Massimo Alessio

ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family

In human cells, Ero1‐Lα and ‐Lβ (hEROs) regulate oxidative protein folding by selectively oxidizing protein disulfide isomerase. Specific protein–protein interactions are probably crucial for regulating the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (ER). To identify molecules involved in ER redox control, we searched for proteins interacting with Ero1‐Lα. Here, we characterize a novel ER resident protein (ERp44), which contains a thioredoxin domain with a CRFS motif and is induced during ER stress. ERp44 forms mixed disulfides with both hEROs and cargo folding intermediates. Whilst the interaction with transport‐competent Ig‐K chains is transient, ERp44 binds more stably with J chains, which are retained in the ER and eventually degraded by proteasomes. ERp44 does not bind a short‐lived ribophorin mutant lacking cysteines. Its overexpression alters the equilibrium of the different Ero1‐Lα redox isoforms, suggesting that ERp44 may be involved in the control of oxidative protein folding.

Thiol-mediated protein retention in the endoplasmic reticulum: the role of ERp44

Formation of disulfide bonds, an essential step for the maturation and exit of secretory proteins from the endoplasmic reticulum (ER), is controlled by specific ER‐resident enzymes. A pivotal element in this process is Ero1α, an oxidoreductin that lacks known ER retention motifs. Here we show that ERp44 mediates Ero1α ER localization through the formation of reversible mixed disulfides. ERp44 also prevents the secretion of an unassembled cargo protein with unpaired cysteines. We conclude that ERp44 is a key element in thiol‐mediated retention. It might also favour the maturation of disulfide‐linked oligomeric proteins and their quality control.

Hemozoin (Malarial Pigment) Inhibits Differentiation and Maturation of Human Monocyte-Derived Dendritic Cells: A Peroxisome Proliferator-Activated Receptor-γ-Mediated Effect

Acute and chronic Plasmodium falciparum malaria are accompanied by severe immunodepression possibly related to subversion of dendritic cells (DC) functionality. Phagocytosed hemozoin (malarial pigment) was shown to inhibit monocyte functions related to immunity. Hemozoin-loaded monocytes, frequently found in circulation and adherent to endothelia in malaria, may interfere with DC development and play a role in immunodepression. Hemozoin-loaded and unloaded human monocytes were differentiated in vitro to immature DC (iDC) by treatment with GM-CSF and IL-4, and to mature DC (mDC) by LPS challenge. In a second setting, hemozoin was fed to iDC further cultured to give mDC. In both settings, cells ingested large amounts of hemozoin undegraded during DC maturation. Hemozoin-fed monocytes did not apoptose but their differentiation and maturation to DC was severely impaired as shown by blunted expression of MHC class II and costimulatory molecules CD83, CD80, CD54, CD40, CD1a, and lower levels of CD83-specific mRNA in hemozoin-loaded iDC and mDC compared with unfed or latex-loaded DC. Further studies indicated activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) in hemozoin-loaded iDC and mDC, associated with increased expression of PPAR-γ mRNA, without apparent involvement of NF-κB. Moreover, expression of PPAR-γ was induced and up-regulation of CD83 was inhibited by supplementing iDC and mDC with plausible concentrations of 15(S)-hydroxyeicosatetraenoic acid, a PPAR-γ ligand abundantly produced by hemozoin via heme-catalyzed lipoperoxidation.

Circulating Autoantibodies to Phosphorylated α-Enolase are a Hallmark of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and no diagnostic markers have, as of yet, been defined. In PDAC patients, α-enolase (ENOA) is up-regulated and elicits the production of autoantibodies. Here, we analyzed the autoantibody response to post-translational modifications of ENOA in PDAC patients. ENOA isolated from PDAC tissues and cell lines was characterized by two-dimensional electrophoresis (2-DE) Western blot (WB), revealing the expression of six different isoforms (named ENOA1,2,3,4,5,6) whereas only 4 isoforms (ENOA3,4,5,6) were detectable in normal tissues. As assessed by 2-DE WB, 62% of PDAC patients produced autoantibodies to the two more acidic isoforms (ENOA1,2) as opposed to only 4% of controls. Mass spectrometry showed that ENOA1,2 isoforms were phosphorylated on serine 419. ROC analysis demonstrated that autoantibodies to ENOA1,2 usefully complement the diagnostic performance of serum CA19.9 levels, achieving approximately 95% diagnostic accuracy in both advanced and resectable PDAC. Moreover, the presence of autoantibodies against ENOA1,2 correlated with a significantly better clinical outcome in advanced patients treated with standard chemotherapy. In conclusion, our results demonstrate that ENOA phosphorylation is associated with PDAC and induces specific autoantibody production in PDAC patients that may have diagnostic value.

