.

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.
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http://dx.doi.org/10.3390/ijms22042172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926307PMC
February 2021

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

Int J Mol Sci 2020 May 14;21(10). Epub 2020 May 14.

Department of Molecular Cell Biology, Institute of Biochemistry, Splaiul Independentei 296, 060031 Bucharest 17, Romania.

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.
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http://dx.doi.org/10.3390/ijms21103468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279049PMC
May 2020

Profiling Optimal Conditions for Capturing EDEM Proteins Complexes in Melanoma Using Mass Spectrometry.

Adv Exp Med Biol 2019 ;1140:155-167

Department of Molecular Cell Biology, Institute of Biochemistry, Bucharest, Romania.

Endoplasmic reticulum (ER) resident and secretory proteins that fail to reach their native conformation are selected for degradation through the ER-Associated Degradation (ERAD) pathway. The ER degradation-enhancing alpha-mannosidase-like proteins (EDEMs) were shown to be involved in this pathway but their precise role is still under investigation. Mass spectrometry analysis has contributed significantly to the characterization of protein complexes in the last years. The recent advancements in instrumentation, especially within resolution and speed can provide unique insights concerning the molecular architecture of protein-protein interactions in systems biology. Previous reports have suggested that several protein complexes in ERAD are sensitive to the extraction conditions. Indeed, whilst EDEM proteins can be recovered in most detergents, some of their partners are not solubilized, which further emphasizes the importance of the experimental setup. Here, we define such dynamic interactions of EDEM proteins by employing offline protein fractionation, nanoLC-MS/MS and describe how mass spectrometry can contribute to the characterization of such complexes, particularly within a disease context like melanoma.
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http://dx.doi.org/10.1007/978-3-030-15950-4_9DOI Listing
September 2019

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

Electrophoresis 2016 06 3;37(11):1448-60. Epub 2016 Feb 3.

Institute of Biochemistry, Romanian Academy, Bucharest, Romania.

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.
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http://dx.doi.org/10.1002/elps.201500449DOI Listing
June 2016

Combinatorial MAPLE gradient thin film assemblies signalling to human osteoblasts.

Biofabrication 2014 Sep 28;6(3):035010. Epub 2014 May 28.

Lasers Department, National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor Street, Magurele, Ilfov RO-77125, Romania.

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.
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http://dx.doi.org/10.1088/1758-5082/6/3/035010DOI Listing
September 2014

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

Melanoma Res 2014 Jun;24(3):219-36

Departments of aMolecular Cell Biology bBioinformatics and Structural Biochemistry, Institute of Biochemistry, Romanian Academy cDepartment of Pathology, Colentina University Hospital, Bucharest, Romania.

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.
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http://dx.doi.org/10.1097/CMR.0000000000000066DOI Listing
June 2014

Expression and subcellular localization of RAGE in melanoma cells.

Biochem Cell Biol 2014 Apr 31;92(2):127-36. Epub 2014 Mar 31.

Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, Bucharest 060031, Romania.

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.
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http://dx.doi.org/10.1139/bcb-2013-0064DOI Listing
April 2014

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

PLoS One 2012 29;7(10):e48142. Epub 2012 Oct 29.

Department of Cell Biology, University Medical Center Utrecht, Utrecht, The Netherlands.

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 β3 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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048142PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3483219PMC
August 2014

C-terminus glycans with critical functional role in the maturation of secretory glycoproteins.

PLoS One 2011 18;6(5):e19979. Epub 2011 May 18.

Department Molecular Cell Biology, Institute of Biochemistry of Romanian Academy, Bucharest, Romania.

The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs--one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019979PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097235PMC
October 2011

Levan nanostructured thin films by MAPLE assembling.

Biomacromolecules 2011 Jun 9;12(6):2251-6. Epub 2011 May 9.

Lasers Department, National Institute for Lasers, Plasma and Radiation Physics, Ilfov, RO-77125, Romania.

