Aberrant phase separation and nucleolar dysfunction in rare genetic diseases.

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.

Cancer Mutations of the Tumor Suppressor SPOP Disrupt the Formation of Active, Phase-Separated Compartments.

Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.

Small molecule inhibits α-synuclein aggregation, disrupts amyloid fibrils, and prevents degeneration of dopaminergic neurons.

Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons, a process that current therapeutic approaches cannot prevent. In PD, the typical pathological hallmark is the accumulation of intracellular protein inclusions, known as Lewy bodies and Lewy neurites, which are mainly composed of α-synuclein. Here, we exploited a high-throughput screening methodology to identify a small molecule (SynuClean-D) able to inhibit α-synuclein aggregation. SynuClean-D significantly reduces the in vitro aggregation of wild-type α-synuclein and the familiar A30P and H50Q variants in a substoichiometric molar ratio. This compound prevents fibril propagation in protein-misfolding cyclic amplification assays and decreases the number of α-synuclein inclusions in human neuroglioma cells. Computational analysis suggests that SynuClean-D can bind to cavities in mature α-synuclein fibrils and, indeed, it displays a strong fibril disaggregation activity. The treatment with SynuClean-D of two PD Caenorhabditis elegans models, expressing α-synuclein either in muscle or in dopaminergic neurons, significantly reduces the toxicity exerted by α-synuclein. SynuClean-D-treated worms show decreased α-synuclein aggregation in muscle and a concomitant motility recovery. More importantly, this compound is able to rescue dopaminergic neurons from α-synuclein-induced degeneration. Overall, SynuClean-D appears to be a promising molecule for therapeutic intervention in Parkinson's disease.

An Adhesive Peptide from the C-Terminal Domain of α-Synuclein for Single-Layer Adsorption of Nanoparticles onto Substrates.

The two-dimensional (2D) homogeneous assembly of nanoparticle monolayer arrays onto a broad range of substrates constitutes an important challenge for chemistry, nanotechnology, and material science. α-Synuclein (αS) is an intrinsically disordered protein associated with neuronal protein complexes and has a high degree of structural plasticity and chaperone activity. The C-terminal domain of αS has been linked to the noncovalent interactions of this protein with biological targets and the activity of αS in presynaptic connections. Herein, we have systematically studied peptide fragments of the chaperone-active C-terminal sequence of αS and identified a 17-residue peptide that preserves the versatile binding nature of αS. Attachment of this short peptide to gold nanoparticles afforded colloidally stable nanoparticle suspensions that allowed the homogeneous 2D adhesion of the conjugates onto a wide variety of surfaces, including the formation of crystalline nanoparticle superlattices. The peptide sequence and the strategy reported here describe a new adhesive molecule for the controlled monolayer adhesion of metal nanoparticles and sets a stepping-stone toward the potential application of the adhesive properties of αS.

Utilizing dipole-dipole cross-correlated relaxation for the measurement of angles between pairs of opposing CαHα-CαHα bonds in anti-parallel ß-sheets.

Dipole-dipole cross-correlated relaxation (CCR) between two spin pairs is rich with macromolecular structural and dynamic information on inter-nuclear bond vectors. Measurement of short range dipolar CCR rates has been demonstrated for a variety of inter-nuclear vector spin pairs in proteins and nucleic acids, where the multiple quantum coherence necessary for observing the CCR rate is created by through-bond scalar coupling. In principle, CCR rates can be measured for any pair of inter-nuclear vectors where coherence can be generated between one spin of each spin pair, regardless of both the distance between the two spin pairs and the distance of the two spins forming the multiple quantum coherence. In practice, however, long range CCR (lrCCR) rates are challenging to measure due to difficulties in linking spatially distant spin pairs. By utilizing through-space relaxation allowed coherence transfer (RACT), we have developed a new method for the measurement of lrCCR rates involving CαHα bonds on opposing anti-parallel ß-strands. The resulting lrCCR rates are straightforward to interpret since only the angle between the two vectors modulates the strength of the interference effect. We applied our lrCCR measurement to the third immunoglobulin-binding domain of the streptococcal protein G (GB3) and utilize published NMR ensembles and static NMR/X-ray structures to highlight the relationship between the lrCCR rates and the CαHα-CαHα inter-bond angle and bond mobility. Furthermore, we employ the lrCCR rates to guide the selection of sub-ensembles from the published NMR ensembles for enhancing the structural and dynamic interpretation of the data. We foresee this methodology for measuring lrCCR rates as improving the generation of structural ensembles by providing highly accurate details concerning the orientation of CαHα bonds on opposing anti-parallel ß-strands.

