Folding or holding?-Hsp70 and Hsp90 chaperoning of misfolded proteins in neurodegenerative disease.

The toxic accumulation of misfolded proteins as inclusions, fibrils, or aggregates is a hallmark of many neurodegenerative diseases. However, how molecular chaperones, such as heat shock protein 70 kDa (Hsp70) and heat shock protein 90 kDa (Hsp90), defend cells against the accumulation of misfolded proteins remains unclear. The ATP-dependent foldase function of both Hsp70 and Hsp90 actively transitions misfolded proteins back to their native conformation. By contrast, the ATP-independent holdase function of Hsp70 and Hsp90 prevents the accumulation of misfolded proteins. Foldase and holdase functions can protect against the toxicity associated with protein misfolding, yet we are only beginning to understand the mechanisms through which they modulate neurodegeneration. This review compares recent structural findings regarding the binding of Hsp90 to misfolded and intrinsically disordered proteins, such as tau, α-synuclein, and Tar DNA-binding protein 43. We propose that Hsp90 and Hsp70 interact with these proteins through an extended and dynamic interface that spans the surface of multiple domains of the chaperone proteins. This contrasts with many other Hsp90-client protein interactions for which only a single bound conformation of Hsp90 is proposed. The dynamic nature of these multidomain interactions allows for polymorphic binding of multiple conformations to vast regions of Hsp90. The holdase functions of Hsp70 and Hsp90 may thus allow neuronal cells to modulate misfolded proteins more efficiently by reducing the long-term ATP running costs of the chaperone budget. However, it remains unclear whether holdase functions protect cells by preventing aggregate formation or can increase neurotoxicity by inadvertently stabilizing deleterious oligomers.

Stress-inducible phosphoprotein 1 (HOP/STI1/STIP1) regulates the accumulation and toxicity of α-synuclein in vivo.

The predominantly pre-synaptic intrinsically disordered protein α-synuclein is prone to misfolding and aggregation in synucleinopathies, such as Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Molecular chaperones play important roles in protein misfolding diseases and members of the chaperone machinery are often deposited in Lewy bodies. Here, we show that the Hsp90 co-chaperone STI1 co-immunoprecipitated α-synuclein, and co-deposited with Hsp90 and Hsp70 in insoluble protein fractions in two mouse models of α-synuclein misfolding. STI1 and Hsp90 also co-localized extensively with filamentous S129 phosphorylated α-synuclein in ubiquitin-positive inclusions. In PD human brains, STI1 transcripts were increased, and in neurologically healthy brains, STI1 and α-synuclein transcripts correlated. Nuclear Magnetic Resonance (NMR) analyses revealed direct interaction of α-synuclein with STI1 and indicated that the STI1 TPR2A, but not TPR1 or TPR2B domains, interacted with the C-terminal domain of α-synuclein. In vitro, the STI1 TPR2A domain facilitated S129 phosphorylation by Polo-like kinase 3. Moreover, mice over-expressing STI1 and Hsp90ß presented elevated α-synuclein S129 phosphorylation accompanied by inclusions when injected with α-synuclein pre-formed fibrils. In contrast, reduced STI1 function decreased protein inclusion formation, S129 α-synuclein phosphorylation, while mitigating motor and cognitive deficits as well as mesoscopic brain atrophy in α-synuclein-over-expressing mice. Our findings reveal a vicious cycle in which STI1 facilitates the generation and accumulation of toxic α-synuclein conformers, while α-synuclein-induced proteostatic stress increased insoluble STI1 and Hsp90.

AlphaFold2: A Role for Disordered Protein/Region Prediction?

