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

Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimer's 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 Alzheimer's disease.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

The microtubule-associated protein tumor overexpressed gene binds to the RNA trafficking protein heterogeneous nuclear ribonucleoprotein A2

In neural cells, such as oligodendrocytes and neurons, transport of certain RNAs along microtubules is mediated by the cis-acting heterogeneous nuclear ribonucleoprotein A2 response element (A2RE) trafficking element and the cognate trans-acting heterogeneous nuclear ribonucleoprotein (hnRNP) A2 trafficking factor. Using a yeast two-hybrid screen, we have identified a microtubule-associated protein, tumor overexpressed gene (TOG)2, as an hnRNP A2 binding partner. The C-terminal third of TOG2 is sufficient for hnRNP A2 binding. TOG2, the large protein isoform of TOG, is the only isoform detected in oligodendrocytes in culture. TOG coimmunoprecipitates with hnRNP A2 present in the cytoskeleton (CSK) fraction of neural cells, and both coprecipitate with microtubule stabilized pellets. Staining with anti-TOG reveals puncta that are localized in proximity to microtubules, often at the plus ends. TOG is colocalized with hnRNP A2 and A2RE-mRNA in trafficking granules that remain associated with CSK-insoluble tissue. These data suggest that TOG mediates the association of hnRNP A2-positive granules with microtubules during transport and/or localization.     

Mapmodulin/leucine-rich acidic nuclear protein binds the light chain of microtubule-associated protein 1B and modulates neuritogenesis

We had previously described the leucine-rich acidic nuclear protein (LANP) as a candidate mediator of toxicity in the polyglutamine disease, spinocerebellar ataxia type 1 (SCA1). This was based on the observation that LANP binds ataxin-1, the protein involved in this disease, in a glutamine repeat-dependent manner. Furthermore, LANP is expressed abundantly in purkinje cells, the primary site of ataxin-1 pathology. Here we focused our efforts on understanding the neuronal properties of LANP. In undifferentiated neuronal cells LANP is predominantly a nuclear protein, requiring a bona fide nuclear localization signal to be imported into the nucleus. LANP translocates from the nucleus to the cytoplasm during the process of neuritogenesis, interacts with the light chain of the microtubule-associated protein 1B (MAP1B), and modulates the effects of MAP1B on neurite extension. LANP thus could play a key role in neuronal development and/or neurodegeneration by its interactions with microtubule associated proteins.     

MPB2C,a microtubule-associated plant protein binds to and interferes with cell-to-cell transport of tobacco mosaic virus movement protein

The movement protein of tobacco mosaic virus, MP30, mediates viral cell-to-cell transport via plasmodesmata. The complex MP30 intra- and intercellular distribution pattern includes localization to the endoplasmic reticulum, cytoplasmic bodies, microtubules, and plasmodesmata and likely requires interaction with plant endogenous factors. We have identified and analyzed an MP30-interacting protein, MPB2C, from the host plant Nicotiana tabacum. MPB2C constitutes a previously uncharacterized microtubule-associated protein that binds to and colocalizes with MP30 at microtubules. In vivo studies indicate that MPB2C mediates accumulation of MP30 at microtubules and interferes with MP30 cell-to-cell movement. In contrast, intercellular transport of a functionally enhanced MP30 mutant, which does not accumulate and colocalize with MP30 at microtubules, is not impaired by MPB2C. Together, these data support the concept that MPB2C is not required for MP30 cell-to-cell movement but may act as a negative effector of MP30 cell-to-cell transport activity.     

Epstein-Barr virus BZLF1 protein binds to mitotic chromosomes

Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and is associated with several types of cancers, including nasopharyngeal carcinoma and Burkitt's lymphoma. An EBV protein that plays an integral role during lytic replication is the immediate-early protein BZLF1. Our laboratory has found that BZLF1 (Z) localizes to host chromosomes during mitosis. Two Z-interacting proteins are also found localized to mitotic chromosomes in the presence of Z. The association between Z and mitotic chromosomes may lead to the sequestering of Z-interacting proteins within the cell and potentially cause an alteration of chromosome compaction or chromatin structure.     

Purification of brain microtubule-associated protein MAP1A

Brain microtubule-associated protein MAP1A has been purified until homogeneity by using a novel procedure involving copolymerization with microtubules, treatment with poly-l-aspartic acid and FPLC. The purified protein retains its capacity to facilitate microtubule assembly.     

Molecular genetic approaches to microtubule-associated protein function

Protein function in vivo can be studied by deleting (knock-out) the gene that encodes it, and search for the consequences. This procedure involves different technologies, including recombinant DNA procedures, cell biology methods and histological and immunocytochemical analysis. In this work we have reviewed these procedures when they have been applied to ascertain the function of several microtubule-associated proteins. These proteins have been previously involved, through in vitro experiments, in having a role in the microtubule stabilization. Here, we will summarize the generation and characterization of different microtubule-associated protein knock-out mice. Special attention will be paid to MAP1B knock-out mice. Amongst the different MAPs knock-out mice these show the strongest phenotype, the most likely for being MAP1B, the MAP that is expressed earliest in neurogenesis. Molecular genetics could be considered as a valid and useful procedure to truly establish the in vivo functions of a protein, although it is necessary to be aware of possible artifacts such as the generation of some kinds of RNA alternative splicing. To avoid this the best strategy to be used must consider the deletion of the exon that contains the functional domains of the protein.     

