Redox State Controls Phase Separation of the Yeast Ataxin-2 Protein via Reversible Oxidation of Its Methionine-Rich Low-Complexity Domain.

Yeast ataxin-2, also known as Pbp1, senses the activity state of mitochondria in order to regulate TORC1. A domain of Pbp1 required to adapt cells to mitochondrial activity is of low sequence complexity. The low-complexity (LC) domain of Pbp1 forms labile, cross-ß polymers that facilitate phase transition of the protein into liquid-like or gel-like states. Phase transition for other LC domains is reliant upon widely distributed aromatic amino acids. In place of tyrosine or phenylalanine residues prototypically used for phase separation, Pbp1 contains 24 similarly disposed methionine residues. Here, we show that the Pbp1 methionine residues are sensitive to hydrogen peroxide (H2O2)-mediated oxidation in vitro and in living cells. Methionine oxidation melts Pbp1 liquid-like droplets in a manner reversed by methionine sulfoxide reductase enzymes. These observations explain how reversible formation of labile polymers by the Pbp1 LC domain enables the protein to function as a sensor of cellular redox state.

Yeast Ataxin-2 Forms an Intracellular Condensate Required for the Inhibition of TORC1 Signaling during Respiratory Growth.

Yeast ataxin-2, also known as Pbp1 (polyA binding protein-binding protein 1), is an intrinsically disordered protein implicated in stress granule formation, RNA biology, and neurodegenerative disease. To understand the endogenous function of this protein, we identify Pbp1 as a dedicated regulator of TORC1 signaling and autophagy under conditions that require mitochondrial respiration. Pbp1 binds to TORC1 specifically during respiratory growth, but utilizes an additional methionine-rich, low complexity (LC) region to inhibit TORC1. This LC region causes phase separation, forms reversible fibrils, and enables self-association into assemblies required for TORC1 inhibition. Mutants that weaken phase separation in vitro exhibit reduced capacity to inhibit TORC1 and induce autophagy. Loss of Pbp1 leads to mitochondrial dysfunction and reduced fitness during nutritional stress. Thus, Pbp1 forms a condensate in response to respiratory status to regulate TORC1 signaling.

A Solid-State Conceptualization of Information Transfer from Gene to Message to Protein.

In this review, we describe speculative ideas and early stage research concerning the flow of genetic information from the nuclear residence of genes to the disparate, cytoplasmic sites of protein synthesis. We propose that this process of information transfer is meticulously guided by transient structures formed from protein segments of low sequence complexity/intrinsic disorder. These low complexity domains are ubiquitously associated with regulatory proteins that control gene expression and RNA biogenesis, but they are also found in the central channel of nuclear pores, the nexus points of intermediate filament assembly, and the locations of action of other well-studied cellular proteins and pathways. Upon being organized into localized cellular positions via mechanisms utilizing properly folded protein domains, thereby facilitating elevated local concentration, certain low complexity domains adopt cross-ß interactions that are both structurally specific and labile to disassembly. These weakly tethered assemblies, we propose, are built to relay the passage of genetic information from one site to another within a cell, ensuring that the process is of extreme fidelity.

Structure of FUS Protein Fibrils and Its Relevance to Self-Assembly and Phase Separation of Low-Complexity Domains.

Polymerization and phase separation of proteins containing low-complexity (LC) domains are important factors in gene expression, mRNA processing and trafficking, and localization of translation. We have used solid-state nuclear magnetic resonance methods to characterize the molecular structure of self-assembling fibrils formed by the LC domain of the fused in sarcoma (FUS) RNA-binding protein. From the 214-residue LC domain of FUS (FUS-LC), a segment of only 57 residues forms the fibril core, while other segments remain dynamically disordered. Unlike pathogenic amyloid fibrils, FUS-LC fibrils lack hydrophobic interactions within the core and are not polymorphic at the molecular structural level. Phosphorylation of core-forming residues by DNA-dependent protein kinase blocks binding of soluble FUS-LC to FUS-LC hydrogels and dissolves phase-separated, liquid-like FUS-LC droplets. These studies offer a structural basis for understanding LC domain self-assembly, phase separation, and regulation by post-translational modification.

Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers.

Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.

The LC Domain of hnRNPA2 Adopts Similar Conformations in Hydrogel Polymers, Liquid-like Droplets, and Nuclei.

Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino acids and have been termed prion-like, intrinsically disordered or low-complexity (LC) domains. When incubated at high concentration, certain of these LC domains polymerize into labile, amyloid-like fibers. Here, we report methods allowing the generation of a molecular footprint of the polymeric state of the LC domain of hnRNPA2. By deploying this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, we offer evidence that its LC domain exists in a similar conformation as that described for recombinant polymers of the protein. These observations favor biologic utility to the polymerization of LC domains in the pathway of information transfer from gene to message to protein.

Phosphorylation-regulated binding of RNA polymerase II to fibrous polymers of low-complexity domains.

The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD.

Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies.

Cellular granules lacking boundary membranes harbor RNAs and their associated proteins and play diverse roles controlling the timing and location of protein synthesis. Formation of such granules was emulated by treatment of mouse brain extracts and human cell lysates with a biotinylated isoxazole (b-isox) chemical. Deep sequencing of the associated RNAs revealed an enrichment for mRNAs known to be recruited to neuronal granules used for dendritic transport and localized translation at synapses. Precipitated mRNAs contain extended 3' UTR sequences and an enrichment in binding sites for known granule-associated proteins. Hydrogels composed of the low complexity (LC) sequence domain of FUS recruited and retained the same mRNAs as were selectively precipitated by the b-isox chemical. Phosphorylation of the LC domain of FUS prevented hydrogel retention, offering a conceptual means of dynamic, signal-dependent control of RNA granule assembly.

Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.

Oxidative regulation of TDP-43 self-association by a ß-to-α conformational switch.

An evolutionarily conserved region of the TDP-43 low-complexity domain (LCD) twenty residues in length can adopt either an α-helical or ß-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-ß structure. Self-association can be monitored via the formation of phase-separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the LCD. Oxidation disassembles the cross-ß structure, thus eliminating both self-association and phase separation. Here, we demonstrate that this process reciprocally enables formation of α-helical structure in precisely the same region formerly functioning to facilitate ß-strand-mediated self-association. We further observe that the α-helical conformation allows interaction with a lipid-like detergent and that exposure to lipids enhances the ß-to-α conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen-bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of LCDs.

How do protein domains of low sequence complexity work?

This review covers research findings reported over the past decade concerning the ability of low complexity (LC) domains to self-associate in a manner leading to their phase separation from aqueous solution. We focus our message upon the reductionist use of two forms of phase separation as biochemical assays to study how LC domains might function in living cells. Cells and their varied compartments represent extreme examples of material condensates. Over the past half century, biochemists, structural biologists, and molecular biologists have resolved the mechanisms driving innumerable forms of macromolecular condensation. In contrast, we remain largely ignorant as to how 10%-20% of our proteins actually work to assist in cell organization. This enigmatic 10%-20% of the proteome corresponds to gibberish-like LC sequences. We contend that many of these LC sequences move in and out of a structurally ordered, self-associated state as a means of offering a combination of organizational specificity and dynamic pliability to living cells. Finally, we speculate that ancient proteins may have behaved similarly, helping to condense, organize, and protect RNA early during evolution.

Observation of the Liquid-Liquid Phase Separation of FUS-LC Using Vacuum-Ultraviolet Circular Dichroism Spectroscopy.

To reveal the structural mechanism by which the low-complexity domain of the fused in sarcoma protein (FUS-LC) mediates liquid-liquid phase separation (LLPS), we conducted a vacuum-ultraviolet circular dichroism (VUV-CD) spectroscopic study, a technique to analyze the secondary structures of proteins. The VUV-CD measurements were performed at the BL12 VUV-CD station at the Hiroshima Synchrotron Radiation Center (HiSOR) in Japan. CD spectra were measured between 180 and 260 nm while controlling the temperature of samples from 37°C to 5°C to obtain the LLPS of FUS-LC. The CD spectrum obtained at 37°C exhibited a large negative peak at 195 nm and a small negative shoulder near 220 nm. The peak intensity around 195 nm decreased as the sample temperature decreased. The spectral changes originated from the LLPS formation.