Proteome Study of Human Cerebrospinal Fluid following Traumatic Brain Injury Indicates Fibrin(ogen) Degradation Products as Trauma-Associated Markers

Traumatic brain injury (TBI), like other central nervous system pathologies, causes changes in the composition of cerebrospinal fluid (CSF). Consequently analysis of the CSF components is important to better understand the pathological processes involved in such diseases. The aim of this work was to identify specific markers of severe TBI. Proteomic analysis including two-dimensional gel electrophoresis combined with mass spectrometry analysis was used to compare the CSF protein profile of severe TBI patients and controls. Proteins (alpha 1 antitrypsin, haptoglobin 1 alpha1, alpha2, and beta) belonging to the acute phase response showed an increased expression in severe TBI patients. Two other proteins, identified as proteolytic degradation products of the carboxyl-terminal portion of the fibrinogen beta, were present only in TBI patients. The presence of these markers could correlate with a post-traumatic local increase in fibrinolysis as well as to an inflammatory event following CNS tissue injury.

Identification of CD36 molecular features required for its in vitro angiostatic activity.

Thrombospondin‐1 (TSP‐1), a natural inhibitor of angiogenesis, acts directly on endothelial cells (EC) via CD36 to inhibit their migration and morphogenesis induced by basic fibroblast growth factor. Here we show that CD36 triggered by TSP‐1 inhibits in vitro angiogenesis stimulated by vascular endothelial growth factor‐A (VEGF‐A). To demonstrate that the TSP‐1 inhibitory signal was mediated by CD36, we transduced CD36 in CD36‐deficient endothelial cells. Both TSP‐1 and the agonist anti‐CD36 mAb SMO, which mimics TSP‐1 activity, reduced the VEGF‐A165‐induced migration and sprouting of CD36‐ECs. To address the mechanisms by which CD36 may exert its angiostatic function, we investigated the functional components of the C‐terminal cytoplasmic tail by site‐directed mutagenesis. Our results indicate that C464, R467, and K469 of CD36 are required for the inhibitory activity of TSP‐1. In contrast, point mutation of C466 did not alter TSP‐1 ability to inhibit EC migration and sprouting. Moreover, we show that activation of CD36 by TSP‐1 down‐modulates the VEGF receptor‐2 (VEGFR‐2) and p38 mitogen‐associated protein kinase phosphorylation induced by VEGF‐A165, and this effect was specifically abolished by point mutation at C464. These results identify specific amino acids of the C‐terminal cytoplasmic tail of CD36 crucial for the in vitro angiostatic activity of TSP‐1 and extend our knowledge of regulation of VEGFR‐2‐mediated biological activities on ECs.

Molecular Cloning of hMena (ENAH) and Its Splice Variant hMena+11a: Epidermal Growth Factor Increases Their Expression and Stimulates hMena+11a Phosphorylation in Breast Cancer Cell Lines

hMena (ENAH), an actin regulatory protein involved in the control of cell motility and adhesion, is modulated during human breast carcinogenesis. In fact, whereas undetectable in normal mammary epithelium, hMena becomes overexpressed in high-risk benign lesions and primary and metastatic tumors. In vivo, hMena overexpression correlates with the HER-2(+)/ER(-)/Ki67(+) unfavorable prognostic phenotype. In vitro, neuregulin-1 up-regulates whereas Herceptin treatment down-modulates hMena expression, suggesting that it may couple tyrosine kinase receptor signaling to the actin cytoskeleton. Herein, we report the cloning of hMena and of a splice variant, hMena(+11a), which contains an additional exon corresponding to 21 amino acids located in the EVH2 domain, from a breast carcinoma cell line of epithelial phenotype. Whereas hMena overexpression consistently characterizes the transformed phenotype of tumor cells of different lineages, hMena(+11a) isoform is concomitantly present only in epithelial tumor cell lines. In breast cancer cell lines, epidermal growth factor (EGF) treatment promotes concomitant up-regulation of hMena and hMena(+11a), resulting in an increase of the fraction of phosphorylated hMena(+11a) isoform only. hMena(+11a) overexpression and phosphorylation leads to increased p42/44 mitogen-activated protein kinase (MAPK) activation and cell proliferation as evidenced in hMena(+11a)-transfected breast cancer cell lines. On the contrary, hMena knockdown induces reduction of p42/44 MAPK phosphorylation and of the proliferative response to EGF. The present data provide new insight into the relevance of actin cytoskeleton regulatory proteins and, in particular, of hMena isoforms in coupling multiple signaling pathways involved in breast cancer.

