Biochemical and spectroscopic studies of human melanotransferrin (MTf): electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.     

Involvement of CD45 in DNA fragmentation in apoptosis induced by mitochondrial perturbing agents

CD45 is a type I transmembrane molecule with phosphatase activity which comprises up to 10% of the cell surface area in nucleated haematopoietic cells. We have previously demonstrated the absence of nuclear apoptosis in CD45-negative T cells after chemical-induced apoptosis. The aim of this study was to characterize the role of CD45 in nuclear apoptosis. In contrast to wild type CD45-positive T cells, the CD45-deficient T cell lines are resistant to the induction of DNA fragmentation and chromatin condensation following tributyltin (TBT) or H₂O₂ exposure, but not to cycloheximide-induced apoptosis. CD45 transfection in deficient cell lines led to the restoration of chromatin condensation and DNA fragmentation following TBT exposure. In both CD45-positive and negative T cell lines, TBT exposure mediates intracellular calcium mobilization, caspase-3 activation and DFF45 cleavage. Moreover, DNA fragmentation was also induced by TBT in cells deficient in expression of p56lck, ZAP-70 and SHP-1. Subcellular partitioning showed a decrease in nuclear localisation of caspase-3 and DFF40. Together, these results demonstrate for the first time, that CD45 expression plays a key role in internucleosomal DNA fragmentation and chromatin condensation processes during apoptosis. CD45 activity or its substrates’ activity, appears to be located downstream of caspase-3 activation and plays a role in retention of DFF40 in the nucleus. Philippe Desharnais and Geneviève Dupéré-Minier have contributed equally to this work.     

CD1a and MHC class I follow a similar endocytic recycling pathway

CD1 proteins are a family of major histocompatibility complex (MHC) class I-like antigen-presenting molecules that present lipids to T cells. The cytoplasmic tails (CTs) of all human CD1 isoforms, with the exception of CD1a, contain tyrosine-based sorting motifs, responsible for the internalization of proteins by the clathrin-mediated pathway. The role of the CD1a CT, which does not possess any sorting motifs, as well as its mode of internalization are not known. We investigated the internalization and recycling pathways followed by CD1a and the role of its CT. We found that CD1a can be internalized by a clathrin- and dynamin-independent pathway and that it follows a Rab22a- and ADP ribosylation factor (ARF)6-dependent recycling pathway, similar to other cargo internalized independent of clathrin. We also found that the CD1a CT is S-acylated. However, this posttranslational modification does not determine the rate of internalization or recycling of the protein or its localization to detergent-resistant membrane microdomains (DRMs) where we found CD1a to be enriched. We also show that plasma membrane DRMs are essential for efficient CD1a-mediated antigen presentation. These findings place CD1a closer to MHC class I in its trafficking and potential antigen-loading compartments among CD1 isoforms. Furthermore, we identify CD1a as a new marker for the clathrin- and dynamin-independent and DRM-dependent pathway of internalization as well as the Rab22a- and ARF6-dependent recycling pathway.     

Oxidation as a crucial reaction for cholesterol to induce tissue degeneration: CD36 overexpression in human promonocytic cells treated with a biologically relevant oxysterol mixture

Oxidative stress, inflammation and altered cholesterol metabolism and levels are among the pathogenetic mechanisms of cognitive impairment that may accompany aging. Within the research area of hypercholesterolemia and age-related disease processes, the molecular mechanisms of cholesterol interaction with the inflammatory cells of the macrophage lineage are yet to be elucidated. We thus investigated the effect of both non-oxidized and oxidized cholesterol on monocytic cell differentiation and foam cell formation, as it occurs within vascular lesions during progression of atherosclerosis. In vitro experiments performed on human U937 promonocytic cells showed that a biologically representative mixture of oxysterols markedly stimulated CD36 expression and synthesis. In contrast, non-oxidized cholesterol did not exert any effect on CD36 mRNA and protein levels. Furthermore, the oxysterol-induced up-regulation of CD36 appeared to be based on the subsequent activation of protein kinase Cdelta (PKCdelta), extracellular signal-regulated kinase 1/2 (ERK1/2) and peroxisome proliferator-activated receptor gamma (PPARgamma). Cells overexpressing CD36 were indeed able to actively take up oxidized low-density lipoproteins, and become foam cells. The essential role of ERK pathway and CD36 receptor in oxysterol-induced foam cell formation was proved by the prevention of the latter event when monocytic cells were incubated in the presence of MEK1/2 selective inhibitor or anti-CD36 specific antibody. These experimental findings point to cholesterol oxidation as an essential reaction for this sterol to exert cellular stress and tissue damage in age-related diseases in which inflammation represents a main driving force.     

