Colette Michalak

Amyloid-β peptide binds to microtubule-associated protein 1B (MAP1B)

Abstract Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimers disease. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of targets have deleterious effects on cellular functions. In the present study we have shown for the first time that amyloid-beta 1–42 bound to a peptide comprising the microtubule binding domain of the heavy chain of microtubule-associated protein 1B by the screening of a human brain cDNA library expressed on M13 phage. This interaction may explain, in part, the loss of neuronal cytoskeletal integrity, impairment of microtubule-dependent transport and synaptic dysfunction observed previously in Alzheimers disease.

Human phagosome processing of Mycobacterium tuberculosis antigens is modulated by interferon-γ and interleukin-10

Intracellular pathogens, such as Mycobacterium tuberculosis, reside in the phagosomes of macrophages where antigenic processing is initiated. Mycobacterial antigen–MHC class II complexes are formed within the phagosome and are then trafficked to the cell surface. Interferon‐γ (IFN‐γ) and interleukin‐10 (IL‐10) influence the outcome of M. tuberculosis infection; however, the role of these cytokines with regard to the formation of M. tuberculosis peptide–MHC‐II complexes remains unknown. We analysed the kinetics and subcellular localization of M. tuberculosis peptide–MHC‐II complexes in M. tuberculosis‐infected human monocyte‐derived macrophages (MDMs) using autologous M. tuberculosis‐specific CD4+ T cells. The MDMs were pre‐treated with either IFN‐γ or IL‐10 and infected with M. tuberculosis. Cells were mechanically homogenized, separated on Percoll density gradients and manually fractionated. The fractions were incubated with autologous M. tuberculosis ‐specific CD4+ T cells. Our results demonstrated that in MDMs pre‐treated with IFN‐γ, M. tuberculosis peptide–MHC‐II complexes were detected early mainly in the phagosomal fractions, whereas in the absence of IFN‐γ, the complexes were detected in the endosomal fractions. In MDMs pre‐treated with IL‐10, the M. tuberculosis peptide–MHC‐II complexes were retained in the endosomal fractions, and these complexes were not detected in the plasma membrane fractions. The results of immunofluorescence microscopy demonstrated the presence of Ag85B associated with HLA‐DR at the cell surface only in the IFN‐γ‐treated MDMs, suggesting that IFN‐γ may accelerate M. tuberculosis antigen processing and presentation at the cell membrane, whereas IL‐10 favours the trafficking of Ag85B to vesicles that do not contain LAMP‐1. Therefore, IFN‐γ and IL‐10 play a role in the formation and trafficking of M. tuberculosis peptide–MHC‐II complexes.

Mannose 6-phosphate-independent endocytosis of β-glucuronidase: II. Purification of a cation-dependent receptor from bovine liver

Abstract A new binding protein, which recognizes a specific peptide sequence from pronase digested bovine β-glucuronidase, has been isolated from bovine liver membranes. Prior work has shown that this peptide (IIIb2) contains a Ser–X–Ser sequence, where X might be a posttranslational modified Trp. This receptor was detergent-extracted from total bovine liver membranes and purified by affinity chromatography on a bovine β-glucuronidase–Sepharose and a IIIb2 peptide–Sepharose column. Binding of bovine β-glucuronidase to the isolated receptor requires divalent cations, and their presence was necessary to maintain the receptor–ligand complex. Only the peptide sequence containing the fraction IIIb2 was able to impair the binding of the bovine enzyme to the receptor, no other peptide from bovine β-glucuronidase had an effect on binding. When analyzed by SDS–PAGE under reducing conditions, two bands were observed, a major band of 78 kDa and a faint band of 72 kDa. Rabbit antibodies against this binding protein revealed the presence of the 78 kDa protein in membranes from bovine liver, human and bovine fibroblasts. These antibodies impaired human fibroblasts endocytosis of the bovine but not of the human β-glucuronidase, which is taken up by a 300 kDa receptor that recognizes phosphomannosyl moieties in the enzyme.

Mannose 6-phosphate-independent endocytosis of β-glucuronidase by human fibroblasts: I. evidence for the existence of a membrane-binding activity

Abstract Prior work has shown that endocytosis of bovine β-glucuronidase by human fibroblasts can be mediated by the existence of a Man6P-independent receptor for the recapture and targeting to lysosomes. In this study, we have isolated a peptide (IIIb2) from pronase digested bovine β-glucuronidase that behaved as competitive inhibitor of the endocytosis of bovine β-glucuronidase by human fibroblasts. This peptide contained a Ser–X–Ser sequence, where X is probably a posttranslational modified Trp. Antibodies raised against this peptide impaired the endocytosis of the bovine but not the human β-glucuronidase, implying that the new recognition marker for the endocytosis of acid hydrolases might reside in a single discrete stretch of amino acid sequence. On the other hand, bovine β-glucuronidase has been shown to bind specifically to receptors of human fibroblast membranes. The binding was saturable, divalent cation-dependent and was competitively inhibited by the IIIb2 peptide, but not by mannose 6-phosphate. Results presented suggested an interplay between manganese concentrations, temperature and pH on the dissociation of the β-glucuronidase-receptor complexes. All together, these data reinforce the presence of two endocytic systems for the recapture and targeting of β-glucuronidase in human fibroblasts.