The low-complexity domain of the FUS RNA binding protein self-assembles via the mutually exclusive use of two distinct cross-ß cores.

The low-complexity (LC) domain of the fused in sarcoma (FUS) RNA binding protein self-associates in a manner causing phase separation from an aqueous environment. Incubation of the FUS LC domain under physiologically normal conditions of salt and pH leads to rapid formation of liquid-like droplets that mature into a gel-like state. Both examples of phase separation have enabled reductionist biochemical assays allowing discovery of an N-terminal region of 57 residues that assembles into a labile, cross-ß structure. Here we provide evidence of a nonoverlapping, C-terminal region of the FUS LC domain that also forms specific cross-ß interactions. We propose that biologic function of the FUS LC domain may operate via the mutually exclusive use of these N- and C-terminal cross-ß cores. Neurodegenerative disease-causing mutations in the FUS LC domain are shown to imbalance the two cross-ß cores, offering an unanticipated concept of LC domain function and dysfunction.

Transiently structured head domains control intermediate filament assembly.

Low complexity (LC) head domains 92 and 108 residues in length are, respectively, required for assembly of neurofilament light (NFL) and desmin intermediate filaments (IFs). As studied in isolation, these IF head domains interconvert between states of conformational disorder and labile, ß-strand-enriched polymers. Solid-state NMR (ss-NMR) spectroscopic studies of NFL and desmin head domain polymers reveal spectral patterns consistent with structural order. A combination of intein chemistry and segmental isotope labeling allowed preparation of fully assembled NFL and desmin IFs that could also be studied by ss-NMR. Assembled IFs revealed spectra overlapping with those observed for ß-strand-enriched polymers formed from the isolated NFL and desmin head domains. Phosphorylation and disease-causing mutations reciprocally alter NFL and desmin head domain self-association yet commonly impede IF assembly. These observations show how facultative structural assembly of LC domains via labile, ß-strand-enriched self-interactions may broadly influence cell morphology.

Redox-mediated regulation of an evolutionarily conserved cross-ß structure formed by the TDP43 low complexity domain.

A methionine-rich low complexity (LC) domain is found within a C-terminal region of the TDP43 RNA-binding protein. Self-association of this domain leads to the formation of labile cross-ß polymers and liquid-like droplets. Treatment with H2O2 caused phenomena of methionine oxidation and droplet melting that were reversed upon exposure of the oxidized protein to methionine sulfoxide reductase enzymes. Morphological features of the cross-ß polymers were revealed by H2O2-mediated footprinting. Equivalent TDP43 LC domain footprints were observed in polymerized hydrogels, liquid-like droplets, and living cells. The ability of H2O2 to impede cross-ß polymerization was abrogated by the prominent M337V amyotrophic lateral sclerosis-causing mutation. These observations may offer insight into the biological role of TDP43 in facilitating synapse-localized translation as well as aberrant aggregation of the protein in neurodegenerative diseases.

Dynamic structural order of a low-complexity domain facilitates assembly of intermediate filaments.

The coiled-coil domains of intermediate filament (IF) proteins are flanked by regions of low sequence complexity. Whereas IF coiled-coil domains assume dimeric and tetrameric conformations on their own, maturation of eight tetramers into cylindrical IFs is dependent on either "head" or "tail" domains of low sequence complexity. Here we confirm that the tail domain required for assembly of Drosophila Tm1-I/C IFs functions by forming labile cross-ß interactions. These interactions are seen in polymers made from the tail domain alone, as well as in assembled IFs formed by the intact Tm1-I/C protein. The ability to visualize such interactions in situ within the context of a discrete cellular assembly lends support to the concept that equivalent interactions may be used in organizing other dynamic aspects of cell morphology.

Dosimetric effect of six degrees of freedom couch top with rotational setup error corrections in proton therapy.

