Molecular basis for benzimidazole resistance from a novel β-tubulin binding site model

ABSTRACTTubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using Chlamydomonas, we attenuated IFT-based tubulin transport of GFP–β-tubulin by altering the IFT74N–IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP–β-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by ∼90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP–β-tubulin in the axoneme was only moderately reduced. In vivo imaging showed more GFP–β-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth.

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Diffusion rather than IFT provides most of the tubulin required for axonemal assembly

10.1101/268573 ◽

2018 ◽

Cited By ~ 1

Author(s):

J. Aaron Harris ◽

Julie C. Van De Weghe ◽

Tomohiro Kubo ◽

George B. Witman ◽

Karl F. Lechtreck

Keyword(s):

Binding Site ◽

Binding Sites ◽

Intraflagellar Transport ◽

Strong Reduction ◽

Tubulin Binding ◽

High Concentrations ◽

Β Tubulin

AbstractTubulin enters the cilia by diffusion and motor-based intraflagellar transport (IFT). The respective contributions of each route in providing tubulin for axonemal assembly are unknown. To attenuate IFT-based transport, we expressed modified GFP-tubulins in strains possessing IFT81 and IFT74 with altered tubulin binding sites. E-hook deficient GFP-β-tubulin normally incorporated into the axonemal microtubules; its transport frequency was reduced by ~90% in control cells and essentially abolished when expressed in a strain possessing IFT81 with an incapacitated tubulin-binding site. Despite the strong reduction in IFT, the share of E-hook deficient GFP-β-tubulin in the axoneme was only moderately reduced indicating that most axonemal tubulin (~80%) enters cilia by diffusion. While not providing the bulk of axonemal tubulin, we propose that motor-based IFT is nevertheless critical for ciliogenesis because it ensures high concentrations of tubulin near the ciliary tip promoting axonemal elongation.

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3D QSAR studies for the β-tubulin binding site of microtubule-stabilizing anticancer agents (MSAAs)

Il Farmaco ◽

10.1016/s0014-827x(03)00099-5 ◽

2003 ◽

Vol 58 (9) ◽

pp. 659-668 ◽

Cited By ~ 10

Author(s):

Laura Maccari ◽

Fabrizio Manetti ◽

Federico Corelli ◽

Maurizio Botta

Keyword(s):

Binding Site ◽

Anticancer Agents ◽

3D Qsar ◽

Qsar Studies ◽

Tubulin Binding ◽

Β Tubulin

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Crystal structure of tubulin folding cofactor A from Arabidopsis thaliana and its β-tubulin binding characterization

FEBS Letters ◽

10.1016/j.febslet.2010.07.017 ◽

2010 ◽

Vol 584 (16) ◽

pp. 3533-3539 ◽

Cited By ~ 4

Author(s):

Lu Lu ◽

Jie Nan ◽

Wei Mi ◽

Lan-Fen Li ◽

Chun-Hong Wei ◽

...

Keyword(s):

Crystal Structure ◽

Arabidopsis Thaliana ◽

Tubulin Binding ◽

Β Tubulin

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Nuclear Import of Adenovirus DNA Involves Direct Interaction of Hexon with an N-Terminal Domain of the Nucleoporin Nup214

Journal of Virology ◽

10.1128/jvi.02639-14 ◽

2014 ◽

Vol 89 (3) ◽

pp. 1719-1730 ◽

Cited By ~ 37

Author(s):

Aurélia Cassany ◽

Jessica Ragues ◽

Tinglu Guan ◽

Dominique Bégu ◽

Harald Wodrich ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

Nuclear Import ◽

Viral Genome ◽

Molecular Basis ◽

Nuclear Pore ◽

Viral Dna ◽

Terminal Domain ◽

Cytoplasmic Face ◽

The Impact

ABSTRACTIn this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the nucleus. We used RNA interference (RNAi) to deplete cells of either Nup214 or Nup358, the two major Phe-Gly (FG) repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin-permeabilized cells in the absence of cytosolic factors. Bothin vitrohexon binding andin vivonuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of protein VII (pVII), indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137-amino-acid segment in the N-terminal domain of Nup214. Together, our results have identified a specific region within the N terminus of Nup214 that acts as a direct NPC binding site for AdV.IMPORTANCEAdVs, which have the largest genome of nonenveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137-amino-acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein, hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers.

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Atomic model of microtubule-bound tau

10.1101/267153 ◽

2018 ◽

Cited By ~ 1

Author(s):

Elizabeth H. Kellogg ◽

Nisreen M.A. Hejab ◽

Simon Poepsel ◽

Kenneth H. Downing ◽

Frank DiMaio ◽

...

