Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization

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Proteins involved in ALS and IBMPFD

IBMPFD was attributed to being caused by mutations in the gene encoding Valosin-Containing Protein (VCP) (Watts et al., 2004). Later on, exome sequencing revealed VCP mutations as a cause of familial ALS as well (Johnson et al., 2010). Inherited forms of ALS and IBMPFD were also found to be often caused by missense mutations impacting heterogeneous ribonucleoproteins (hnRNPs) such as TDP-43, FUS, hnRNPA1 and hnRNPA2B1.


Valosin-containing protein (VCP), also known as p97, is a protein belonging to the ATPase Associated with diverse cellular Activities (AAA+ ATPase) protein family. This family of enzymes is associated with a wide range of cellular processes such as proteolysis, DNA replication and repair, and membrane fusion, which it carries out in an ATP-dependent manner (Ogura and Wilkinson, 2001). VCP is ubiquitously expressed in the cytoplasm and the nucleus and has a tripartite structure consisting of an N-terminal domain and two central D1 and D2 AAA+ domains. It also contains linker regions that join the N-D1 and D1-D2 domains (Figure 3).
The N-terminal domain is necessary for substrate and co-factor association, whereas the D1 and D2 domains are needed for ATP binding and hydrolysis. The D1 domain is primarily responsible for VCP hexamerization but this molecular assembly is not dependent on nucleotide binding (Wang et al., 2003a; Wang et al., 2003b; Zhang et al., 2000). The ATPase activity conferred by the D2 domain is essential for VCP’s function as a molecular chaperone in various cellular processes (Esaki and Ogura, 2010; Song et al., 2003). Six VCP monomers assemble into a macromolecular ring complex with the inner core formed by the D1/D2 domains, with the N-domains radiating outward. Structural changes can occur in the ring, allowing VCP to function as a chaperon that interacts with diverse adaptors. Notably, VCP is a ubiquitin-dependent segregase that can remove some ubiquitinylated proteins from protein complexes due to interactions with various adaptors. Through this mechanism, VCP can mediate a wide variety of essential cellular processes such as: cell cycle progression, DNA replication, protein quality control, and chromosomal decondensation. The C-terminal domain of VCP, following the D2 domain, is poorly characterized, but this region includes the major tyrosine phosphorylation site implicated in the regulation of endoplasmic reticulum (ER assembly) and cell cycle-dependent nuclear localization of VCP (Egerton and Samelson, 1994; Lavoie et al., 2000; Madeo et al., 1998).
Because of its role in many cell processes, deregulation or mutations in VCP cause serious diseases. For example, VCP is upregulated in cancer while missense mutations cause a dominantly inherited degeneration of bone, brain, and muscles. Fourteen mutations in VCP have been associated with diseases including myopathy, dementia, Paget’s disease of bone, ALS, and Parkinsonism. Among these mutations, R115H is the most prevalent disease-associated mutation and A232R causes the most serious symptoms of the diseases (Ju and Weihl, 2010). Interestingly, the amino acids affected by the disease-causing mutations are highly conserved across species. Even though it remains unknown how mutations in VCP cause disease, some studies have shown how they affect the structure of the protein. The majority of the mutations localize to the interface of the N- and D1- domains (Figure 4).
Figure 4: Graphic representation of VCP crystal structure showing the distribution of the IBMPFD-linked mutations. The N domain is represented in green, the D1 domain in blue and the D2 domain in grey. Adapted from (Niwa et al., 2012) Because of this localization, a conformational change occurs, leading to impaired communication between the D1 and N-domains. This change indirectly influences the D1 nucleotide binding pocket (Fernandez-Saiz and Buchberger, 2010), altering the relative affinity for ATP and ADP. The VCP mutations R155P and A232E, found in IBMPFD patients, show an increase in ATPase activity and increased sensitivity to heat-induced upregulation in ATPase activity (Halawani et al., 2009). It has been shown recently that stress granule clearance was impaired when VCP was mutated (Buchan et al., 2013). Yet, it remains unclear how the mutations lead to the pathogenesis in biological systems.


