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Structural Studies of C9orf72-SMCR8-WDR41 Protein Complex.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Structural Studies of C9orf72-SMCR8-WDR41 Protein Complex./
Author:	Shkuratova, Valeria.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2020,
Description:	62 p.
Notes:	Source: Masters Abstracts International, Volume: 82-10.
Contained By:	Masters Abstracts International82-10.
Subject:	Mass spectrometry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28383826
ISBN:	9798708710772

Structural Studies of C9orf72-SMCR8-WDR41 Protein Complex.
Shkuratova, Valeria.

Structural Studies of C9orf72-SMCR8-WDR41 Protein Complex. - Ann Arbor : ProQuest Dissertations & Theses, 2020 - 62 p.

Source: Masters Abstracts International, Volume: 82-10.

Thesis (M.Sc.)--McGill University (Canada), 2020.

This item must not be sold to any third party vendors.

Hexanucleotide repeat expansions in the C9orf72 gene are the leading cause for the development of two neurodegenerative diseases: amyotrophic lateral sclerosis and frontotemporal dementia. Previous studies have identified that C9orf72 binds SMCR8 and WDR41 to form a stable complex, which was shown to be important during autophagy in neuronal cells. However, the exact functions of the complex and its structural features are mainly unknown. Here we established a purification protocol for C9orf72-SMCR8-WDR41 protein complex. We constructed a single plasmid containing all three proteins for efficient expression in Sf9 insect cells. The two-step purification using GFP-nanobody-coupled beads was optimized to yield high quantities of pure trimeric complex (up to 7.5 mg for 1 L of cell culture). Crystallization trials were unsuccessful, suggesting a requirement for other techniques such as cryoEM for solving the complex structure. Studies using HDX-MS revealed that WDR41 binds to SMCR8 DENN domain but not to C9orf72. However, the conformational changes occur in both SMCR8 and C9orf72. Additionally, we identified unstructured regions within the SMCR8 linker region and WDR41 that could be important for complex assembly. All these results are in agreement with the recently published structure of the C9orf72-SMCR8-WDR41 complex.

ISBN: 9798708710772Subjects--Topical Terms:

551172
Mass spectrometry.
Subjects--Index Terms:

C9orf72-SMCR8-WDR41

Structural Studies of C9orf72-SMCR8-WDR41 Protein Complex.
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020 $a 9798708710772
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035 $a (MiAaPQ)McGill_z029p9338
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040 $a MiAaPQ $c MiAaPQ
100 1 $a Shkuratova, Valeria. $3 3686267
245 1 0 $a Structural Studies of C9orf72-SMCR8-WDR41 Protein Complex.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2020
300 $a 62 p.
500 $a Source: Masters Abstracts International, Volume: 82-10.
500 $a Advisor: Gehring, Kalle Burgess.
502 $a Thesis (M.Sc.)--McGill University (Canada), 2020.
506 $a This item must not be sold to any third party vendors.
520 $a Hexanucleotide repeat expansions in the C9orf72 gene are the leading cause for the development of two neurodegenerative diseases: amyotrophic lateral sclerosis and frontotemporal dementia. Previous studies have identified that C9orf72 binds SMCR8 and WDR41 to form a stable complex, which was shown to be important during autophagy in neuronal cells. However, the exact functions of the complex and its structural features are mainly unknown. Here we established a purification protocol for C9orf72-SMCR8-WDR41 protein complex. We constructed a single plasmid containing all three proteins for efficient expression in Sf9 insect cells. The two-step purification using GFP-nanobody-coupled beads was optimized to yield high quantities of pure trimeric complex (up to 7.5 mg for 1 L of cell culture). Crystallization trials were unsuccessful, suggesting a requirement for other techniques such as cryoEM for solving the complex structure. Studies using HDX-MS revealed that WDR41 binds to SMCR8 DENN domain but not to C9orf72. However, the conformational changes occur in both SMCR8 and C9orf72. Additionally, we identified unstructured regions within the SMCR8 linker region and WDR41 that could be important for complex assembly. All these results are in agreement with the recently published structure of the C9orf72-SMCR8-WDR41 complex.
520 $a Les expansions repetees hexanucleotidiques du gene C9orf72 sont la principale cause du developpement de deux maladies neurodegeneratives: la sclerose laterale amyotrophique et la demence fronto-temporale. Des etudes anterieures ont montre que C9orf72 se lie a SMCR8 et WDR41 pour former un complexe stable. Ce complexe s'est revele important lors de l'autophagie dans les cellules neuronales. Cependant, les fonctions exactes du complexe et ses caracteristiques structurelles sont principalement inconnues. Ici, nous avons etabli un protocole de purification pour le complexe proteique C9orf72-SMCR8-WDR41. Nous avons construit un seul plasmide contenant les trois proteines pour une expression efficace dans les cellules d'insectes Sf9. La purification en deux etapes a l'aide de billes couplees a la nanoparticule GFP a ete optimisee pour maximiser le rendement du complexe trimerique pur (jusqu'a 7,5 mg pour 1 L de culture cellulaire). Les essais de cristallisation menes ont echoue, suggerant une exigence pour d'autres techniques tels que cryoEM pour resoudre la structure du complexe. Des etudes utilisant HDX-MS ont revele que WDR41 se lie au domaine DENN du SMCR8 mais pas a C9orf72. Cependant, les changements de conformation se produisent a la fois dans SMCR8 et C9orf72. De plus, nous avons identifie des regions non structurees au sein dans la region de liaison SMCR8 et WDR41 qui peuvent etre importantes pour l'assemblage du complexe. Tous ces resultats sont en accord avec la structure du complexe C9orf72-SMCR8-WDR41 recemment publiee.
590 $a School code: 0781.
650 4 $a Mass spectrometry. $3 551172
650 4 $a Mutation. $3 837917
650 4 $a Cloning. $3 571606
650 4 $a Dementia. $3 568139
650 4 $a Optimization. $3 891104
650 4 $a Chromosomes. $3 638948
650 4 $a Crystallization. $3 608799
650 4 $a Amyotrophic lateral sclerosis. $3 1094680
650 4 $a Scientific imaging. $3 3560377
650 4 $a Autophagy. $3 2045493
650 4 $a Chromatography. $3 1073639
650 4 $a Sedimentation & deposition. $3 3566241
650 4 $a Kinases. $3 3558077
650 4 $a Cell culture. $3 604671
650 4 $a Proteins. $3 558769
653 $a C9orf72-SMCR8-WDR41
653 $a Protein complex
653 $a Structure
690 $a 0487
710 2 $a McGill University (Canada). $3 1018122
773 0 $t Masters Abstracts International $g 82-10.
790 $a 0781
791 $a M.Sc.
792 $a 2020
793 $a English
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28383826