α‐Synuclein stimulation of monoamine oxidase‐B and legumain protease mediates the pathology of Parkinson's disease

Dopaminergic neurodegeneration in Parkinson's disease (PD) is associated with abnormal dopamine metabolism by MAO‐B (monoamine oxidase‐B) and intracellular α‐Synuclein (α‐Syn) aggregates, called the Lewy body. However, the molecular relationship between α‐Syn and MAO‐B remains unclear. Here, we show that α‐Syn directly binds to MAO‐B and stimulates its enzymatic activity, which triggers AEP (asparagine endopeptidase; legumain) activation and subsequent α‐Syn cleavage at N103, leading to dopaminergic neurodegeneration. Interestingly, the dopamine metabolite, DOPAL, strongly activates AEP, and the N103 fragment of α‐Syn binds and activates MAO‐B. Accordingly, overexpression of AEP in SNCA transgenic mice elicits α‐Syn N103 cleavage and accelerates PD pathogenesis, and inhibition of MAO‐B by Rasagiline diminishes α‐Syn‐mediated PD pathology and motor dysfunction. Moreover, virally mediated expression of α‐Syn N103 induces PD pathogenesis in wild‐type, but not MAO‐B‐null mice. Our findings thus support that AEP‐mediated cleavage of α‐Syn at N103 is required for the association and activation of MAO‐B, mediating PD pathogenesis.     

α‐Synuclein sequesters arachidonic acid to modulate SNARE‐mediated exocytosis

α‐Synuclein is a synaptic modulatory protein implicated in the pathogenesis of Parkinson disease. The precise functions of this small cytosolic protein are still under investigation. α‐Synuclein has been proposed to regulate soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins involved in vesicle fusion. Interestingly, α‐synuclein fails to interact with SNARE proteins in conventional protein‐binding assays, thus suggesting an indirect mode of action. As the structural and functional properties of both α‐synuclein and the SNARE proteins can be modified by arachidonic acid, a common lipid regulator, we analysed this possible tripartite link in detail. Here, we show that the ability of arachidonic acid to stimulate SNARE complex formation and exocytosis can be controlled by α‐synuclein, both in vitro and in vivo. α‐Synuclein sequesters arachidonic acid and thereby blocks the activation of SNAREs. Our data provide mechanistic insights into the action of α‐synuclein in the modulation of neurotransmission.     

Guanidine hydrochloride denaturation of dopamine‐induced α‐synuclein oligomers: A small‐angle X‐ray scattering study

Alpha‐synuclein (α‐syn) forms the amyloid‐containing Lewy bodies found in the brain in Parkinson's disease. The neurotransmitter dopamine (DA) reacts with α‐syn to form SDS‐resistant soluble, non‐amyloid, and melanin‐containing oligomers. Their toxicity is debated, as is the nature of their structure and their relation to amyloid‐forming conformers of α‐syn. The small‐angle X‐ray scattering technique in combination with modeling by the ensemble optimization method showed that the un‐reacted native protein populated three broad classes of conformer, while reaction with DA gave a restricted ensemble range suggesting that the rigid melanin molecule played an important part in their structure. We found that 6 M guanidine hydrochloride did not dissociate α‐syn DA‐reacted dimers and trimers, suggesting covalent linkages. The pathological significance of covalent association is that if they are non‐toxic, the oligomers would act as a sink for toxic excess DA and α‐syn; if toxic, their stability could enhance their toxicity. We argue it is essential, therefore, to resolve the question of whether they are toxic or not. Proteins 2014; 82:10–21. © 2013 Wiley Periodicals, Inc.     

