The Novel KIF1A Missense Variant (R169T) Strongly Reduces Microtubule Stimulated ATPase Activity and Is Associated With NESCAV Syndrome

Aguilera, Cinthia; Hümmer, Stefan; Masanas, Marc; Gabau, Elisabeth; Guitart, Míriam; Jeyaprakash, A. Arockia; Segura, Miguel F.; Santamaría, Anna; Ruiz, Anna

doi:10.3389/fnins.2021.618098

Cited by 13 publications

(10 citation statements)

References 33 publications

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“…Similar to R167C, the ATPase analysis of K161A and K163A mutants also showed a significant decrease in the ATP hydrolysis rates and affinity for the microtubules (Additional file 1: Fig. S7H, I), consistent with the previous work that conversion of all the positively charged residues in loop8 to alanine (K161A/R167A/R169A/K183A) strongly affected MT-binding affinity [15,62,71]. We did not measure the ATPase activity of the R169A mutant because the motor showed very low expression levels even after 2 days in mammalian cells and failed to express in Sf9 cells despite several attempts.…”

Section: Kinesin-3 Motor Velocities Inversely Correlate To Their Micr...supporting

confidence: 88%

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Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs

et al. 2022

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Background Kinesin-3 family motors drive diverse cellular processes and have significant clinical importance. The ATPase cycle is integral to the processive motility of kinesin motors to drive long-distance intracellular transport. Our previous work has demonstrated that kinesin-3 motors are fast and superprocessive with high microtubule affinity. However, chemomechanics of these motors remain poorly understood. Results We purified kinesin-3 motors using the Sf9-baculovirus expression system and demonstrated that their motility properties are on par with the motors expressed in mammalian cells. Using biochemical analysis, we show for the first time that kinesin-3 motors exhibited high ATP turnover rates, which is 1.3- to threefold higher compared to the well-studied kinesin-1 motor. Remarkably, these ATPase rates correlate to their stepping rate, suggesting a tight coupling between chemical and mechanical cycles. Intriguingly, kinesin-3 velocities (KIF1A > KIF13A > KIF13B > KIF16B) show an inverse correlation with their microtubule-binding affinities (KIF1A < KIF13A < KIF13B < KIF16B). We demonstrate that this differential microtubule-binding affinity is largely contributed by the positively charged residues in loop8 of the kinesin-3 motor domain. Furthermore, microtubule gliding and cellular expression studies displayed significant microtubule bending that is influenced by the positively charged insert in the motor domain, K-loop, a hallmark of kinesin-3 family. Conclusions Together, we propose that a fine balance between the rate of ATP hydrolysis and microtubule affinity endows kinesin-3 motors with distinct mechanical outputs. The K-loop, a positively charged insert in the loop12 of the kinesin-3 motor domain promotes microtubule bending, an interesting phenomenon often observed in cells, which requires further investigation to understand its cellular and physiological significance.

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Section: Kinesin-3 Motor Velocities Inversely Correlate To Their Micr...supporting

confidence: 88%

“…S7G), the mutant exhibited a significant decrease in the rate of ATP hydrolysis (~ 33%) and microtubule affinity (~ 1.5 times), compared to wild-type motors (Additional file 1 : Fig. S7H, I) [ 71 ]. The residue R167 establishes multiple stable interactions with MT in ATP and ADP states [ 72 ].…”

Section: Resultsmentioning

confidence: 99%

Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs

et al. 2022

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“…KIF1A(P305L) reduces the microtubule association rate of the motor (27). KIF1A(R169T) disrupts the microtubule-dependent ATPase activity of the motor domain (28). KIF1A(R254W) reduces the velocity and run length of the motor protein (24) .…”

Section: Introductionmentioning

confidence: 99%

“…KIF1A(V8M) causes defects in force generation, while KIF1A(R254W), KIF1A(A255V), and KIF1A(R350G) have a shorter run length ( 27 , 31 , 32 ). The KIF1A(P305L) mutation strongly reduces the microtubule association rate of the motor, while KIF1A(R169T) disrupted the microtubule-dependent ATPase activity of the motor domain ( 33 , 34 ). On the other hand, we have suggested that KIF1A(V8M), KIF1A(A255V), and KIF1A(R350G) mutations (all of them are familial) result in a gain of function ( 25 ).…”

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confidence: 99%

De novo mutations in KIF1A-associated neuronal disorder (KAND) dominant-negatively inhibit motor activity and axonal transport of synaptic vesicle precursors

Anazawa

Kita

Iguchi

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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KIF1A is a kinesin superfamily motor protein that transports synaptic vesicle precursors in axons. Cargo binding stimulates the dimerization of KIF1A molecules to induce processive movement along microtubules. Mutations in human Kif1a lead to a group of neurodegenerative diseases called KIF1A-associated neuronal disorder (KAND). KAND mutations are mostly de novo and autosomal dominant; however, it is unknown if the function of wild-type KIF1A motors is inhibited by heterodimerization with mutated KIF1A. Here, we have established Caenorhabditis elegans models for KAND using CRISPR-Cas9 technology and analyzed the effects of human KIF1A mutation on axonal transport. In our C. elegans models, both heterozygotes and homozygotes exhibited reduced axonal transport. Suppressor screening using the disease model identified a mutation that recovers the motor activity of mutated human KIF1A. In addition, we developed in vitro assays to analyze the motility of heterodimeric motors composed of wild-type and mutant KIF1A. We find that mutant KIF1A significantly impaired the motility of heterodimeric motors. Our data provide insight into the molecular mechanism underlying the dominant nature of de novo KAND mutations.

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“…KIF1A(P305L) mutation reduces microtubule association rate of the motor (32). KIF1A(R169T) disrupted the microtubule-dependent ATPase activity of the motor domain (33). KIF1A(R254W) mutation reduces the velocity and run length of the motor protein (27).…”

Section: Introductionmentioning

confidence: 99%

De novo mutations in KIF1A-associated neuronal disorder (KAND) dominant-negatively inhibit motor activity and axonal transport of synaptic vesicle precursors

Anazawa

Kita

Hayashi

2021

Preprint

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KIF1A is a kinesin superfamily molecular motor that transports synaptic vesicle precursors in axons. Mutations in Kif1a lead to a group of neuronal diseases called KIF1A-associated neuronal disorder (KAND). KIF1A forms a homodimer and KAND mutations are mostly de novo and autosomal dominant; however, it is not known whether the function of wild-type KIF1A is inhibited by disease-associated KIF1A. No reliable in vivo model systems to analyze the molecular and cellular biology of KAND have been developed; therefore, here, we established Caenorhabditis elegans models for KAND using CRISPR/cas9 technology and analyzed defects in axonal transport. In the C. elegans models, heterozygotes and homozygotes exhibited reduced axonal transport phenotypes. In addition, we developed in vitro assays to analyze the motility of single heterodimers composed of wild-type KIF1A and disease-associated KIF1A. Disease-associated KIF1A significantly inhibited the motility of wild-type KIF1A when heterodimers were formed. These data indicate the molecular mechanism underlying the dominant nature of de novo KAND mutations.

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The Novel KIF1A Missense Variant (R169T) Strongly Reduces Microtubule Stimulated ATPase Activity and Is Associated With NESCAV Syndrome

Cited by 13 publications

References 33 publications

Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs

Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs

De novo mutations in KIF1A-associated neuronal disorder (KAND) dominant-negatively inhibit motor activity and axonal transport of synaptic vesicle precursors

De novo mutations in KIF1A-associated neuronal disorder (KAND) dominant-negatively inhibit motor activity and axonal transport of synaptic vesicle precursors

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