TIA1 variant drives myodegeneration in multisystem proteinopathy with SQSTM1 mutations.

Multisystem proteinopathy (MSP) involves disturbances of stress granule (SG) dynamics and autophagic protein degradation that underlie the pathogenesis of a spectrum of degenerative diseases that affect muscle, brain, and bone. Specifically, identical mutations in the autophagic adaptor SQSTM1 can cause varied penetrance of 4 distinct phenotypes: amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Paget's disease of the bone, and distal myopathy. It has been hypothesized that clinical pleiotropy relates to additional genetic determinants, but thus far, evidence has been lacking. Here, we provide evidence that a TIA1 (p.N357S) variant dictates a myodegenerative phenotype when inherited, along with a pathogenic SQSTM1 mutation. Experimentally, the TIA1-N357S variant significantly enhances liquid-liquid-phase separation in vitro and impairs SG dynamics in living cells. Depletion of SQSTM1 or the introduction of a mutant version of SQSTM1 similarly impairs SG dynamics. TIA1-N357S-persistent SGs have increased association with SQSTM1, accumulation of ubiquitin conjugates, and additional aggregated proteins. Synergistic expression of the TIA1-N357S variant and a SQSTM1-A390X mutation in myoblasts leads to impaired SG clearance and myotoxicity relative to control myoblasts. These findings demonstrate a pathogenic connection between SG homeostasis and ubiquitin-mediated autophagic degradation that drives the penetrance of an MSP phenotype.

TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are age-related neurodegenerative disorders with shared genetic etiologies and overlapping clinical and pathological features. Here we studied a novel ALS/FTD family and identified the P362L mutation in the low-complexity domain (LCD) of T cell-restricted intracellular antigen-1 (TIA1). Subsequent genetic association analyses showed an increased burden of TIA1 LCD mutations in ALS patients compared to controls (p = 8.7 × 10-6). Postmortem neuropathology of five TIA1 mutations carriers showed a consistent pathological signature with numerous round, hyaline, TAR DNA-binding protein 43 (TDP-43)-positive inclusions. TIA1 mutations significantly increased the propensity of TIA1 protein to undergo phase transition. In live cells, TIA1 mutations delayed stress granule (SG) disassembly and promoted the accumulation of non-dynamic SGs that harbored TDP-43. Moreover, TDP-43 in SGs became less mobile and insoluble. The identification of TIA1 mutations in ALS/FTD reinforces the importance of RNA metabolism and SG dynamics in ALS/FTD pathogenesis.

C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles.

Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.

An osmolyte mitigates the destabilizing effect of protein crowding.

Most theories predict that macromolecular crowding stabilizes globular proteins, but recent studies show that weak attractive interactions can result in crowding-induced destabilization. Osmolytes are ubiquitous in biology and help protect cells against stress. Given that dehydration stress adds to the crowded nature of the cytoplasm, we speculated that cells might use osmolytes to overcome the destabilization caused by the increased weak interactions that accompany desiccation. We used NMR-detected amide proton exchange experiments to measure the stability of the test protein chymotrypsin inhibitor 2 under physiologically relevant crowded conditions in the presence and absence of the osmolyte glycine betaine. The osmolyte overcame the destabilizing effect of the cytosol. This result provides a physiologically relevant explanation for the accumulation of osmolytes by dehydration-stressed cells.

Protein crowder charge and protein stability.

Macromolecular crowding effects arise from steric repulsions and weak, nonspecific, chemical interactions. Steric repulsions stabilize globular proteins, but the effect of chemical interactions depends on their nature. Repulsive interactions such as those between similarly charged species should reinforce the effect of steric repulsions, increasing the equilibrium thermodynamic stability of a test protein. Attractive chemical interactions, on the other hand, counteract the effect of hard-core repulsions, decreasing stability. We tested these ideas by using the anionic proteins from Escherichia coli as crowding agents and assessing the stability of the anionic test protein chymotrypsin inhibitor 2 at pH 7.0. The anionic protein crowders destabilize the test protein despite the similarity of their net charges. Thus, weak, nonspecific, attractive interactions between proteins can overcome the charge-charge repulsion and counterbalance the stabilizing effect of steric repulsion.