Expression of γ‐tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal‐induced oxidative stresses by reducing reactive oxygen species

The γ‐tocopherol methyltransferase (γ‐TMT) is an important enzyme regulating synthesis of four tocopherols (α, γ, β and δ). In this report, we investigated the role of γ‐TMT in regulating abiotic stress within chloroplasts. The At γ‐tmt overexpressed via the tobacco chloroplast genome accumulated up to 7.7% of the total leaf protein, resulting in massive proliferation of the inner envelope membrane (IEM, up to eight layers). Such high‐level expression of γ‐TMT converted most of γ‐tocopherol to α‐tocopherol in transplastomic seeds (~10‐fold higher) in the absence of abiotic stress. When grown in 400 mm NaCl, α‐tocopherol content in transplastomic TMT leaves increased up to 8.2‐fold and 2.4‐fold higher than wild‐type leaves. Likewise, under heavy metal stress, α‐tocopherol content in the TMT leaves increased up to 7.5‐fold, twice higher than in the wild type. Under extreme salt stress, the wild type accumulated higher starch and total soluble sugars, but TMT plants were able to regulate sugar transport. Hydrogen peroxide and superoxide content in wild type increased up to 3‐fold within 48 h of NaCl stress when compared to TMT plants. The ion leakage from TMT leaves was significantly less than wild‐type plants under abiotic stress and with less malondialdehyde, indicating lower lipid peroxidation. Taken together, these studies show that α‐tocopherol plays a crucial role in the alleviation of salt and heavy metal stresses by decreasing ROS, lipid peroxidation and ion leakage, in addition to enhancing vitamin E conversion. Increased proliferation of the IEM should facilitate studies on retrograde signalling from chloroplast to the nucleus.     

MUC1 is a substrate for γ‐secretase

Understanding the underlying mechanisms by which a normal cell avoids the oncogenic potential of MUC1 signaling requires further definition of the pathways by which the MUC1 cytoplasmic tail is processed in both normal and tumor‐derived cells. In the present study we describe the processing pathway initiated by TACE/ADAM17 cleavage of MUC1. Utilizing the human uterine epithelial cell line, HES, derived from normal endometrium, we show that endogenous full length MUC1 undergoes regulated intramembranous proteolysis mediated by presenillin‐dependent γ‐secretase. Cytokine‐stimulated HES cells exposed to γ‐secretase inhibitors accumulated a membrane‐associated 15 kDa fragment of the MUC1 C‐terminal subunit (CTF15). Inhibitors of TACE/ADAM17‐mediated shedding inhibited accumulation of MUC1‐CTF15 and MUC1 ectodomain release to a similar extent consistent with MUC1‐CTF15 being a product of TACE/ADAM17 action. Reduction of catalytically active γ‐secretase complex by nicastrin siRNA treatment also resulted in CTF15 accumulation. Furthermore, mature nicastrin, the substrate receptor for γ‐secretase, co‐immunoprecipitated with CTF15 in the presence of γ‐secretase inhibitors indicating the formation of CTF15: nicastrin complexes. MUC1‐CTF15 accumulation in response to γ‐secretase inhibition was demonstrated in both normal and tumor‐derived cells from humans and mice indicating that this processing pathway exists in many cell contexts. We did not detect products of MUC1 cleavage by γ‐secretase in the presence of various proteasomal inhibitors indicating that subsequent degradation is either non‐proteasomal or extremely efficient. We suggest that this efficient pathway attenuates potential signaling mediated by cytoplasmic tail fragments. J. Cell. Biochem. 108: 802–815, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of acidic pH on the stability of α‐synuclein dimers

Environmental factors, such as acidic pH, facilitate the assembly of α‐synuclein (α‐Syn) in aggregates, but the impact of pH on the very first step of α‐Syn aggregation remains elusive. Recently, we developed a single‐molecule approach that enabled us to measure directly the stability of α‐Syn dimers. Unlabeled α‐Syn monomers were immobilized on a substrate, and fluorophore‐labeled monomers were added to the solution to allow them to form dimers with immobilized α‐Syn monomers. The dimer lifetimes were measured directly from the fluorescence bursts on the time trajectories. Herein, we applied the single‐molecule tethered approach for probing of intermolecular interaction to characterize the effect of acidic pH on the lifetimes of α‐Syn dimers. The experiments were performed at pH 5 and 7 for wild‐type α?Syn and for two mutants containing familial type mutations E46K and A53T. We demonstrate that a decrease of pH resulted in more than threefold increase in the α‐Syn dimers lifetimes with some variability between the α‐Syn species. We hypothesize that the stabilization effect is explained by neutralization of residues 96–140 of α‐Syn and this electrostatic effect facilitates the association of the two monomers. Given that dimerization is the first step of α‐Syn aggregation, we posit that the electrostatic effect thereby contributes to accelerating α‐Syn aggregation at acidic pH. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 715–724, 2016.     