Synthesis of nanostructured thin films of pure and oxidized levan exopolysaccharide by matrix-assisted pulsed laser evaporation is reported. Solutions of pure exopolysaccharides in dimethyl sulfoxide were frozen in liquid nitrogen to obtain solid cryogenic pellets that have been used as targets in pulsed laser evaporation experiments with a KrF* excimer source. The expulsed material was collected and assembled onto glass slides and Si wafers. The contact angle studies evidenced a higher hydrophilic behavior in the case of oxidized levan structures because of the presence of acidic aldehyde-hydrogen bonds of the coating formed after oxidation. The obtained films preserved the base material composition as confirmed by Fourier transform infrared spectroscopy. They were compact with high specific surface areas, as demonstrated by scanning electron and atomic force microscopy investigations. In vitro colorimetric assays revealed a high potential for cell proliferation for all coatings with certain predominance for oxidized levan.
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http://dx.doi.org/10.1021/bm200340bDOI Listing
June 2011

Biocompatibility and bioactivity enhancement of Ce stabilized ZrO(2) doped HA coatings by controlled porosity change of Al(2) O(3) substrates.

J Biomed Mater Res B Appl Biomater 2011 Feb 17;96(2):218-24. Epub 2010 Nov 17.

Laser Department, National Institute for Lasers, Plasma, and Radiation Physics, Magurele, Ilfov, Romania.

Al(2) O(3) substrates with controlled porosity were manufactured from nanosized powders obtained by plasma processing. It was observed that when increasing the sintering temperature the overall porosity was decreasing, but the pores got larger. In a second step, Ce stabilized ZrO(2) doped hydroxyapatite coatings were pulsed laser deposited onto the Al(2) O(3) substrates. It was shown that the surface morphology, consisting of aggregates and particulates in micrometric range, was altered by the substrate porosity and interface properties, respectively. TEM studies evidenced that Ce stabilized ZrO(2) doped HA particulates ranged from 10 to 50 nm, strongly depending on the Al(2) O(3) porosity. The coatings consisted of HA nanocrystals embedded in an amorphous matrix quite similar to the bone structure. These findings were congruent with the increased biocompatibility and bioactivity of these layers confirmed by enhanced growing and proliferation of human mesenchymal stem cells.
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http://dx.doi.org/10.1002/jbm.b.31755DOI Listing
February 2011

Tailoring immobilization of immunoglobulin by excimer laser for biosensor applications.

J Biomed Mater Res A 2011 Feb 8;96(2):384-94. Epub 2010 Dec 8.

National Institute for Lasers, Plasma and Radiation Physics, Magurele, Ilfov, Romania.

The sheltered transfer and immobilization of rabbit anti-human antiserum immunoglobulin G (IgG) by matrix-assisted pulsed laser evaporation (MAPLE) are reported. The iced targets submitted to laser irradiation consisted of 0.2-2 mg/mL IgG blended or not with lipid (L-α-phosphatidylcholine dipalmitoyl) dissolved in distilled water-based saline buffer. Thin IgG coatings were obtained at room temperature onto glass, fused silica, or silicon substrates. Ten thousand subsequent laser pulses of 0.33, 0.5, or 0.67 J/cm(2) fluence were applied for the synthesis of each sample. Morphology and composition of the thin films were studied by optical, scanning, and atomic force microscopy and Fourier transformed infrared spectrometry. Optical labeling methods such as spectrofluorimetry and fluorescence microscopy were selected to verify the biosensor transduction principle because of their high sensitivity for detecting low amounts of antigen (IgG). Protein immobilization to the substrate surface was demonstrated for all obtained structures after immersion in the donkey anti-rabbit secondary antibody solution. The IgG transfer and immobilization onto substrates were improved by addition of lipid to MAPLE solutions.
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http://dx.doi.org/10.1002/jbm.a.32991DOI Listing
February 2011

Differentiation of mesenchymal stem cells onto highly adherent radio frequency-sputtered carbonated hydroxylapatite thin films.