Keyword(s):

High Resolution ◽

Physiological Role ◽

Biochemical Data ◽

Disease Pathogenesis ◽

Developmentally Regulated ◽

Computational Approaches ◽

Selective Stabilization ◽

Tubulin Binding ◽

Future Treatments ◽

Β Tubulin

AbstractTau is a developmentally regulated protein found in axons, whose physiological role is to stabilize and bundle microtubules (MTs). Hyper-phosphorylation of tau is thought to cause its detachment from MTs and subsequent aggregation into pathological fibrils that have been implicated in Alzheimer’s disease pathogenesis. Despite its known MT binding role, there is no consensus regarding which tau residues are crucial for tau-MT interactions, where on the MT tau binds, and how binding results in MT stabilization. We have used cryo-EM to visualize the interaction of different tau constructs with MTs at high resolution (3.2-4.8 Å) and used computational approaches to generate atomic models of tau-tubulin interactions. Our work shows that the highly conserved tubulin-binding repeats within tau adopt very similar structures in their interactions with the MT. Each tau repeat binds the MT exterior and adopts an extended structure along the crest of the protofilament (PF), interacting with both α- and β-tubulin, thus stabilizing the interface between tubulin dimers. Our structures agree with and explain previous biochemical data concerning the effect of phosphorylation on MT affinity and lead to a model in which tau repeats bind in tandem along a PF, tethering together tubulin dimers and stabilizing longitudinal polymerization interfaces. These structural findings could establish a basis of future treatments aiming at the selective stabilization of tau-MT interactions.

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Perinuclear localization of huntingtin as a consequence of its binding to microtubules through an interaction with β-tubulin: relevance to Huntington's disease

Journal of Cell Science ◽

10.1242/jcs.115.5.941 ◽

2002 ◽

Vol 115 (5) ◽

pp. 941-948 ◽

Cited By ~ 4

Author(s):

Guylaine Hoffner ◽

Pascal Kahlem ◽

Philippe Djian

Keyword(s):

Electron Microscopy ◽

Huntington's Disease ◽

Huntington’S Disease ◽

Binding Property ◽

Perinuclear Region ◽

Β Subunit ◽

Tubulin Binding ◽

Β Tubulin

Huntington's disease results from an expansion of a series of glutamine repeats in the protein huntingtin. We have discovered from immunopurification studies that huntingtin combines specifically with the β subunit of tubulin. This binding explains why huntingtin can be shown on assembled microtubules by electron microscopy. Immunostaining shows that most of the huntingtin in the cytoplasm is associated with microtubules. Huntingtin is particularly abundant in the perinuclear region, where it is also associated with microtubules and in the centrosomal region, where it co-localizes withγ-tubulin. In Huntington's disease, inclusions are often nuclear or perinuclear. Since the perinuclear concentration of huntingtin does not depend on the number of its glutamine repeats, we propose that inclusions are found in perinuclear and intranuclear locations because the β-tubulin binding property of huntingtin brings it to the perinuclear region, from which it readily gains access to the nucleus. The mutational glutamine expansion then promotes insolubility and results in an inclusion.

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Crystal structure and interactions of the Tof1–Csm3 (Timeless–Tipin) fork protection complex

Nucleic Acids Research ◽

10.1093/nar/gkaa456 ◽

2020 ◽

Vol 48 (12) ◽

pp. 6996-7004 ◽

Cited By ~ 1

Author(s):

Daniel B Grabarczyk

Keyword(s):

Crystal Structure ◽

Circadian Rhythm ◽

Dna Replication ◽

Binding Site ◽

Molecular Basis ◽

Replication Forks ◽

Repeat Structure ◽

Chaetomium Thermophilum ◽

Fork Protection Complex ◽

Human Orthologue

Abstract The Tof1–Csm3 fork protection complex has a central role in the replisome—it promotes the progression of DNA replication forks and protects them when they stall, while also enabling cohesion establishment and checkpoint responses. Here, I present the crystal structure of the Tof1–Csm3 complex from Chaetomium thermophilum at 3.1 Å resolution. The structure reveals that both proteins together form an extended alpha helical repeat structure, which suggests a mechanical or scaffolding role for the complex. Expanding on this idea, I characterize a DNA interacting region and a cancer-associated Mrc1 binding site. This study provides the molecular basis for understanding the functions of the Tof1–Csm3 complex, its human orthologue the Timeless–Tipin complex and additionally the Drosophila circadian rhythm protein Timeless.

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