(C) PlosOne
This story was originally published on plosone.org. The content has not been altered[1]
Licensed under Creative Commons Attribution (CC BY) license .
url:https://journals.plos.org/plosone/s/licenses-and-copyright
--------------------



Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture

['Julia Nörpel', 'Friedrich Miescher Institute For Biomedical Research', 'Basel', 'University Of Basel', 'Simone Cavadini', 'Andreas D. Schenk', 'Alexandra Graff-Meyer', 'Daniel Hess', 'Jan Seebacher', 'Jeffrey A. Chao']
Date: None

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G 4 C 2 repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain of function by repeat G 4 C 2 RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex.

In this study, we report the structure of the C9orf72-SMCR8 complex using single-particle cryo-electron microscopy (cryo-EM). The cryo-EM structure sheds light onto the global architecture of the C9orf72-SMCR8 complex, revealing the overall arrangement of individual Denn domains in each protein and the extensive dimerization interface. Furthermore, this large interaction surface between the 2 proteins helps to rationalize why protein expression levels of C9orf72 and SMCR8 are positively correlated in cells [ 16 , 17 , 29 – 32 ]. Structural analysis of the core Denn domains of C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2) reveals a high similarity between the 2 complexes. Structural superposition of the SMCR8 and FLCN uDenn domains enabled the identification of a conserved arginine finger in SMCR8 that suggests a potential GAP activity for the C9orf72-SMCR8 complex [ 28 ]. Furthermore, structural analysis of a coiled-coil region in SMCR8 identifies it as a potential interaction platform for other coiled-coil proteins. The interaction of FIP200, which contains a coiled coil, with C9orf72-SMCR8 was reduced upon deletion of the coiled coil within SMCR8 in starved condition. Interestingly, loss of the SMCR8 coiled coil also increased its interaction with WDR41, which highlights the potential interplay between distinct protein binding regions of the multifunctional C9orf72-SMCR8 complex.

Sequence analysis of C9orf72 and SMCR8 identified the presence of differently expressed in normal and neoplastic cells (Denn) module in these proteins [ 24 ]. Denn module proteins are frequently implicated in vesicular pathways in cells and act as either guanosine exchange factors (GEFs) or GTPase activating proteins (GAPs) for small GTPases [ 25 , 26 ]. Previously, the C9orf72-SMCR8 complex was shown to have GEF activity for Rab8a and Rab39b [ 16 ]. Structurally, Denn modules are characterized by the presence of a tripartite Denn domain that includes the upstream Denn (uDenn) domain, the central Denn (cDenn) domain, and the downstream Denn (dDenn) domain [ 26 – 28 ]. The uDenn domain adopts a longin-like fold, and the cDenn domain contains a mixed α/β fold [ 26 – 28 ].

The C9orf72 gene is transcribed into 3 transcript variants resulting in production of 2 isoforms. The longer C9orf72 isoform (481 AA) associates with SMCR8 and WDR41 and assembles into a trimeric complex that is implicated in regulation of endosomal and autophagic pathways [ 16 – 22 ]. Specifically, the C9orf72-SMCR8 heterodimer is an effector of Rab1a GTPase and interacts with the ULK1 complex to regulate autophagy initiation [ 17 – 19 ]. Furthermore, the C9orf72-SMCR8 complex was shown to localize to lysosomes and is involved in mTORC1 signaling [ 20 – 22 ]. Interestingly, loss of C9orf72 has been shown to increase the autophagic flux [ 22 ]. Additionally, C9orf72 is also suggested to modulate actin dynamics in axons, thereby influencing axon extension and growth of cones [ 23 ]. Finally, the loss of this complex has been shown to reduce the clearance of the dipeptide repeat proteins in patient iPS neurons [ 6 , 7 ].

A hexanucleotide G 4 C 2 repeat expansion in the first intron of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder [ 1 , 2 ]. Healthy individuals carry less than 30 G 4 C 2 repeats, whereas patients have expansions ranging from 30 to 7,000 repeats [ 1 , 2 ]. While the exact mechanisms leading to disease are still being studied, multiple mutually nonexclusive mechanisms have been proposed including the toxic gain of function of the G 4 C 2 repeat RNAs, dipeptide repeat protein products generated by translation of these repeat expansions [ 1 – 5 ], and reduction of the C9orf72 protein [ 1 , 6 , 7 ]. Early work in zebrafish and worms showed that reduced protein levels of the C9orf72 orthologs lead to motor neuron degeneration, indicating a possible role of C9orf72 loss of function in disease phenotype [ 8 , 9 ]. In contrast, studies in C9orf72 null mouse models detected only mild cognitive and behavioral deficits related to the ALS–FTD phenotype; however, loss of C9orf72 in combination with expression of G 4 C 2 repeat RNAs resulted in an exacerbated disease phenotype in mice and patient iPS neurons [ 7 , 10 – 14 ]. Additionally, mice models with reduced C9orf72 levels developed splenomegaly and showed altered immune responses [ 11 , 12 , 15 ]. While loss of the C9orf72 gene product in different genetic backgrounds and model systems shows distinct effects, the cellular function of the C9orf72 protein is not fully understood.

Results and discussion

[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001344

(C) GlobalVoices
Licensed under Creative Commons Attribution 3.0 Unported (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/

You are viewing proxied material from magical.fish. The copyright of proxied material belongs to its original authors. Any comments or complaints in relation to proxied material should be directed to the original authors of the content concerned. Please see the disclaimer for more details.