Sequence Context Influences the Structure and Aggregation Behavior of a PolyQ Tract.

Expansions of polyglutamine (polyQ) tracts in nine different proteins cause a family of neurodegenerative disorders called polyQ diseases. Because polyQ tracts are potential therapeutic targets for these pathologies there is great interest in characterizing the conformations that they adopt and in understanding how their aggregation behavior is influenced by the sequences flanking them. We used solution NMR to study at single-residue resolution a 156-residue proteolytic fragment of the androgen receptor that contains a polyQ tract associated with the disease spinobulbar muscular atrophy, also known as Kennedy disease. Our findings indicate that a Leu-rich region preceding the polyQ tract causes it to become α-helical and appears to protect the protein against aggregation, which represents a new, to our knowledge, mechanism by which sequence context can minimize the deleterious properties of these repetitive regions. Our results have implications for drug discovery for polyQ diseases because they suggest that the residues flanking these repetitive sequences may represent viable therapeutic targets.

Toward an atomistic description of the urea-denatured state of proteins.

We present here the characterization of the structural, dynamics, and energetics of properties of the urea-denatured state of ubiquitin, a small prototypical soluble protein. By combining state-of-the-art molecular dynamics simulations with NMR and small-angle X-ray scattering data, we were able to: (i) define the unfolded state ensemble, (ii) understand the energetics stabilizing unfolded structures in urea, (iii) describe the dedifferential nature of the interactions of the fully unfolded proteins with urea and water, and (iv) characterize the early stages of protein refolding when chemically denatured proteins are transferred to native conditions. The results presented herein are unique in providing a complete picture of the chemically unfolded state of proteins and contribute to deciphering the mechanisms that stabilize the native state of proteins, as well as those that maintain them unfolded in the presence of urea.

Kinetics of the Antibody Recognition Site in the Third IgG-Binding Domain of Protein G.

Protein dynamics occurring on a wide range of timescales play a crucial role in governing protein function. Particularly, motions between the globular rotational correlation time (τc ) and 40 µs (supra-τc window), strongly influence molecular recognition. This supra-τc window was previously hidden, owing to a lack of experimental methods. Recently, we have developed a high-power relaxation dispersion (RD) experiment for measuring kinetics as fast as 4 µs. For the first time, this method, performed under super-cooled conditions, enabled us to detect a global motion in the first ß-turn of the third IgG-binding domain of protein G (GB3), which was extrapolated to 371±115 ns at 310 K. Furthermore, the same residues show the plasticity in the model-free residual dipolar coupling (RDC) order parameters and in an ensemble encoding the supra-τc dynamics. This ß-turn is involved in antibody binding, exhibiting the potential link of the observed supra-τc motion with molecular recognition.

Structure-Free Validation of Residual Dipolar Coupling and Paramagnetic Relaxation Enhancement Measurements of Disordered Proteins.

Residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs) have emerged as valuable parameters for defining the structures and dynamics of disordered proteins by nuclear magnetic resonance (NMR) spectroscopy. Procedures for their measurement, however, may lead to conformational perturbations because of the presence of the alignment media necessary for recording RDCs, or of the paramagnetic groups that must be introduced for measuring PREs. We discuss here experimental methods for quantifying these effects by considering the case of the 40-residue isoform of the amyloid ß peptide (Aß40), which is associated with Alzheimer's disease. By conducting RDC measurements over a range of concentrations of certain alignment media, we show that perturbations arising from transient binding of Aß40 can be characterized, allowing appropriate corrections to be made. In addition, by using NMR experiments sensitive to long-range interactions, we show that it is possible to identify relatively nonperturbing sites for attaching nitroxide radicals for PRE measurements. Thus, minimizing the conformational perturbations introduced by RDC and PRE measurements should facilitate their use for the rigorous determination of the conformational properties of disordered proteins.

Correlated inter-domain motions in adenylate kinase.

Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK), using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs) measured under steric alignment by nuclear magnetic resonance (NMR). We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes.

Human serum albumin, systemic inflammation, and cirrhosis.