J Biomed Mater Res A 2010 Dec 11;95(4):1203-14. Epub 2010 Oct 11.

Department of Molecular Cell Biology, Institute of Biochemistry, Romanian Academy, Bucharest, Romania.

In this work, an improved version of the radio frequency magnetron sputtering (RF-MS) technique was used to prepare highly adherent B-type carbonated hydroxylapatite (B-CHA) thin films. Fourier transform infrared spectroscopy (FTIR) and grazing incidence X-ray diffraction studies proved that the coatings maintained the composition and revealed the polycrystalline structure of HA. Scanning electron microscopy analysis showed that the CHA films are rough and exhibit a homogeneous microstructure. Energy-dispersive X-ray spectroscopy (EDX) mapping demonstrated a uniform distribution of the Ca and P cations while a Ca/P ratio of 1.8 was found. In addition, the FTIR experiments showed a remarkable reproducibility of the nanostructures. Human mesenchymal stem cells (hMSCs), in vitro differentiated osteoblasts, and explanted bone cells were grown over the surface of CHA coatings for periods between a few hours and 21 days. Osteoprogenitor cells maintained viability and characteristic morphology after adhesion on CHA coatings. Ki67-positive osteoblasts were the evidence of cell proliferation events. Cells showed positive staining for markers of osteoblast phenotype such as collagen type I, bone sialoprotein and osteonectin. Our data showed the formation of mineralized foci by differentiation of hMSCs to human primary osteoblasts after cultivation in osteogenic media on RF-sputtered films. The results demonstrate the capacity of B-type CHA coating to support MSCs adhesion and osteogenic differentiation ability.
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http://dx.doi.org/10.1002/jbm.a.32947DOI Listing
December 2010

Encapsulated cargo internalized by fusogenic liposomes partially overlaps the endoplasmic reticulum.

J Cell Mol Med 2009 Sep 2;13(9B):3110-21. Epub 2009 Apr 2.

Department of Molecular Cell Biology, Institute of Biochemistry of Romanian Academy, Splaiul Independentei, Bucharest, Romania.

Few endocytosed ligands, including bacterial toxins and simian virus 40 (SV40) have been shown to reach the endoplasmic reticulum (ER) in mammalian cells. Using calcein and fluorescently labelled lactoferrin encapsulated in fusogenic liposomes we found that the cargo uses a microtubule-based pathway with ER delivery. Endocytic uptake of the lipid vesicles was cholesterol dependent in all cell lines tested, including the caveolin-1-deficient human hepatoma 7 cell line. The ligand was transported in non-caveosome organelles requiring acidic pH for maturation, but able to escape the lysosomal route. These organelles were not recycling endosomes either, as shown by the lack of co-localization with recycling transferrin. Co-localization with the ER-tracker, orange fluorescent protein with KDEL signal retention and cholera toxin in live microscopy revealed an ER distribution of the fluorescent ligand. Brefeldin A, which prevents Golgi-dependent retrograde trafficking, does not disrupt the cargo delivery to the ER. This new endocytic pathway making use of acidic endosome-like organelles is an alternative to the reported SV40 caveolae pathways. Exploiting a cellular route linking the cell surface to the ER, fusogenic liposomes may become efficient drug delivery vehicles for ER stress and diseases.
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http://dx.doi.org/10.1111/j.1582-4934.2009.00724.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516470PMC
September 2009

Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.

Glycobiology 2007 Dec 20;17(12):1344-56. Epub 2007 Sep 20.

Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford, UK.