TAR DNA-binding protein 43 (TDP-43) belongs to the hnRNP (heterogeneous nuclear ribonucleoprotein) family of proteins (Chaudhury et al., 2010). TDP-43 is a 414 amino acid nuclear protein encoded by the TARDBP gene on chromosome 1. It is composed of two N-terminal RNA-recognition motifs (RRM), nuclear localization (NLS) and nuclear export (NES) sequences, and a C-terminal domain enriched in glycines (Gly-rich) that may be required for the exon skipping and splicing inhibitory activity as well as for binding to nuclear proteins (Figure 5).
Figure 5: Domain structure of TDP-43. (shown in black) and ubiquitin-positive FTD 2011).
Known mutations are associated with ALS (in blue). Adapted from (Dormann and Haass, TDP-43 is a nuclear DNA- and RNA-binding protein with multiple functions in transcriptional repression, pre-mRNA splicing, and translational regulation (Da Cruz and Cleveland, 2011; Lagier-Tourenne et al., 2010). TDP-43 is highly conserved through evolution and it associates with other members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family by interactions through its C-terminal domain (Brundin et al., 2010).
TDP-43 was initially identified as the major constituent of neuronal inclusions in sporadic ALS and in the largest subset of FTD (Neumann et al., 2006). In the degenerating neurons and glial cells of the central nervous system (CNS) of ALS patients, TDP-43 is relocated from the nucleus to large cytoplasmic aggregates. Additional studies showed that TDP-43 abnormally accumulates in the sarcoplasm of IBMPFD patients (Salajegheh et al., 2009). This accumulation is accompanied by its nuclear depletion, suggesting its redistribution from the myonucleus to the sarcoplasm is similar to the nuclear to cytoplasmic redistribution seen in affected cortical regions.
Mutations in TDP-43 were identified in both sporadic ALS and familial ALS (Sreedharan et al., 2008). However, these rare mutations are associated with less than 5% of the familial cases. It is important to note that most of the disease-causing mutations are localized in the C-terminal glycine-rich region of the protein (Figure 5).

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Identification of hnRNPA1 and hnRNPA2B1 as disease-causing proteins

Identification of VCP-mutations negative families

Some, but not all, cases of IBMPFD associated with ALS are caused by mutations in the VCP gene. Work from Kim et al. (Kim et al., 2013) (See annex) identified two families with dominantly inherited degeneration of muscle, bone, brain, and motor neurons. The affected members of these families had identical clinical symptoms compared to patients presenting VCP mutations. Sequencing of the affected patients revealed that VCP was not mutated. Genetic analysis of this family by exome sequencing and linkage analysis identified mutations in two genes: hnRNPA1 and hnRNPA2B1 (Figure 6).
Figure 6: Identification of previously unknown disease mutations in IBMPFD/ALS. a), Family 1 pedigree indicating individuals affected by dementia, myopathy, PDB and ALS. The causative mutation was p.D290V/D302V in hnRNPA2B1. Roman numerals denote generation and Arabic numbers denote family member within a generation. b), Family 2 pedigree indicating individuals affected by myopathy and PDB. The causative mutation was p.D262V/D314V in hnRNPA1. Adapted from (Kim et al., 2013)  The two mutations described corresponded to a substitution of a valine residue in the place of a highly conserved aspartate residue in human paralogs of the hnRNP A/B family (Figure 7). The mutations identified for hnRNPA2B1 and hnRNPA1 were D290V and D262V, respectively.
Figure 7: Sequence alignment of hnRNPA2B1 and hnRNPA1 orthologues. There is an evolutionary conservation of the mutated aspartate and surrounding residues.