Lipid peroxidation is essential for α‐synuclein‐induced cell death

Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α‐synuclein (α‐Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α‐Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co‐cultures of neurons and astrocytes. We found that oligomeric but not monomeric α‐Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre‐incubation of cells with isotope‐reinforced polyunsaturated fatty acids (D‐PUFAs) completely prevented the effect of oligomeric α‐Syn on lipid peroxidation. Inhibition of lipid peroxidation with D‐PUFAs further protected cells from cell death induced by oligomeric α‐Syn. Thus, lipid peroxidation induced by misfolding of α‐Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease.

     

Manipulation of two α‐endo‐β‐1,4‐glucanase genes,AtCel6 and GmCel7, reduces susceptibility to Heterodera glycines in soybean roots

Plant endo‐β‐1,4‐glucanases (EGases) include cell wall‐modifying enzymes that are involved in nematode‐induced growth of syncytia (feeding structures) in nematode‐infected roots. EGases in the α‐ and β‐subfamilies contain signal peptides and are secreted, whereas those in the γ‐subfamily have a membrane‐anchoring domain and are not secreted. The Arabidopsis α‐EGase At1g48930, designated as AtCel6, is known to be down‐regulated by beet cyst nematode (Heterodera schachtii) in Arabidopsis roots, whereas another α‐EGase, AtCel2, is up‐regulated. Here, we report that the ectopic expression of AtCel6 in soybean roots reduces susceptibility to both soybean cyst nematode (SCN; Heterodera glycines) and root knot nematode (Meloidogyne incognita). Suppression of GmCel7, the soybean homologue of AtCel2, in soybean roots also reduces the susceptibility to SCN. In contrast, in studies on two γ‐EGases, both ectopic expression of AtKOR2 in soybean roots and suppression of the soybean homologue of AtKOR3 had no significant effect on SCN parasitism. Our results suggest that secreted α‐EGases are likely to be more useful than membrane‐bound γ‐EGases in the development of an SCN‐resistant soybean through gene manipulation. Furthermore, this study provides evidence that Arabidopsis shares molecular events of cyst nematode parasitism with soybean, and confirms the suitability of the Arabidopsis–H. schachtii interaction as a model for the soybean–H. glycines pathosystem.     

The stress response neuropeptide CRF increases amyloid‐β production by regulating γ‐secretase activity

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid‐β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ‐secretase internalization. Co‐immunoprecipitation studies establish that γ‐secretase associates with CRFR1; this is mediated by β‐arrestin binding motifs. Additionally, CRFR1 and γ‐secretase co‐localize in lipid raft fractions, with increased γ‐secretase accumulation upon CRF treatment. CRF treatment also increases γ‐secretase activity in vitro, revealing a second, receptor‐independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ‐secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ‐secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ‐secretase.     

Chaperone proteostasis in Parkinson's disease: stabilization of the Hsp70/α‐synuclein complex by Hip

The ATP‐dependent protein chaperone heat‐shock protein 70 (Hsp70) displays broad anti‐aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of α‐synuclein (αSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation‐prone polypeptides. We show a nucleotide‐dependent interaction between Hsp70 and αSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with αSyn. Such a co‐aggregation phenomenon can be prevented in vitro by the co‐chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of αSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.     