Impact of reconstituted cytosol on protein stability.

Protein stability is usually studied in simple buffered solutions, but most proteins function inside cells, where the heterogeneous and crowded environment presents a complex, nonideal system. Proteins are expected to behave differently under cellular crowding owing to two types of contacts: hard-core repulsions and weak, chemical interactions. The effect of hard-core repulsions is purely entropic, resulting in volume exclusion owing to the mere presence of the crowders. The weak interactions can be repulsive or attractive, thus enhancing or diminishing the excluded volume, respectively. We used a reductionist approach to assess the effects of intracellular crowding. Escherichia coli cytoplasm was dialyzed, lyophilized, and resuspended at two concentrations. NMR-detected amide proton exchange was then used to quantify the stability of the globular protein chymotrypsin inhibitor 2 (CI2) in these crowded solutions. The cytosol destabilizes CI2, and the destabilization increases with increasing cytosol concentration. This observation shows that the cytoplasm interacts favorably, but nonspecifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. The effects of the cytosol are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, nonspecific interactions.

Amide proton exchange of a dynamic loop in cell extracts.

Intrinsic rates of exchange are essential parameters for obtaining protein stabilities from amide (1) H exchange data. To understand the influence of the intracellular environment on stability, one must know the effect of the cytoplasm on these rates. We probed exchange rates in buffer and in Escherichia coli lysates for the dynamic loop in the small globular protein chymotrypsin inhibitor 2 using a modified form of the nuclear magnetic resonance experiment, SOLEXSY. No significant changes were observed, even in 100 g dry weight L(-1) lysate. Our results suggest that intrinsic rates from studies conducted in buffers are applicable to studies conducted under cellular conditions.

Soft interactions and crowding.

The intracellular milieu is complex, heterogeneous and crowded-an environment vastly different from dilute solutions in which most biophysical studies are performed. The crowded cytoplasm excludes about a third of the volume available to macromolecules in dilute solution. This excluded volume is the sum of two parts: steric repulsions and chemical interactions, also called soft interactions. Until recently, most efforts to understand crowding have focused on steric repulsions. Here, we summarize the results and conclusions from recent studies on macromolecular crowding, emphasizing the contribution of soft interactions to the equilibrium thermodynamics of protein stability. Despite their non-specific and weak nature, the large number of soft interactions present under many crowded conditions can sometimes overcome the stabilizing steric, excluded volume effect.

Macromolecular crowding and protein stability.

An understanding of cellular chemistry requires knowledge of how crowded environments affect proteins. The influence of crowding on protein stability arises from two phenomena, hard-core repulsions and soft (i.e., chemical) interactions. Most efforts to understand crowding effects on protein stability, however, focus on hard-core repulsions, which are inherently entropic and stabilizing. We assessed these phenomena by measuring the temperature dependence of NMR-detected amide proton exchange and used these data to extract the entropic and enthalpic contributions of crowding to the stability of ubiquitin. Contrary to expectations, the contribution of chemical interactions is large and in many cases dominates the contribution from hardcore repulsions. Our results show that both chemical interactions and hard-core repulsions must be considered when assessing the effects of crowding and help explain previous observations about protein stability and dynamics in cells.

Protein crowding tunes protein stability.

Thirty percent of a cell's volume is filled with macromolecules, and protein chemistry in a crowded environment is predicted to differ from that in dilute solution. We quantified the effect of crowding by globular proteins on the equilibrium thermodynamic stability of a small globular protein. Theory has long predicted that crowding should stabilize proteins, and experiments using synthetic polymers as crowders show such stabilizing effects. We find that protein crowders can be mildly destabilizing. The destabilization arises from a competition between stabilizing excluded-volume effects and destabilizing nonspecific interactions, including electrostatic interactions. This competition results in tunable stability, which could impact our understanding of the spatial and temporal roles of proteins in living systems.