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      Axotrophin/MARCH7 acts as an E3 ubiquitin ligase and ubiquitinates tau protein in vitro impairing microtubule binding

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          Abstract

          Tau is the major microtubule-associated protein in neurons involved in microtubule stabilization in the axonal compartment. Changes in tau gene expression, alternative splicing and posttranslational modification regulate tau function and in tauopathies can result in tau mislocalization and dysfunction, causing tau aggregation and cell death. To uncover proteins involved in the development of tauopathies, a yeast two-hybrid system was used to screen for tau-interacting proteins. We show that axotrophin/MARCH7, a RING-variant domain containing protein with similarity to E3 ubiquitin ligases interacts with tau. We defined the tau binding domain to amino acids 552–682 of axotrophin comprising the RING-variant domain. Co-immunoprecipitation and co-localization confirmed the specificity of the interaction. Intracellular localization of axotrophin is determined by an N-terminal nuclear targeting signal and a C-terminal nuclear export signal. In AD brain nuclear localization is lost and axotrophin is rather associated with neurofibrillary tangles. We find here that tau becomes mono-ubiquitinated by recombinant tau-interacting RING-variant domain, which diminishes its microtubule-binding. In vitro ubiquitination of four-repeat tau results in incorporation of up to four ubiquitin molecules compared to two molecules in three-repeat tau. In summary, we present a novel tau modification occurring preferentially on 4-repeat tau protein which modifies microtubule-binding and may impact on the pathogenesis of tauopathies.

          Highlights

          • We search for tau-interacting proteins using a cytotrap yeast two-hybrid assay.

          • MARCH7 was identified as a tau-binding protein and confirmed by several methods.

          • Recombinant MARCH7 Ring-variant domain uses Ubc5 for E3 self-ubiquitinating activity.

          • MARCH7 Ring-variant domain mono-ubiquitinates tau protein at multiple sites including the microtubule-binding domain.

          • Mono-ubiquitination of tau protein diminishes its microtubule-binding.

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          Most cited references46

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          Tau protein isoforms, phosphorylation and role in neurodegenerative disorders.

          Tau proteins belong to the family of microtubule-associated proteins. They are mainly expressed in neurons where they play an important role in the assembly of tubulin monomers into microtubules to constitute the neuronal microtubules network. Microtubules are involved in maintaining the cell shape and serve as tracks for axonal transport. Tau proteins also establish some links between microtubules and other cytoskeletal elements or proteins. Tau proteins are translated from a single gene located on chromosome 17. Their expression is developmentally regulated by an alternative splicing mechanism and six different isoforms exist in the human adult brain. Tau proteins are the major constituents of intraneuronal and glial fibrillar lesions described in Alzheimer's disease and numerous neurodegenerative disorders referred to as 'tauopathies'. Molecular analysis has revealed that an abnormal phosphorylation might be one of the important events in the process leading to their aggregation. Moreover, a specific set of pathological tau proteins exhibiting a typical biochemical pattern, and a different regional and laminar distribution could characterize each of these disorders. Finally, a direct correlation has been established between the progressive involvement of the neocortical areas and the increasing severity of dementia, suggesting that pathological tau proteins are reliable marker of the neurodegenerative process. The recent discovery of tau gene mutations in frontotemporal dementia with parkinsonism linked to chromosome 17 has reinforced the predominant role attributed to tau proteins in the pathogenesis of neurodegenerative disorders, and underlined the fact that distinct sets of tau isoforms expressed in different neuronal populations could lead to different pathologies.
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            "Stemness": transcriptional profiling of embryonic and adult stem cells.

            The transcriptional profiles of mouse embryonic, neural, and hematopoietic stem cells were compared to define a genetic program for stem cells. A total of 216 genes are enriched in all three types of stem cells, and several of these genes are clustered in the genome. When compared to differentiated cell types, stem cells express a significantly higher number of genes (represented by expressed sequence tags) whose functions are unknown. Embryonic and neural stem cells have many similarities at the transcriptional level. These results provide a foundation for a more detailed understanding of stem cell biology.
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              Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms.

              Neurofibrillary tangles are composed of insoluble aggregates of the microtubule-associated protein tau. In Alzheimer's disease the accumulation of neurofibrillary tangles occurs in the absence of tau mutations. Here we present mice that develop pathology from non-mutant human tau, in the absence of other exogenous factors, including beta-amyloid. The pathology in these mice is Alzheimer-like, with hyperphosphorylated tau accumulating as aggregated paired helical filaments. This pathologic tau accumulates in the cell bodies and dendrites of neurons in a spatiotemporally relevant distribution.
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                Author and article information

                Contributors
                Journal
                Biochim Biophys Acta
                Biochim. Biophys. Acta
                Biochimica et Biophysica Acta
                Elsevier Pub. Co
                0006-3002
                1 September 2014
                September 2014
                : 1842
                : 9
                : 1527-1538
                Affiliations
                [a ]Paul Flechsig Institute of Brain Research, Department of Molecular and Cellular Mechanisms of Neurodegeneration, University of Leipzig, 04109 Leipzig, Germany
                [b ]MRC, Laboratory of Molecular Biology, Neurobiology Division, Francis Crick Avenue, Cambridge CB2 0QH, UK
                Author notes
                [* ]Corresponding author at: Paul Flechsig Institute of Brain Research, Jahnallee 59, 04109 Leipzig, Germany. Tel.: + 49 3419725759; fax: + 49 3419725729. holm@ 123456medizin.uni-leipzig.de
                Article
                S0925-4439(14)00157-4
                10.1016/j.bbadis.2014.05.029
                4311138
                24905733
                3c633869-fc35-4a20-a2e5-d618d719f2cb
                © 2014 Elsevier B.V. All rights reserved.
                Categories
                Article

                Biochemistry
                alzheimer's disease,tau protein,mono-ubiquitination,ring-variant domain,yeast two-hybrid assay,microtubule binding

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