Holocarboxylase synthetase acts as a biotin-independent transcriptional repressor interacting with HDAC1, HDAC2 and HDAC7

In human cells, HCS catalyzes the biotinylation of biotin-dependent carboxylases and mediates the transcriptional control of genes involved in biotin metabolism through the activation of a cGMP-dependent signal transduction pathway. HCS also targets to the cell nucleus in association with lamin-B suggesting additional gene regulatory functions. Studies from our laboratory in Drosophila melanogaster showed that nuclear HCS is associated with heterochromatin bands enriched with the transcriptionally repressive mark histone 3 trimethylated at lysine 9. Further, HCS was shown to be recruited to the core promoter of the transcriptionally inactive hsp70 gene suggesting that it may participate in the repression of gene expression, although the mechanism involved remained elusive. In this work, we expressed HCS as a fusion protein with the DNA-binding domain of GAL4 to evaluate its effect on the transcription of a luciferase reporter gene. We show that HCS possesses transcriptional repressor activity in HepG2 cells. The transcriptional function of HCS was shown by in vitro pull down and in vivo co-immunoprecipitation assays to depend on its interaction with the histone deacetylases HDAC1, HDAC2 and HDAC7. We show further that HCS interaction with HDACs and its function in transcriptional repression is not affected by mutations impairing its biotin-ligase activity. We propose that nuclear HCS mediates events of transcriptional repression through a biotin-independent mechanism that involves its interaction with chromatin-modifying protein complexes that include histone deacetylases.

Annexin VI is a mannose-6-phosphate-independent endocytic receptor for bovine β-glucuronidase.

Endocytosis and transport of bovine liver β-glucuronidase to lysosomes in human fibroblasts are mediated by two receptors: the well-characterized cation-independent mannose 6-phosphate receptor (IGF-II/Man6PR) and an IGF-II/Man6PR-independent receptor, which recognizes a Ser-Trp*-Ser sequence present on the ligand. The latter receptor was detergent extracted from bovine liver membranes and purified. LC/ESI-MS/MS analysis revealed that this endocytic receptor was annexin VI (AnxA6). Several approaches were used to confirm this finding. First, the binding of bovine β-glucuronidase to the purified receptor from bovine liver membranes and His-tagged recombinant human AnxA6 protein was confirmed using ligand-blotting assays. Second, western blot analysis using antibodies raised against IGF-II/Man6PR-independent receptor as well as commercial antibodies against AnxA6 confirmed that the receptor and AnxA6 were indeed the same protein. Third, double immunofluorescence experiments in human fibroblasts confirmed a complete colocalization of the bovine β-glucuronidase and the AnxA6 receptor on the plasma membrane. Lastly, two cell lines were stably transfected with a plasmid containing the cDNA for human AnxA6. In both transfected cell lines, an increase in cell surface AnxA6 and in mannose 6-phosphate-independent endocytosis of bovine β-glucuronidase was detected. These results indicate that AnxA6 is a novel receptor that mediates the endocytosis of the bovine β-glucuronidase.

Two translation initiation codons direct the expression of annexin VI 64 kDa and 68 kDa isoforms

Annexin A6 is a multicompetent, multifunctional protein involved in several biological processes within and outside of the cell. Whereas HeLa cells express annexin A6 only as a 68/67-kDa doublet, indicating alternative splicing (Smith PD et al. (1994) Proc Natl Acad Sci USA 91, 2713-2717), the GMO2784 human fibroblast cell line expresses two additional isoforms at 64 and 58kDa. In both cell lines, annexin A6 is located intracellularly and on the plasma membrane. In vitro eukaryotic protein synthesis of pIRESneoAnxA6 cDNA and pIRESneoAnxA6/Met1- or Met33- using a reticulocyte lysate coupled transcription/translation system revealed that this gene contains two translation start codons, Met1 and Met33. Immunoprecipitation of the products obtained from the transcription/translation system using various anti-annexin A6 antibodies confirmed the presence of several isoforms and suggested that this protein might be present in different configurations.