Ovarian cancer is the fourth most common cancer in women in the Western world. In a pilot scale study, we highlight changes in the total serum glycome of patients with advanced ovarian cancer that might shed insight into disease pathogenesis. These changes include increases in levels of core fucosylated, agalactosyl biantennary glycans (FA2) and sialyl Lewis x (SLe(x)). To investigate further which proteins contribute to these alterations, we developed technology to analyze simultaneously the glycosylation of protein glycoforms contained in single spots excised from a 2D gel (<1 ng protein). The acute-phase proteins, haptoglobin, alpha1-acid glycoprotein, and alpha1-antichymotrypsin from patients contained elevated levels of subsets of glycoforms containing SLe(x). We also established that IgG heavy chains from patients contained twice the level of FA2 compared with healthy controls. Serum CA125 is the only biomarker that is used routinely, and there is a need for complementary markers that will improve both sensitivity and specificity. There was some preliminary indication that combinations of changes in the serum glycome might improve the separation of ovarian cancer and benign tumors; however, a larger study using data receiver operating characteristic curves will be required to draw any firm conclusions.
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http://dx.doi.org/10.1093/glycob/cwm100DOI Listing
December 2007

An N-linked glycan modulates the interaction between the CD1d heavy chain and beta 2-microglobulin.

J Biol Chem 2006 Dec 27;281(52):40369-78. Epub 2006 Oct 27.

Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA.

Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with beta(2)-microglobulin (beta(2)m). Assembly occurs in the endoplasmic reticulum (ER) and involves the initial glycan-dependent association of the free heavy chain with calreticulin and calnexin and the thiol oxidoreductase ERp57. Folding and disulfide bond formation within the heavy chain occurs prior to beta(2)m binding. There are four N-linked glycans on the CD1d heavy chain, and we mutated them individually to ascertain their importance for the assembly and function of CD1d-beta(2)m heterodimers. None of the four were indispensable for assembly or the ability to bind alpha-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present alpha-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(alpha1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d-beta(2)m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with beta(2)m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from beta(2)m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from beta(2)m upon exposure to the detergent Triton X-100, whereas a mutant expressing only glycan 2 at Asn-42 was more stable. In addition, glycan 2 was not processed efficiently to the complex form in mature wild-type CD1d molecules. Modeling the glycans on the published structure indicated that glycan 2 interacts significantly with both the CD1d heavy chain and beta(2)m, which may explain these unusual properties.
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http://dx.doi.org/10.1074/jbc.M608518200DOI Listing
December 2006

Productive folding of tyrosinase ectodomain is controlled by the transmembrane anchor.

J Biol Chem 2006 Aug 31;281(31):21682-21689. Epub 2006 May 31.

Institute of Biochemistry, Splaiul Independentei 296, 060031 Bucharest 17, Romania. Electronic address:

Transmembrane domains (TMDs) are known as structural elements required for the insertion into the membrane of integral membrane proteins. We have provided here an example showing that the presence of the TMD is compulsory for the productive folding pathway of a membrane-anchored glycoprotein. Tyrosinase, a type I transmembrane protein whose insertion into the melanosomal membrane initiates melanin synthesis, is misfolded and degraded when expressed as a truncated polypeptide. We used constructs of tyrosinase ectodomain fused with chimeric TMDs or glycosylphosphatidylinositol anchor to gain insights into how the TMD enables the productive folding pathway of the ectodomain. We found that in contrast to the soluble constructs, the membrane-anchored chimeras fold into the native conformation, which allows their endoplasmic reticulum exit. They recruit calnexin to monitor their productive folding pathway characterized by the post-translational formation of buried disulfides. Lacking calnexin assistance, the truncated mutant is arrested in an unstable conformation bearing exposed disulfides. We showed that the transmembrane anchor of a protein may crucially, albeit indirectly, control the folding pathway of the ectodomain.
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http://dx.doi.org/10.1074/jbc.M603841200DOI Listing
August 2006

Do calnexin and calreticulin have a role in melanin formation?

IUBMB Life 2005 Jun;57(6):455-7

Institute of Biochemistry, Bucharest, Romania.

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http://dx.doi.org/10.1080/15216540500147106DOI Listing
June 2005

Tyrosinase-related protein-2 and -1 are trafficked on distinct routes in B16 melanoma cells.