ZAP-70 is expressed by normal and malignant human B-cell subsets of different maturational stage

ZAP-70 tyrosine kinase is involved in signalling pathways following T-cell receptor stimulation and was originally described only in T cells and natural killer cells. ZAP-70 expression has been reported in normal mouse B lineage cells and in human malignant B lymphocytes, mainly in chronic lymphocytic leukemia (CLL) where it correlates with clinical outcome. We analyzed several B-cell lines and ex vivo malignant B cells, ranging from acute lymphoblastic leukemia to multiple myeloma and reflecting different stages of B-cell differentiation, and they showed ZAP-70 expression regardless their maturation stage. We then analyzed by Western blot and flow cytometry different human normal B-lymphocyte subpopulations: naïve, germinal center and memory B cells from tonsils, CD19+ CD5+ cells from cord blood and CD19+ lymphocytes from peripheral blood. All expressed ZAP-70 protein, though at different levels depending on their differentiation, activation and tissue localization. In addition, ZAP-70 expression levels could be modulated following stimulation via the B-cell receptor. These findings implicate a potential role of ZAP-70 in the signalling pathway of B lymphocytes at different maturational stages, indicate that ZAP-70 expression is not a CLL-specific feature among B-cell malignancies and suggest that the absence of ZAP-70 rather than its presence should be considered abnormal for malignant B lymphocytes.

Synthesis, Processing, and Intracellular Transport of CD36 during Monocytic Differentiation

CD36 is an integral membrane glycoprotein expressed by several cell types, including endothelial cells of the microvasculature, erythrocytes, platelets, and monocytes. In the monocytic lineage, CD36 is expressed during the late stages of differentiation in the bone marrow, in circulating monocytes, and in some tissue resident macrophages, and it is thought to mediate the phagocytosis of apoptotic cells and the endocytic uptake of modified lipoproteins. Here we analyze the synthesis, processing, and intracellular transport of CD36 in U937 and THP-1, two human cell lines representing different stages of monocytic maturation. In both cell lines, phorbol 12-myristate 13-acetate induces the expression of CD36. A 74-kDa intracellular precursor is first synthesized that has the hallmarks of a resident protein of the endoplasmic reticulum. The precursor protein is later processed into a mature form of 90-105 kDa which is transported to the cell surface. The kinetics of processing differ significantly in U937 and THP-1. These differences are specific for the CD36, as two unrelated proteins (CD11b and CD45R) are processed and transported to the surface at similar rates in the two cell lines. A 33-kDa endoglycosidase H-sensitive glycoprotein specifically associates with the 74-kDa precursor. Coprecipitation of gp33 correlates with slow processing of CD36 precursor, suggesting that gp33 may play a role in regulating the intracellular transport of CD36, during monocyte maturation.

Human Mena+11a isoform serves as a marker of epithelial phenotype and sensitivity to epidermal growth factor receptor inhibition in human pancreatic cancer cell lines.

Purpose: hMena, member of the enabled/vasodilator-stimulated phosphoprotein family, is a cytoskeletal protein that is involved in the regulation of cell motility and adhesion. The aim of this study was to determine whether or not the expression of hMena isoforms correlated with sensitivity to EGFR tyrosine kinase inhibitors and could serve as markers with potential clinical use. Experimental Design: Human pancreatic ductal adenocarcinoma cell lines were characterized for in vitro sensitivity to erlotinib, expression of HER family receptors, markers of epithelial to mesenchymal transition, and expression of hMena and its isoform hMena+11a. The effects of epidermal growth factor (EGF) and erlotinib on hMena expression as well as the effect of hMena knockdown on cell proliferation were also evaluated. Results: hMena was detected in all of the pancreatic tumor cell lines tested as well as in the majority of the human tumor samples [primary (92%) and metastatic (86%)]. Intriguingly, in vitro hMena+11a isoform was specifically associated with an epithelial phenotype, EGFR dependency, and sensitivity to erlotinib. In epithelial BxPC3 cells, epidermal growth factor up-regulated hMena/hMena+11a and erlotinib down-regulated expression. hMena knockdown reduced cell proliferation and mitogen-activated protein kinase and AKT activation in BxPC3 cells, and promoted the growth inhibitory effects of erlotinib. Conclusions: Collectively, our data indicate that the hMena+11a isoform is associated with an epithelial phenotype and identifies EGFR-dependent cell lines that are sensitive to the EGFR inhibitor erlotinib. The availability of anti-hMena+11a–specific probes may offer a new tool in pancreatic cancer management if these results can be verified prospectively in cancer patients.