The development of AlphaFold2 marked a paradigm-shift in the structural biology community. Herein, we assess the ability of AlphaFold2 to predict disordered regions against traditional sequence-based disorder predictors. We find that AlphaFold2 performs well at discriminating disordered regions, but also note that the disorder predictor one constructs from an AlphaFold2 structure determines accuracy. In particular, a naïve, but non-trivial assumption that residues assigned to helices, strands, and H-bond stabilized turns are likely ordered and all other residues are disordered results in a dramatic overestimation in disorder; conversely, the predicted local distance difference test (pLDDT) provides an excellent measure of residue-wise disorder. Furthermore, by employing molecular dynamics (MD) simulations, we note an interesting relationship between the pLDDT and secondary structure, that may explain our observations and suggests a broader application of the pLDDT for characterizing the local dynamics of intrinsically disordered proteins and regions (IDPs/IDRs).

KEAP1 Cancer Mutants: A Large-Scale Molecular Dynamics Study of Protein Stability.

We have performed 280 µs of unbiased molecular dynamics (MD) simulations to investigate the effects of 12 different cancer mutations on Kelch-like ECH-associated protein 1 (KEAP1) (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C, R470C, R470H, R470S and G476R), one of the frequently mutated proteins in lung cancer. The aim was to provide structural insight into the effects of these mutants, including a new class of ANCHOR (additionally NRF2-complexed hypomorph) mutant variants. Our work provides additional insight into the structural dynamics of mutants that could not be analyzed experimentally, painting a more complete picture of their mutagenic effects. Notably, blade-wise analysis of the Kelch domain points to stability as a possible target of cancer in KEAP1. Interestingly, structural analysis of the R470C ANCHOR mutant, the most prevalent missense mutation in KEAP1, revealed no significant change in structural stability or NRF2 binding site dynamics, possibly indicating an covalent modification as this mutant's mode of action.

Nrf2, the Major Regulator of the Cellular Oxidative Stress Response, is Partially Disordered.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription regulator that plays a pivotal role in coordinating the cellular response to oxidative stress. Through interactions with other proteins, such as Kelch-like ECH-associated protein 1 (Keap1), CREB-binding protein (CBP), and retinoid X receptor alpha (RXRα), Nrf2 mediates the transcription of cytoprotective genes critical for removing toxicants and preventing DNA damage, thereby playing a significant role in chemoprevention. Dysregulation of Nrf2 is linked to tumorigenesis and chemoresistance, making Nrf2 a promising target for anticancer therapeutics. However, despite the physiological importance of Nrf2, the molecular details of this protein and its interactions with most of its targets remain unknown, hindering the rational design of Nrf2-targeted therapeutics. With this in mind, we used a combined bioinformatics and experimental approach to characterize the structure of full-length Nrf2 and its interaction with Keap1. Our results show that Nrf2 is partially disordered, with transiently structured elements in its Neh2, Neh7, and Neh1 domains. Moreover, interaction with the Kelch domain of Keap1 leads to protection of the binding motifs in the Neh2 domain of Nrf2, while the rest of the protein remains highly dynamic. This work represents the first detailed structural characterization of full-length Nrf2 and provides valuable insights into the molecular basis of Nrf2 activity modulation in oxidative stress response.

Modulation of hippocampal neuronal resilience during aging by the Hsp70/Hsp90 co-chaperone STI1.

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749.

Molecular basis for the interaction between stress-inducible phosphoprotein 1 (STIP1) and S100A1.

Stress-inducible phosphoprotein 1 (STIP1) is a cellular co-chaperone, which regulates heat-shock protein 70 (Hsp70) and Hsp90 activity during client protein folding. Members of the S100 family of dimeric calcium-binding proteins have been found to inhibit Hsp association with STIP1 through binding of STIP1 tetratricopeptide repeat (TPR) domains, possibly regulating the chaperone cycle. Here, we investigated the molecular basis of S100A1 binding to STIP1. We show that three S100A1 dimers associate with one molecule of STIP1 in a calcium-dependent manner. Isothermal titration calorimetry revealed that individual STIP1 TPR domains, TPR1, TPR2A and TPR2B, bind a single S100A1 dimer with significantly different affinities and that the TPR2B domain possesses the highest affinity for S100A1. S100A1 bound each TPR domain through a common binding interface composed of α-helices III and IV of each S100A1 subunit, which is only accessible following a large conformational change in S100A1 upon calcium binding. The TPR2B-binding site for S100A1 was predominately mapped to the C-terminal α-helix of TPR2B, where it is inserted into the hydrophobic cleft of an S100A1 dimer, suggesting a novel binding mechanism. Our data present the structural basis behind STIP1 and S100A1 complex formation, and provide novel insights into TPR module-containing proteins and S100 family member complexes.