Biochem Biophys Res Commun 2005 Mar;328(4):914-21

Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 060031 Bucharest 17, Romania.

Tyrosinase related protein (TRP)-1 and -2 regulate the main steps in melanin synthesis and are immune targets in skin cancer or autoimmune pigmentary disorders. We found that ionophore monensin (Mon) and the quaternary amine chloroquine (CQ) discriminate between the traffic routes of TRP-2 and TRP-1. TRP-2 N-glycan processing is interrupted by Mon between ER and trans-Golgi, whereas this process continues for TRP-1. Mature TRP-2 is diverted by CQ treatment to a degradation pathway which depends on functional vacuolar ATPases. Conversely, the subcellular distribution and stability of TRP-1 were not affected by CQ. We propose that TRP-2 is sorted and trafficked in the early secretory pathway with a cargo which does not include TRP-1; post Golgi, TRP-2 intersects the endocytic pathway following a route via early endosomes, possibly by rapid recycling from the plasma membrane. These data show that highly structural homologous glycoproteins use distinct trafficking pathways in the same cell.
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http://dx.doi.org/10.1016/j.bbrc.2005.01.040DOI Listing
March 2005

Soluble tyrosinase is an endoplasmic reticulum (ER)-associated degradation substrate retained in the ER by calreticulin and BiP/GRP78 and not calnexin.

J Biol Chem 2005 Apr 27;280(14):13833-40. Epub 2005 Jan 27.

Institute of Biochemistry, Splaiul Independentei 296, 060031 Bucharest 17, Romania.

Tyrosinase is a type I membrane protein regulating the pigmentation process in humans. Mutations of the human tyrosinase gene cause the tyrosinase negative type I oculocutaneous albinism (OCAI). Some OCAI mutations were shown to delete the transmembrane domain or to affect its hydrophobic properties, resulting in soluble tyrosinase mutants that are retained in the endoplasmic reticulum (ER). To understand the specific mechanisms involved in the ER retention of soluble tyrosinase, we have constructed a tyrosinase mutant truncated at its C-terminal end and investigated its maturation process. The mutant is retained in the ER, and it is degraded through the proteasomal pathway. We determined that the mannose trimming is required for an efficient degradation process. Moreover, this soluble ER-associated degradation substrate is stopped at the ER quality control checkpoint with no requirements for an ER-Golgi recycling pathway. Co-immmunoprecipitation experiments showed that soluble tyrosinase interacts with calreticulin and BiP/GRP78 (and not calnexin) during its ER transit. Expression of soluble tyrosinase in calreticulin-deficient cells resulted in the export of soluble tyrosinase of the ER, indicating the calreticulin role in ER retention. Taken together, these data show that OCAI soluble tyrosinase is an ER-associated degradation substrate that, unlike other albino tyrosinases, associates with calreticulin and BiP/GRP78. The lack of specificity for calnexin interaction reveals a novel role for calreticulin in OCAI albinism.
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http://dx.doi.org/10.1074/jbc.M413087200DOI Listing
April 2005

The glycosylation of tyrosinase in melanoma cells and the effect on antigen presentation.

Adv Exp Med Biol 2003 ;535:257-69

Institute of Biochemistry, Romanian Academy, Splaiul Independentei 296, 77700 Bucharest 17, Romania.

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http://dx.doi.org/10.1007/978-1-4615-0065-0_17DOI Listing
February 2004

Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding.

Glycobiology 2004 Feb 26;14(2):103-14. Epub 2003 Sep 26.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, U.K.