Rational design of amyloid β peptide–binding proteins: Pseudo‐Aβ β‐sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Aβ

Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid β‐peptide (Aβ) is thought to be a toxic agent by self‐assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo‐Aβ β‐sheet surfaces and evaluated their abilities to bind to Aβ and inhibit Aβ oligomerization. Two GFP variants P13H and AP93Q bound tightly to Aβ, K_d = 260 nM and K_d = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Aβ oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Aβ‐derived sequences was designed and bound more tightly to Aβ (K_d = 100 nM) than those having only two pseudo‐Aβ strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Aβ more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo‐Aβ β‐sheet structures onto a protein surface arranged in the β‐barrel structure is useful to construct molecules capable of binding tightly to Aβ and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Microbial production of conjugated γ‐linolenic acid from γ‐linolenic acid by Lactobacillus plantarum AKU 1009a

Aims: Optimal production conditions of conjugated γ‐linolenic acid (CGLA) from γ‐linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated. Methods and Results: Washed cells of Lact. plantarum AKU 1009a exhibiting a high level of CGLA productivity were obtained by cultivation in a nutrient medium supplemented with 0·03% (w/v) α‐linolenic acid as an inducer. Under the optimal reaction conditions with 13 mg ml^?1γ‐linolenic acid as a substrate in 5 ‐ml reaction volume, the washed cells [32% (wet cells, w/v) corresponding to 46 mg ml^?1 dry cells] as the catalysts produced 8·8 mg CGLA per millilitre reaction mixture (68% molar yield) in 27 h. The produced CGLA was a mixture of two isomers, i.e., cis‐6,cis‐9,trans‐11‐octadecatrienoic acid (CGLA1, 40% of total CGLA) and cis‐6,trans‐9,trans‐11‐octadecatrienoic acid (CGLA2, 60% of total CGLA), and accounted for 66% of total fatty acid obtained. The CGLA produced was obtained as free fatty acids adsorbed mostly on the surface of the cells of Lact. plantarum AKU1009a. Conclusion: The practical process of CGLA production from γ‐linolenic acid using washed cells of Lact. plantarum AKU 1009a was successfully established. Significance and Impact of the Study: We presented the first example of microbial production of CGLA. CGLA produced by the process is valuable for evaluating their physiological and nutritional effects, and chemical characteristics.     

Phenolic compounds prevent the oligomerization of α‐synuclein and reduce synaptic toxicity

Lewy bodies, mainly composed of α‐synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low‐order αS oligomers are neurotoxic and critical species in the pathogenesis of α‐synucleinopathies. To develop disease modifying therapies for α‐synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo‐induced cross‐linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N‐terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long‐term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers.

     

Isolation and characterization of a Pseudomonas sp. strain IITR01 capable of degrading α‐endosulfan and endosulfan sulfate

Aim: To isolate bacteria capable of degrading endosulfan (ES) and the more toxic ES sulfate and to characterize their metabolites. Methods and Results: A Pseudomonas sp. strain IITR01 capable of degrading α‐ES and toxic ES sulfate was isolated using technical‐ES through enrichment culture techniques. No growth and no degradation were observed using β‐ES. Thin‐layer chromatography and gas chromatography‐mass spectrum analysis revealed the disappearance of both α‐ES and ES sulfate and the formation of hydroxylated products ES diol, ether and lactone. We show here for the first time the formation of aforementioned metabolites in contrast to ES hemisulfate yielded by an Arthrobacter sp. Metabolism of α‐ES and endosulfate was also observed using the crude cell extract of IITR01. The molecular mass of protein induced during the degradation of α‐ES and sulfate as substrate was found to be approximately 150 kDa as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Conclusion: We describe characterization of bacterium capable of degrading α‐ES and ES sulfate but not β‐ES. Genetic investigation suggests that a gene nonhomologous to the reported esd may be present in the strain IITR01. Significance and Impact of the Study: This study describes toxic ES degradation by a Pseudomonas species that may be utilized for the bioremediation of the industrial soils contaminated with ES residues.     