Domains of STIP1 responsible for regulating PrPC-dependent amyloid-ß oligomer toxicity.

Soluble oligomers of amyloid-beta peptide (AßO) transmit neurotoxic signals through the cellular prion protein (PrP(C)) in Alzheimer's disease (AD). Secreted stress-inducible phosphoprotein 1 (STIP1), an Hsp70 and Hsp90 cochaperone, inhibits AßO binding to PrP(C) and protects neurons from AßO-induced cell death. Here, we investigated the molecular interactions between AßO and STIP1 binding to PrP(C) and their effect on neuronal cell death. We showed that residues located in a short region of PrP (90-110) mediate AßO binding and we narrowed the major interaction in this site to amino acids 91-100. In contrast, multiple binding sites on STIP1 (DP1, TPR1 and TPR2A) contribute to PrP binding. DP1 bound the N-terminal of PrP (residues 23-95), whereas TPR1 and TPR2A showed binding to the C-terminal of PrP (residues 90-231). Importantly, only TPR1 and TPR2A directly inhibit both AßO binding to PrP and cell death. Furthermore, our structural studies reveal that TPR1 and TPR2A bind to PrP through distinct regions. The TPR2A interface was shown to be much more extensive and to partially overlap with the Hsp90 binding site. Our data show the possibility of a PrP, STIP1 and Hsp90 ternary complex, which may influence AßO-mediated cell death.

Molecular effects of cancer-associated somatic mutations on the structural and target recognition properties of Keap1.

Kelch-like ECH-associated protein 1 (Keap1) plays an important regulatory role in the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent oxidative stress response pathway. It functions as a repressor of Nrf2, a key transcription factor that initiates the expression of cytoprotective enzymes during oxidative stress to protect cells from damage caused by reactive oxygen species. Recent studies show that mutations of Keap1 can lead to aberrant activation of the antioxidant pathway, which is associated with different types of cancers. To gain a mechanistic understanding of the links between Keap1 mutations and cancer pathogenesis, we have investigated the molecular effects of a series of mutations (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C and G476R) on the structural and target recognition properties of Keap1 by using nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) and isothermal titration calorimetry (ITC). Depending on their locations in the protein, these mutations are found to exert differential effects on the protein stability and target binding. Together with the proposed hinge-and-latch mechanism of Nrf2-Keap1 binding in the literature, our results provide important insight into the molecular affect of different somatic mutations on Keap1's function as an Nrf2 repressor.

The prion protein ligand, stress-inducible phosphoprotein 1, regulates amyloid-ß oligomer toxicity.