PURPOSE: To investigate the dosimetric effect of six degrees of freedom (6DoF) couch top with rotational corrections in proton therapy (PT). METHODS: The water equivalent thickness (WET) was measured using a proton beam with a 6DoF couch top and patient immobilization base plate (PIBP) placed in front of a motorized water phantom. The accuracy verification was performed with the beam axis set perpendicular to the 6DoF couch top and tilted in 10° steps from 10° to 30°. Up to 3° rotational correction may be added during the actual treatment to correct the rotational setup error on our system. The measured and calculated values using the treatment planning system were compared. Additionally, the effect of the 3° difference was evaluated using actual measurements concerning each angle on the proton beam range. RESULTS: The WET of the 6DoF couch top and PIBP were 8.5 ± 0.1 mm and 6.8 ± 0.1 mm, respectively. The calculation and the actual measurement at each angle agreed within 0.2 mm at the maximum. A maximum difference of approximately 0.6 mm was confirmed when tilted at 3° following 30° with the 6DoF couch top plus PIBP. CONCLUSIONS: The dosimetric effect of the 6DoF couch top with rotational corrections in PT differs depending on the incidence angle on the couch top, and it increased with the increased oblique angle of incidence. However, the effect on the range was as small as 0.6 mm at the maximum. The amount of rotational correction, the angle of incidence of the beam, and the effect of rotational corrections on the proton beam range may differ depending on the structure of the couch top. Therefore, sufficient prior confirmation, and subsequent periodical quality assurance management are important.

Pressure ulcer induced by discontinuation of levodopa: Case report of an older patient with Parkinson's disease.

We report the case of a pressure ulcer that developed consequent to the discontinuation of levodopa (L-3,4-dihydroxyphenylalanine) administration. The 86-year-old female patient had a 5-year history of Parkinson's disease treated with levodopa. She developed a sacral pressure ulcer due to unanticipated immobilization induced by the discontinuation of levodopa. Discontinuation of mandatory drugs is therefore a risk factor for the development of pressure ulcers in patients with Parkinson's disease.

Parametric wavelength conversion with bidirectional utilization of a multiple QPM device.

A configuration for wavelength conversion and optical amplification by parametric interaction using a nonlinear optical device is proposed. It enables pump generation through second harmonic generation (SHG), difference frequency generation (DFG), and optical parametric amplification (OPA) using a multiple-quasi-phase-matched (M-QPM) LiNbO3 waveguide in a bidirectional manner. Wavelength conversion for the 1.4-1.6 µm band is experimentally demonstrated. In addition, it is demonstrated that the parametric gain band can be changed using various detunings between the pump and QPM wavelengths used for the DFG/OPA process. The proposed method would be useful for enabling high-capacity optical transmission outside the 1550-nm band.

Structural characterization of the D290V mutation site in hnRNPA2 low-complexity-domain polymers.

Human genetic studies have given evidence of familial, disease-causing mutations in the analogous amino acid residue shared by three related RNA binding proteins causative of three neurological diseases. Alteration of aspartic acid residue 290 of hnRNPA2 to valine is believed to predispose patients to multisystem proteinopathy. Mutation of aspartic acid 262 of hnRNPA1 to either valine or asparagine has been linked to either amyotrophic lateral sclerosis or multisystem proteinopathy. Mutation of aspartic acid 378 of hnRNPDL to either asparagine or histidine has been associated with limb girdle muscular dystrophy. All three of these aspartic acid residues map to evolutionarily conserved regions of low-complexity (LC) sequence that may function in states of either intrinsic disorder or labile self-association. Here, we present a combination of solid-state NMR spectroscopy with segmental isotope labeling and electron microscopy on the LC domain of the hnRNPA2 protein. We show that, for both the wild-type protein and the aspartic acid 290-to-valine mutant, labile polymers are formed in which the LC domain associates into an in-register cross-ß conformation. Aspartic acid 290 is shown to be charged at physiological pH and immobilized within the polymer core. Polymers of the aspartic acid 290-to-valine mutant are thermodynamically more stable than wild-type polymers. These observations give evidence that removal of destabilizing electrostatic interactions may be responsible for the increased propensity of the mutated LC domains to self-associate in disease-causing conformations.