Characterizing the inhibition of α‐synuclein oligomerization by a pharmacological chaperone that prevents prion formation by the protein PrP

Aggregation of the disordered protein α‐synuclein into amyloid fibrils is a central feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease. Small, pre‐fibrillar oligomers of misfolded α‐synuclein are thought to be the key toxic entities, and α‐synuclein misfolding can propagate in a prion‐like way. We explored whether a compound with anti‐prion activity that can bind to unfolded parts of the protein PrP, the cyclic tetrapyrrole Fe‐TMPyP, was also active against α‐synuclein aggregation. Observing the initial stages of aggregation via fluorescence cross‐correlation spectroscopy, we found that Fe‐TMPyP inhibited small oligomer formation in a dose‐dependent manner. Fe‐TMPyP also inhibited the formation of mature amyloid fibrils in vitro, as detected by thioflavin T fluorescence. Isothermal titration calorimetry indicated Fe‐TMPyP bound to monomeric α‐synuclein with a stoichiometry of 2, and two‐dimensional heteronuclear single quantum coherence NMR spectra revealed significant interactions between Fe‐TMPyP and the C‐terminus of the protein. These results suggest commonalities among aggregation mechanisms for α‐synuclein and the prion protein may exist that can be exploited as therapeutic targets.     

γ‐Secretase Inhibition Induces Muscle Hypertrophy in a Notch‐Independent Mechanism

A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ‐secretase. Notch‐1 is one of the substrates of γ‐secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch‐1, a number of proteins have been identified to undergo proteolysis by γ‐secretase. To date, the specific role of γ‐secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ‐secretase inhibitor DAPT (N‐N[‐(3,5‐Difluorophenacetyl‐l ‐alanyl)]‐S‐328 phenylglycine‐t‐butyl‐ester) induces muscle hypertrophy. Knockdown of Notch‐1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ‐secretase‐dependent effects on muscle hypertrophy in chick myogenic cells are Notch‐1‐independent. We also investigate the effects of γ‐secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label‐free proteomic approach. Data overview of interaction network obtained from STRING show that after γ‐secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ‐secretase in skeletal muscle hypertrophy.     

DJ‐1 deficiency impairs autophagy and reduces alpha‐synuclein phagocytosis by microglia

Parkinson's disease (PD) is a progressive neurodegenerative disorder, of which 1% of the hereditary cases are linked to mutations in DJ‐1, an oxidative stress sensor. The pathological hallmark of PD is intercellular inclusions termed Lewy Bodies, composed mainly of α‐Synuclein (α‐Syn) protein. Recent findings have shown that α‐Syn can be transmitted from cell to cell, suggesting an important role of microglia, as the main scavenger cells of the brain, in clearing α‐Syn. We previously reported that the knock down (KD) of DJ‐1 in microglia increased cells’ neurotoxicity to dopaminergic neurons. Here, we discovered that α‐Syn significantly induced elevated secretion of the proinflammatory cytokines IL‐6 and IL‐1β and a significant dose‐dependent elevation in the production of nitric oxide in DJ‐1 KD microglia, compared to control microglia. We further investigated the ability of DJ‐1 KD microglia to uptake and degrade soluble α‐Syn, and discovered that DJ‐1 KD reduces cell‐surface lipid raft expression in microglia and impairs their ability to uptake soluble α‐Syn. Autophagy is an important mechanism for degradation of intracellular proteins and organelles. We discovered that DJ‐1 KD microglia exhibit an impaired autophagy‐dependent degradation of p62 and LC3 proteins, and that manipulation of autophagy had less effect on α‐Syn uptake and clearance in DJ‐1 KD microglia, compared to control microglia. Further studies of the link between DJ‐1, α‐Syn uptake and autophagy may provide useful insights into the role of microglia in the etiology of the PD.

     

NMR secondary structure and interactions of recombinant human MOZART1 protein,a component of the gamma‐tubulin complex

Mitotic‐spindle organizing protein associated with a ring of γ‐tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ‐tubulin ring complexes (γTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC‐MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha‐helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N‐terminus (residues 1–250) of GCP3 (γ‐tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.