In Alzheimer's disease (AD), soluble amyloid-ß oligomers (AßOs) trigger neurotoxic signaling, at least partially, via the cellular prion protein (PrP(C)). However, it is unknown whether other ligands of PrP(C) can regulate this potentially toxic interaction. Stress-inducible phosphoprotein 1 (STI1), an Hsp90 cochaperone secreted by astrocytes, binds to PrP(C) in the vicinity of the AßO binding site to protect neurons against toxic stimuli. Here, we investigated a potential role of STI1 in AßO toxicity. We confirmed the specific binding of AßOs and STI1 to the PrP and showed that STI1 efficiently inhibited AßO binding to PrP in vitro (IC50 of ∼70 nm) and also decreased AßO binding to cultured mouse primary hippocampal neurons. Treatment with STI1 prevented AßO-induced synaptic loss and neuronal death in mouse cultured neurons and long-term potentiation inhibition in mouse hippocampal slices. Interestingly, STI1-haploinsufficient neurons were more sensitive to AßO-induced cell death and could be rescued by treatment with recombinant STI1. Noteworthy, both AßO binding to PrP(C) and PrP(C)-dependent AßO toxicity were inhibited by TPR2A, the PrP(C)-interacting domain of STI1. Additionally, PrP(C)-STI1 engagement activated α7 nicotinic acetylcholine receptors, which participated in neuroprotection against AßO-induced toxicity. We found an age-dependent upregulation of cortical STI1 in the APPswe/PS1dE9 mouse model of AD and in the brains of AD-affected individuals, suggesting a compensatory response. Our findings reveal a previously unrecognized role of the PrP(C) ligand STI1 in protecting neurons in AD and suggest a novel pathway that may help to offset AßO-induced toxicity.

Characterization of disordered proteins with ENSEMBLE.

UNLABELLED: ENSEMBLE is a computational approach for determining a set of conformations that represents the structural ensemble of a disordered protein based on input experimental data. The disordered protein can be an unfolded or intrinsically disordered state. Here, we introduce the latest version of the program, which has been enhanced to facilitate its general release and includes an intuitive user interface, as well as new approaches to treat data and analyse results. AVAILABILITY AND IMPLEMENTATION: ENSEMBLE is a program implemented in C and embedded in a Perl wrapper. It is supported on main Linux distributions. Source codes and installation files, including a detailed example, can be freely downloaded at http://abragam.med.utoronto.ca/∼JFKlab.

Molecular basis of the interactions between the disordered Neh4 and Neh5 domains of Nrf2 and CBP/p300 in oxidative stress response.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a major transcription factor that functions in maintaining redox homeostasis in cells. It mediates the transcription of cytoprotective genes in response to environmental and endogenous stresses to prevent oxidative damage. Thus, Nrf2 plays a significant role in chemoprevention. However, aberrant activation of Nrf2 has been shown to protect cancer cells from apoptosis and contribute to their chemoresistance. The interaction between Nrf2 and CBP is critical for the gene transcription activation. CBP and its homologue p300 interact with two transactivation domains in Nrf2, Neh4, and Neh5 domains through their TAZ1 and TAZ2 domains. To date, the molecular basis of this crucial interaction is not known, hindering a more detailed understanding of the regulation of Nrf2. To close this knowledge gap, we have used a set of biophysical experiments to dissect the Nrf2-CBP/p300 interactions. Structural properties of Neh4 and Neh5 and their binding with the TAZ1 and TAZ2 domains of CBP/p300 were characterized. Our results show that the Neh4 and Neh5 domains of Nrf2 are intrinsically disordered, and they both can bind the TAZ1 and TAZ2 domains of CBP/p300 with micromolar affinities. The findings provide molecular insight into the regulation of Nrf2 by CBP/p300 through multi-domain interactions.

Solution structure and dynamics of human hemoglobin in the carbonmonoxy form.

The solution structure of human adult carbonmonoxy hemoglobin (HbCO A) was refined using stereospecifically assigned methyl groups and residual dipolar couplings based on our previous nuclear magnetic resonance structure. The tertiary structures of individual chains were found to be very similar to the X-ray structures, while the quaternary structures in solution at low salt concentrations resembled the X-ray R structure more than the R2 structure. On the basis of chemical shift perturbation by inositol hexaphosphate (IHP) titration and docking, we identified five possible IHP binding sites in HbCO A. Amide-water proton exchange experiments demonstrated that αThr38 located in the α1ß2 interface and several loop regions in both α- and ß-chains were dynamic on the subsecond time scale. Side chain methyl dynamics revealed that methyl groups in the α1ß2 interface were dynamic, but those in the α1ß1 interface were quite rigid on the nanosecond to picosecond and millisecond to microsecond time scales. All the data strongly suggest a dynamic α1ß2 interface that allows conformational changes among different forms (like T, R, and R2) easily in solution. Binding of IHP to HbCO A induced small structural and dynamic changes in the α1ß2 interface and the regions around the hemes but did not increase the conformational entropy of HbCO A. The binding also caused conformational changes on the millisecond time scale, very likely arising from the relative motion of the α1ß1 dimer with respect to the α2ß2 dimer. Heterotropic effectors like IHP may change the oxygen affinity of Hb through modulating the relative motion of the two dimers and then further altering the structure of heme binding regions.

CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices.

The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.

A novel yeast model detects Nrf2 and Keap1 interactions with Hsp90.

Nrf2 is the master transcriptional regulator of cellular responses against oxidative stress. It is chiefly regulated by Keap1, a substrate adaptor protein that mediates Nrf2 degradation. Nrf2 activity is also influenced by many other protein interactions that provide Keap1-independent regulation. To study Nrf2 regulation, we established and characterized yeast models expressing human Nrf2 (also known as NFE2L2), Keap1 and other proteins that interact with and regulate Nrf2. Yeast models have been well established as powerful tools to study protein function and genetic and physical protein-protein interactions. In this work, we recapitulate previously described Nrf2 interactions in yeast and discover that Nrf2 interacts with the molecular chaperone Hsp90. Our work establishes yeast as a useful tool to study Nrf2 interactions and provides new insight into the crosstalk between the antioxidant response and the heat shock response.

Oxidative Stress-Induced Misfolding and Inclusion Formation of Nrf2 and Keap1.

Cells that experience high levels of oxidative stress respond by inducing antioxidant proteins through activation of the protein transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 is negatively regulated by the E3 ubiquitin ligase Kelch-like ECH-associated protein 1 (Keap1), which binds to Nrf2 to facilitate its ubiquitination and ensuing proteasomal degradation under basal conditions. Here, we studied protein folding and misfolding in Nrf2 and Keap1 in yeast, mammalian cells, and purified proteins under oxidative stress conditions. Both Nrf2 and Keap1 are susceptible to protein misfolding and inclusion formation upon oxidative stress. We propose that the intrinsically disordered regions within Nrf2 and the high cysteine content of Keap1 contribute to their oxidation and the ensuing misfolding. Our work reveals previously unexplored aspects of Nrf2 and Keap1 regulation and/or dysregulation by oxidation-induced protein misfolding.

Structural characterization of partially disordered human Chibby: insights into its function in the Wnt-signaling pathway.

The Wnt/ß-catenin signaling pathway is critical to embryonic development as well as adult tissue regeneration. Dysregulation of this pathway can lead to a variety of human diseases, in particular cancers. Chibby (Cby), a small and highly conserved protein, plays an antagonistic role in Wnt signaling by inhibiting the binding of ß-catenin to Tcf/Lef family proteins, a protein interaction that is essential for the transcriptional activation of Wnt target genes. Cby is also involved in regulating intracellular distribution of ß-catenin. Phosphorylated Cby forms a ternary complex with 14-3-3 protein and ß-catenin, facilitating the export of ß-catenin from the nucleus. On the other hand, the antagonistic function of Cby is inhibited upon binding to thyroid cancer-1 (TC-1). To dissect the structure-function relationship of Cby, we have used NMR spectroscopy, ESI-MS, CD, and DLS to extensively characterize the structure of human Cby. Our results show that the 126-residue Cby is partially disordered under nondenaturing conditions. While the N-terminal portion of the protein is predominantly unstructured in solution, the C-terminal half of Cby adopts a coiled-coil structure through self-association. Initial data for the binding studies of Cby to 14-3-3ζ (one of the isoforms in the 14-3-3 family) and TC-1 via these two distinct structural modules have also been obtained. It is noteworthy that in a recent large-scale analysis of the intrinsically disordered proteome of mouse, a substantial number of disordered proteins are predicted to have coiled-coil motif presence in their sequences. The combination of these two molecular recognition features could facilitate disordered Cby in assembling protein complexes via different modes of interaction.

Dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor.

Intrinsically disordered proteins play critical but often poorly understood roles in mediating protein interactions. The interactions of disordered proteins studied to date typically entail structural stabilization, whether as a global disorder-to-order transition or minimal ordering of short linear motifs. The disordered cyclin-dependent kinase (CDK) inhibitor Sic1 interacts with a single site on its receptor Cdc4 only upon phosphorylation of its multiple dispersed CDK sites. The molecular basis for this multisite-dependent interaction with a single receptor site is not known. By NMR analysis, we show that multiple phosphorylated sites on Sic1 interact with Cdc4 in dynamic equilibrium with only local ordering around each site. Regardless of phosphorylation status, Sic1 exists in an intrinsically disordered state but is surprisingly compact with transient structure. The observation of this unusual binding mode between Sic1 and Cdc4 extends the understanding of protein interactions from predominantly static complexes to include dynamic ensembles of intrinsically disordered states.

Exploring the Conformational Landscape of the Neh4 and Neh5 Domains of Nrf2 Using Two Different Force Fields and Circular Dichroism.

The nuclear factor erythroid 2-related factor 2 (Nrf2)-ARE transcriptional response pathway plays a critical role in protecting the cell from oxidative stresses via the upregulation of cytoprotective genes. Aberrant activation of Nrf2 in cancer cells can confer this cytoprotectivity, thereby reducing the efficacy of both chemotherapeutics and radiotherapies. Key to this antioxidant pathway is the interaction between Nrf2 and CREB binding protein (CBP), mediated by the Neh4 and Neh5 domains of Nrf2. Disruption of this interaction via small-molecule therapeutics could negate the effects of aberrant Nrf2 upregulation. Due to the disordered nature of these domains, there remains no three-dimensional structure of Neh4 or Neh5, making structure-based drug design a challenge. Here, we performed 48 µs of unbiased molecular dynamics (MD) simulations with the Amber99SB*-ILDNP and CHARMM36m force fields and circular dichroism (CD) spectropolarimetry experiments to elucidate the free-state structures of these domains; no previous data regarding their conformational landscapes exists. There are two main findings: First, we find Neh5 to be markedly more disordered than Neh4, which has nine residues in the middle of the domain showing α-helical propensity, thus pointing to Neh4 and Neh5 having different binding mechanisms. Second, the two force fields show strong differences for the glutamic acid-rich Neh5 peptide but are in reasonable agreement for Neh4, which has no glutamic acid. The CHARMM36m force field agrees more closely with the CD results.

Matrin3: Disorder and ALS Pathogenesis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of both upper and lower motor neurons in the brain and spinal cord. ALS is associated with protein misfolding and inclusion formation involving RNA-binding proteins, including TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS). The 125-kDa Matrin3 is a highly conserved nuclear DNA/RNA-binding protein that is implicated in many cellular processes, including binding and stabilizing mRNA, regulating mRNA nuclear export, modulating alternative splicing, and managing chromosomal distribution. Mutations in MATR3, the gene encoding Matrin3, have been identified as causal in familial ALS (fALS). Matrin3 lacks a prion-like domain that characterizes many other ALS-associated RNA-binding proteins, including TDP-43 and FUS, however, our bioinformatics analyses and preliminary studies document that Matrin3 contains long intrinsically disordered regions that may facilitate promiscuous interactions with many proteins and may contribute to its misfolding. In addition, these disordered regions in Matrin3 undergo numerous post-translational modifications, including phosphorylation, ubiquitination and acetylation that modulate the function and misfolding of the protein. Here we discuss the disordered nature of Matrin3 and review the factors that may promote its misfolding and aggregation, two elements that might explain its role in ALS pathogenesis.