We recently reported statistical analysis of structural data on glycosidic linkages. Here we extend this analysis to the glycan-protein linkage, and the peptide primary, secondary, and tertiary structures around N-glycosylation sites. We surveyed 506 glycoproteins in the Protein Data Bank crystallographic database, giving 2592 glycosylation sequons (1683 occupied) and generated a database of 626 nonredundant sequons with 386 occupied. Deviations in the expected amino acid composition were seen around occupied asparagines, particularly an increased occurrence of aromatic residues before the asparagine and threonine at position +2. Glycosylation alters the asparagine side chain torsion angle distribution and reduces its flexibility. There is an elevated probability of finding glycosylation sites in which secondary structure changes. An 11-class taxonomy was developed to describe protein surface geometry around glycosylation sites. Thirty-three percent of the occupied sites are on exposed convex surfaces, 10% in deep recesses and 20% on the edge of grooves with the glycan filling the cleft. A surprisingly large number of glycosylated asparagine residues have a low accessibility. The incidence of aromatic amino acids brought into close contact with the glycan by the folding process is higher than their normal levels on the surface or in the protein core. These data have significant implications for control of sequon occupancy and evolutionary selection of glycosylation sites and are discussed in relation to mechanisms of protein fold stabilization and regional quality control of protein folding. Hydrophobic protein-glycan interactions and the low accessibility of glycosylation sites in folded proteins are common features and may be critical in mediating these functions.
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http://dx.doi.org/10.1093/glycob/cwh008DOI Listing
February 2004

The inhibition of early N-glycan processing targets TRP-2 to degradation in B16 melanoma cells.

J Biol Chem 2003 Jul 28;278(29):27035-42. Epub 2003 Apr 28.

Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 77700 Bucharest, Romania.

Tyrosinase-related protein-2 (TRP-2) is a DOPAchrome tautomerase catalyzing a distal step in the melanin synthesis pathway. Similar to the other two melanogenic enzymes belonging to the TRP gene family, tyrosinase and TRP-1, TRP-2 is expressed in melanocytes and melanoma cells. Despite the increasing evidence of its efficiency as a melanoma antigen, little is known about the maturation and intracellular trafficking of TRP-2. Here we show that TRP-2 is mainly distributed in the TGN of melanoma cells instead of being confined solely to melanosomes. This, together with the plasma membrane occasional localization observed by immunofluorescence, suggest the TRP-2 participation in a recycling pathway, which could include or not the melanosomes. Using pulse-chase experiments we show that the TRP-2 polypeptide folds in the endoplasmic reticulum (ER) in the presence of calnexin, until it reaches a dithiothreitol-resistant conformation enabling its ER exit to the Golgi. If N-glycosylation inhibitors prevent the association with calnexin, the TRP-2 nascent chain undergoes an accelerated degradation process. This process is delayed in the presence of proteasomal inhibitors, indicating that the misfolded chain is retro-translocated from the ER into the cytosol and degraded in proteasomes. This is a rare example in which calnexin although indispensable for the nascent chain folding is not required for its targeting to degradation. Therefore TRP-2 may prove to be a good model to document the calnexin-independent retro-translocation process of proteasomally degraded proteins. Clearly, TRP-2 has a distinct maturation pathway from tyrosinase and TRP-1 and possibly a second regulatory function within the cell.
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http://dx.doi.org/10.1074/jbc.M303167200DOI Listing
July 2003

pH-sensitive liposomes are efficient carriers for endoplasmic reticulum-targeted drugs in mouse melanoma cells.

Biochem Biophys Res Commun 2002 May;293(3):918-23

Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 77700 Bucharest, Romania.

Tyrosinase, the key enzyme of melanin biosynthesis, is inactivated in melanoma cells following the incubation with the imino-sugar N-butyldeoxynojirimycin, an inhibitor of the endoplasmic reticulum N-glycosylation processing. We have previously shown that tyrosinase inhibition requires high NB-DNJ concentrations, suggesting an inefficient cellular uptake of the drug. Here we show that the use of pH-sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesteryl hemisuccinate for the delivery of NB-DNJ reduced the required dose for tyrosinase inhibition by a factor of 1000. The results indicate that these pH-sensitive liposomes are efficient carriers for imino-sugars delivery in the endoplasmic reticulum of mammalian cells.
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http://dx.doi.org/10.1016/S0006-291X(02)00317-0DOI Listing
May 2002
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