Energetic rationale for an unexpected and abrupt reversal of guanidinium chloride-induced unfolding of peptide deformylase

Peptide deformylase (PDF) catalyzes the removal of formyl group from the N-terminal methionine residues of nascent proteins in prokaryotes, and this enzyme is a high priority target for antibiotic design. In pursuit of delineating the structural-functional features of Escherichia coli PDF (EcPDF), we investigated the mechanistic pathway for the guanidinium chloride (GdmCl)-induced unfolding of the enzyme by monitoring the secondary structural changes via CD spectroscopy. The experimental data revealed that EcPDF is a highly stable enzyme, and it undergoes slow denaturation in the presence of varying concentrations of GdmCl. The most interesting aspect of these studies has been the abrupt reversal of the unfolding pathway at low to moderate concentrations of the denaturant, but not at high concentration. An energetic rationale for such an unprecedented feature in protein chemistry is offered.     

Secretion of active membrane type 1 matrix metalloproteinase (MMP-14) into extracellular space in microvesicular exosomes

Membrane type 1 matrix metalloproteinase (MT1-MMP, MMP14) is an efficient extracellular matrix (ECM) degrading enzyme that plays important roles in tissue homeostasis and cell invasion. Like a number of type I membrane proteins, MT1-MMP can be internalized from the cell surface through early and recycling endosomes to late endosomes, and recycled to the plasma membrane. Late endosomes participate in the biogenesis of small (30-100 nm) vesicles, exosomes, which redirect plasma membrane proteins for extracellular secretion. We hypothesized that some of the endosomal MT1-MMP could be directed to exosomes for extracellular release. Using cultured human fibrosarcoma (HT-1080) and melanoma (G361) cells we provide evidence that both the full-length 60 kDa and the proteolytically processed 43 kDa forms of MT1-MMP are secreted in exosomes. The isolated exosomes were identified by their vesicular structure in electron microscopy and by exosomal marker proteins CD9 and tumor susceptibility gene (TSG101). Furthermore, exosomes contained beta1-integrin (CD29). The exosomes were able to activate pro-MMP-2 and degrade type 1 collagen and gelatin, suggesting that the exosomal MT1-MMP was functionally active. The targeting of MT1-MMP in exosomes represents a novel mechanism for cancer cells to secrete membrane type metalloproteolytic activity into the extracellular space.     

CD38 is constitutively expressed in the nucleus of human hematopoietic cells

CD38 is a type II glycoprotein that acts both as a bifunctional enzyme, responsible for the synthesis and hydrolysis of cyclic ADP-ribose, and as a signal-transducing surface receptor. Although CD38 was originally described as a plasma membrane molecule, several reports indicate that CD38 is expressed in the nucleus, even in cells known to be CD38 surface-negative. In this study, firstly we investigated the presence of nuclear CD38 by immunofluorescence and confocal microscopy using a panel of hematopoietic cell lines that exhibit different levels of CD38 plasma membrane expression. Our second aim was to explore the relationship between the nuclear and plasma membrane forms of CD38 in human cell lines which represent discrete early maturation stages of the human lymphoid and myeloid compartments. Our results indicate that CD38 is constitutively present in the nucleus of cells belonging to distinct lineages. Furthermore, nuclear CD38 appears to be independent of the plasma membrane pool. The presence of nuclear CD38 during different stages of hematopoietic differentiation suggests that it may play a role in the control of nuclear Ca(2+) homeostasis and NAD levels.