hnRNPA1 and hnRNPA2B1

hnRNPA2B1 and hnRNPA1 belong to the A/B subfamily of ubiquitously expressed hnRNPs. hnRNPA2B1 is expressed as two alternatively spliced isoforms: A2 and B1. The shorter isoform, hnRNPA2, lacks 12 amino acids in the N-terminal region and is the major isoform, accounting for ~90% of the protein in most tissues. The hnRNPA2 and hnRNPA1 proteins have two repeats of RNA-recognition motifs (RRMs) that bind RNAs. They are one of the most abundant core proteins of hnRNP complexes (Krecic and Swanson, 1999). These complexes are made of predominantly nuclear RNA-binding proteins that form complexes with RNA polymerase II transcripts. These proteins function in multiple cellular activities, ranging from transcription and pre-mRNA processing in the nucleus to cytoplasmic mRNA translation and turnover . hnRNPA1 and hnRNPA2B1 are involved in the packaging of pre-mRNA into hnRNP particles, transport of poly A+ mRNA from the nucleus to the cytoplasm, and may modulate splice site selection (Han et al., 2010). They are mainly present in the nucleus but can also shuttle between the nucleus and the cytoplasm (Pinol-Roma and Dreyfuss, 1992). Indeed, they are exported from the nucleus, along with other hnRNP proteins and probably bound to mRNA, to then be re-imported.
Their M9 domain acts as both a nuclear localization and nuclear export signal (Izaurralde et al., 1997). hnRNPA1 and hnRNPA2 also contain a carboxy terminal domain enriched in glycines that clusters the disease-causing mutations (Figure 8).

Table of contents :

Chapter I: Introduction
I.1 Pathogenesis of different neuronal and muscle disorders
I.1.1 Introduction of ALS and IBMPFD
I.1.1.1 Amyotrophic lateral sclerosis (ALS)
I.1.1.2 Inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia (IBMPFD)
I.1.1.3 RNA-binding proteins aggregates are a hallmark of ALS and IBMPFD
I.1.2 Proteins involved in ALS and IBMPFD
I.1.2.1 VCP
I.1.2.2 TDP-43
I.1.2.3 Identification of hnRNPA1 and hnRNPA2B1 as disease-causing proteins
I. Identification of VCP-mutations negative families
I. hnRNPA1 and hnRNPA2B1
I.1.2.4 Relationship between previously described disease-implicated proteins
I. Interaction between VCP, TDP-43, hnRNPA1 and hnRNPA2
I. hnRNPs contain a low complexity sequence domain clustering the disease-causing mutations
I.2 Stress granules as crucibles of these pathogeneses
I.2.1 What are stress granules?
I.2.2 Stress Granule dynamics
I.2.2.1 Assembly of Stress Granules
I.2.2.2 Disassembly of Stress Granules
I.2.3 Stress granule function
I.2.4 Core component of stress granules
I.2.5 Implication of disease-causing mutations in stress granule dynamics
I.3 Does fibrillization mediate stress granule assembly?
I.4 Recruitment of stress signaling proteins to stress granules
I.4.1 Compartmentalization of membrane-less organelles in cells
I.4.2 Liquid-like behavior of cellular structures
I.4.3 Liquid-liquid phase separation: a mechanism that allows cellular compartmentalization
I.4.3.1 Definition of liquid-liquid phase separation
I.4.3.2 Phase diagrams
I.4.3.3 Liquid-liquid phase separation: a mechanism for subcompartmentalization
I.4.3.4 Proteins harboring low complexity domain sequences can mediate LLPS
Chapter II: Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization
Chapter III: Discussion
III.1 LLPS as a mechanism commonly used by cells in order to accomplish their biological processes
III.2 From stress granules to pathological inclusions: is there a unifying model?
III.3 Compartmentalization via LLPS: a code in low complexity sequence domains?
Chapter IV: Annexes
IV.1 Annex I: VCP is essential for mitochondrial quality control by PINK1/Parkin and this function is impaired by VCP mutations
IV.2 Annex II: Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS
IV.3 Annex III: No mutations in hnRNPA1 and hnRNPA2B1 in Dutch patients with amyotrophic lateral sclerosis, frontotemporal dementia, and inclusion body myopathy


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