Solution NMR assignment of the cryptic sixth TOG domain of mini spindles

Burgess, Selena G.; Bayliss, Richard; Pfuhl, Mark

doi:10.1007/s12104-015-9620-4

Cited by 5 publications

(3 citation statements)

References 13 publications

(22 reference statements)

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“…8B , right). The domain organization of the conserved C-terminus still remains to be characterized, but the existence of a sixth TOG domain has been speculated 45 , 46 , which could be a potential γ-tubulin interaction site. In the N-terminus, TOG domains 1 and 2 could primarily recruit αβ-tubulin dimers, whereas the later TOG domains stabilize the newly forming MT.…”

Section: Discussionmentioning

confidence: 99%

XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex

2018

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How microtubules (MT) are generated in the cell is a major question in understanding how the cytoskeleton is assembled. For several decades, γ-tubulin has been accepted as the cell’s universal MT nucleator. Although there is evidence that γ-tubulin complexes are not the sole MT nucleators, identification of other nucleation factors has proven difficult. Here, we report that the well-characterized MT polymerase XMAP215 (chTOG/Msps/Stu2p/Alp14/Dis1 homologue) is essential for MT nucleation in Xenopus egg extracts. The concentration of XMAP215 determines the extent of MT nucleation. Even though XMAP215 and γ-tubulin ring complex (γ-TuRC) possess minimal nucleation activity individually, together these factors synergistically stimulate MT nucleation in vitro. The N-terminal TOG domains 1–5 of XMAP215 bind αβ-tubulin and promote MT polymerization, while the conserved C-terminus is required for efficient MT nucleation and directly binds γ-tubulin. In sum, XMAP215 and γ-TuRC together function as the principal nucleation module that generates MTs in cells.

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Section: Discussionmentioning

confidence: 99%

XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex

2018

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“…Protein for a 15 N labelled sample was expressed in modified minimal media as described previously (42,43) with purification and sample preparation as described for the unlabelled samples. NMR spectra were recorded on Bruker Avance III 700 and 800 MHz spectrometers equipped with cryoprobes.…”

Section: Bacterial Expression and Purification Of Recombinant T Parva Schizont Antigen Tp2mentioning

confidence: 99%

Development of a Potential Yeast-Based Vaccine Platform for Theileria parva Infection in Cattle

et al. 2021

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East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called “Infection and Treatment Method” (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 induced IFNγ responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induced Tp2-specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen-specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cattle will further contribute to the development of a yeast-based vaccine for ECF.

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“…Protein for a 15 N labelled sample was expressed in modified minimal media as described previously 41,42 with purification and sample preparation as described for the unlabelled samples. NMR spectra were recorded on Bruker Avance III 700 and 800 MHz spectrometers equipped with cryoprobes.…”

Section: Bacterial Expression and Purification Of Recombinant T Parva Schizont Antigen Tp2mentioning

confidence: 99%

Development of a yeast-based vaccine forTheileria parvainfection in cattle

Goh

Kolakowski

Holder

et al. 2021

Preprint

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East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called “Infection and Treatment Method” (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain specific, MHC class I restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length native Tp2 and Tp9 as well as fragments of native Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNy responses using PBMCs from ITM-animals, while Tp2 and Tp9 induced IFNy responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induce Tp2 specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cows will further contribute to the development of a yeast-based vaccine for ECF.

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Solution NMR assignment of the cryptic sixth TOG domain of mini spindles

Cited by 5 publications

References 13 publications

XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex

XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex

Development of a Potential Yeast-Based Vaccine Platform for Theileria parva Infection in Cattle

Development of a yeast-based vaccine forTheileria parvainfection in cattle

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