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

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.

Role of Post‐translational Modifications in Alzheimer's Disease

“…These ubiquitylated oligomers are associated with proteasome and synaptic dysfunction in AD pathology . Axotrophin/MARCH7 is a protein similar to that of E3 ligase, which interacts with tau, ubiquitylates and impairs its binding to microtubules, and accelerates aggregation (Figure D) …”

Signaling pathways and posttranslational modifications of tau in Alzheimer's disease: the humanization of yeast cells

“…Since ubiquitinated tau is mainly found in the insoluble NFTs which accumulate late in AD rather than in the soluble oligomers, ubiquitination may be a way of detoxification. A specific E3 ubiquitin ligase, Axotropin/MARCH7, has been identified in a yeast two-hybrid screen and shown to impair binding of the modified tau to microtubules [54]. Whilst only four lysine residues of tau were found to be ubiquitinated in paired helical filaments of human tau [11], a recent work detected 15 residues in wild-type mouse tau [21].…”

“…Thus, although aggregates of hyperphosphorylated tau do form [78] could be used for studies of effects on tau phosphorylation and aggregation upon co-expression PP2A function in double mutant pph21Δ pph22Δ upon co-expression with tau PIN1 function in ess1Δ upon co-expression with tau [61,79] Use of deletions to study specific modifications of tau use of rim11Δ/mds1Δ to reduce tau phosphorylation and study its aggregation [76] sod2Δ to study oxidative stress [77] and its effect on tau aggregation temperature-sensitive mutants in proteasomal proteases to investigate their effect on tau processing Heterologous gene expression tau for purposes of protein production and biochemical/immunological approaches [77,81] expression of tau and annexin A2 for Co-IPs [66] expression of all three heterologous genes encoding human AMPK subunits [81] to study their effect on tau phosphorylation and aggregation expression of genes for modifying enzymes (e.g. acetyltransferase p300 [55] to study the role of these modifications for tau aggregation Chimeric gene expression expression of tau and human tubulin or a hybrid yeast/human tubulin gene to investigate the interaction with microtubules in yeast Yeast two-hybrid assays (YTH) identification of E3 ubiquitin ligase modifying tau [54] and other new interactors Protein aggregation assays application of colony-colour assay for prion functions to tau aggregation [80] use of fluorescence labels either in conjunction with FACS as demonstrated for Aß [83] or with FRET to study tau aggregation and its interaction with other proteins Synthetic lethal screens use of a platform yeast strain co-expressing the genes for tau and the Aß peptide to screen for suppressors from cDNA libraries or for high-throughput drug screens use of tau overexpression strain to screen for cDNA clones enhancing/provoking toxicity [4] Chronological aging test the effects of tau variants on long-term survival of yeast transformants in synthetic medium [9] to obtain a phenotype useful for genetic screens of the tau interactome [9] Synthetic biology establish entire human modification or signaling pathways in yeast platform strains (lacking the yeast orthologous genes if required and complemented by the human counterparts) to establish a "humanized yeast physiology" * aspects also applicable to the alternative non-conventional yeast K. lactis (but not yet examined for tau-related functions) are underlined. Approaches without references are either unpublished results from our laboratory or suggestions for future investigations (see text for details).…”

“…the E3 ubiquitin ligase Axotropin/MARCH7 [54]. As a complementary strategy, we have expressed tagged versions of wildtype and a mutant tau (R406W) together with annexin A2 in yeast to confirm their interaction by coimmunoprecipitations, thus evading interferences which might be caused by larger neuron-specific protein complexes [66].…”

N-terminal ubiquitination of amyloidogenic proteins triggers removal of their oligomers by the proteasome holoenzyme

“…In addition, α S in the PDassociated Lewy Bodies is also mainly monoubiquitinated [17,24]. Both tau and α S have dedicated Ub ligases, AXOT/MARCH7 [25] and SIAH1 [26,27], respectively, which preferentially monoubiquitinate their substrates. UBE2W, a Ub-conjugating enzyme that directly monoubiquitinates the N-terminus of intrinsically disordered proteins [28], has also been shown to modify tau [22,23].…”