HIV-1 Nef protein binds to the cellular protein PACS-1 to downregulate class I major histocompatibility complexes

Major-histocompatibility-complex (MHC) proteins are used to display, on the surface of a cell, peptides derived from foreign material - such as a virus - that is infecting that cell. Cytotoxic T lymphocytes then recognize and kill the infected cell. The HIV-1 Nef protein downregulates the cell-surface expression of class I MHC proteins, and probably thereby promotes immune evasion by HIV-1. In the presence of Nef, class I MHC molecules are relocalized from the cell surface to the trans-Golgi network (TGN) through as-yet-unknown mechanisms. Here we show that Nef-induced downregulation of MHC-I expression and MHC-I targeting to the TGN require the binding of Nef to PACS-1, a molecule that controls the TGN localization of the cellular protein furin. This interaction is dependent on Nef's cluster of acidic amino acids. A chimaeric integral membrane protein containing Nef as its cytoplasmic domain localizes to the TGN after internalization, in an acidic-cluster- and PACS-1-dependent manner. These results support a model in which Nef relocalizes MHC-I by acting as a connector between MHC-I's cytoplasmic tail and the PACS-1-dependent protein-sorting pathway.     

Interactions of the HSV-1 UL25 capsid protein with cellular microtubule-associated protein

An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus.     

Characterization of microtubule-associated protein 1-associated protein kinases from rat brain

The microtubule-associated protein (MAP) 1 preparation, MAP1A and 1B, obtained from rat brain microtubules was associated with protein kinases that were insensitive to cAMP, cGMP, calcium, calcium/calmodulin and calcium/phosphatidylserine. The fractionation of highly purified MAP1 by phosphocellulose chromatography revealed that protein kinase activity to phosphorylate phosvitin was separated into three major peaks (MAP1 kinases A, B and C). MAP1 was recovered in the MAP1 kinase A fraction and phosphorylated by the contained kinase. MAP1 kinase A is a novel protein kinase that is remarkably activated by poly- -lysine and poly- -arginine, but very insensitive to heparin among the kinases. Photoaffinity labeling using [-³²P]8-azido ATP indicated that the e65 kDa polypeptide is identified as an ATP-binding protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the highly purified MAP1 and MAP1 kinase A fractions. MAP1 kinases B and C may be identified as casein kinase I- and II-like kinases. The present results show that MAP1 is associated with at least three kinases and provide an insight for understanding thoroughly the MAP1-mediated microtubule functions.     

Heterochromatin protein 1 binds transgene arrays

Heterochromatin protein 1 (HP1) of Drosophila and its homologs in vertebrates are key components of constitutive heterochromatin. Here we provide cytological evidence for the presence of heterochromatin within a euchromatic chromosome arm by immunolocalization of HP1 to the site of a silenced transgene repeat array. The amount of HP1 associated with arrays in polytene chromosomes is correlated with the array size. Inverted transposons within an array or increased proximity of an array to blocks of naturally occurring heterochromatin may increase transgene silencing without increasing HP1 labeling. Less dense anti-HP1 labeling is found at transposon arrays in which there is no transgene silencing. The results indicate that HP1 targets the chromatin of transposon insertions and binds more densely at a site with repeated sequences susceptible to heterochromatin formation. Received: 26 June 1998; in revised form: 6 July 1998 / Accepted: 12 July 1998     

Heterochromatin protein 1 binds to nucleosomes and DNA in vitro

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein primarily associated with the pericentric heterochromatin and telomeres in Drosophila. The molecular mechanism by which HP1 specifically recognizes and binds to chromatin is unknown. The purpose of this study was to test whether HP1 can bind directly to nucleosomes. HP1 binds nucleosome core particles and naked DNA. HP1-DNA complex formation is length-dependent and cooperative but relatively sequence-independent. We show that histone H4 amino-terminal peptides bind to monomeric and dimeric HP1 in vitro. Acetylation of lysine residues had no significant effect on in vitro binding. The C-terminal chromo shadow domain of HP1 specifically binds H4 N-terminal peptide. Neither the chromo domain nor chromo shadow domain alone binds DNA; intact native HP1 is required for such interactions. Together, these observations suggest that HP1 may serve as a cross-linker in chromatin, linking nucleosomal DNA and nonhistone protein complexes to form higher order chromatin structures.     

Prostaglandin E1 binds to Z protein of rat liver

Z protein or fatty-acid-binding protein is abundant in the cytosol of many cell types including liver cells. It is considered to play an important role in intracellular transport and metabolism of long-chain fatty acids and other organic anions. We studied the role of Z protein in the metabolism of prostaglandin E1 (PGE1). Binding of tritiated prostaglandin E1 to this fatty-acid-binding protein (Z protein) purified from rat liver was determined. The binding of [3H]prostaglandin E1 to Z protein is rapid, saturable and reversible. Scatchard analysis of [3H]PGE1 binding to Z protein showed a single class of binding sites with a dissociation constant (Kd) of 37 nM. The binding capacity is 110 nmol/mg Z protein. Optimal [3H]PGE1 binding occurred at pH 7.4. The presence of 3 mM MgCl2 stimulated the prostaglandin E1 binding to Z protein. Competition experiments show that the binding of this autacoid to Z protein is highly specific. It could not be displaced by other prostaglandins (PGA1, PGA2, PGE2, PGB1, PGI2, PGD2, PGF2 alpha, and 6-keto PGF1 alpha). Z protein might be involved in the metabolism of prostaglandins in the cytosol.