Human serum albumin (HSA) is one of the most frequent treatments in patients with decompensated cirrhosis. Prevention of paracentesis-induced circulatory dysfunction, prevention of type-1 HRS associated with bacterial infections, and treatment of type-1 hepatorenal syndrome are the main indications. In these indications treatment with HSA is associated with improvement in survival. Albumin is a stable and very flexible molecule with a heart shape, 585 residues, and three domains of similar size, each one containing two sub-domains. Many of the physiological functions of HSA rely on its ability to bind an extremely wide range of endogenous and exogenous ligands, to increase their solubility in plasma, to transport them to specific tissues and organs, or to dispose of them when they are toxic. The chemical structure of albumin can be altered by some specific processes (oxidation, glycation) leading to rapid clearance and catabolism. An outstanding feature of HSA is its capacity to bind lipopolysaccharide and other bacterial products (lipoteichoic acid and peptidoglycan), reactive oxygen species, nitric oxide and other nitrogen reactive species, and prostaglandins. Binding to NO and prostaglandins are reversible, so they can be transferred to other molecules at different sites from their synthesis. Through these functions, HSA modulates the inflammatory reaction. Decompensated cirrhosis is a disease associated systemic inflammation, which plays an important role in the pathogenesis of organ or system dysfunction/failure. Although, the beneficial effects of HAS have been traditionally attributed to plasma volume expansion, they could also relate to its effects modulating systemic and organ inflammation.

Sorting of secretory proteins at the trans-Golgi network by human TGN46.

Secretory proteins are sorted at the trans-Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. Here, we identified the human transmembrane protein TGN46 as a receptor for the export of secretory cargo protein PAUF in CARTS - a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the lumenal domain of TGN46 encodes for its cargo sorting function. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN.

Thio-2 Inhibits Key Signaling Pathways Required for the Development and Progression of Castration-resistant Prostate Cancer.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BCL-2-associated athanogene-1 (BAG-1) mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies, we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited "on-target" toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment-resistant prostate cancer cell lines and patient-derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation as the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2-mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC.

Identification of fibril-like tertiary contacts in soluble monomeric α-synuclein.

Structural conversion of the presynaptic, intrinsically disordered protein α-synuclein into amyloid fibrils underlies neurotoxicity in Parkinson's disease. The detailed mechanism by which this conversion occurs is largely unknown. Here, we identify a discrete pattern of transient tertiary interactions in monomeric α-synuclein involving amino acid residues that are, in the fibrillar state, part of ß-strands. Importantly, this pattern of pairwise interactions does not correspond to that found in the amyloid state. A redistribution of this network of fibril-like contacts must precede aggregation into the amyloid structure.

Electrostatic effects in filamentous protein aggregation.

Electrostatic forces play a key role in mediating interactions between proteins. However, gaining quantitative insights into the complex effects of electrostatics on protein behavior has proved challenging, due to the wide palette of scenarios through which both cations and anions can interact with polypeptide molecules in a specific manner or can result in screening in solution. In this article, we have used a variety of biophysical methods to probe the steady-state kinetics of fibrillar protein self-assembly in a highly quantitative manner to detect how it is modulated by changes in solution ionic strength. Due to the exponential modulation of the reaction rate by electrostatic forces, this reaction represents an exquisitely sensitive probe of these effects in protein-protein interactions. Our approach, which involves a combination of experimental kinetic measurements and theoretical analysis, reveals a hierarchy of electrostatic effects that control protein aggregation. Furthermore, our results provide a highly sensitive method for the estimation of the magnitude of binding of a variety of ions to protein molecules.

Average conformations determined from PRE data provide high-resolution maps of transient tertiary interactions in disordered proteins.

In the last decade it has become evident that disordered states of proteins play important physiological and pathological roles and that the transient tertiary interactions often present in these systems can play a role in their biological activity. The structural characterization of such states has so far largely relied on ensemble representations, which in principle account for both their local and global structural features. However, these approaches are inherently of low resolution due to the large number of degrees of freedom of conformational ensembles and to the sparse nature of the experimental data used to determine them. Here, we overcome these limitations by showing that tertiary interactions in disordered states can be mapped at high resolution by fitting paramagnetic relaxation enhancement data to a small number of conformations, which can be as low as one. This result opens up the possibility of determining the topology of cooperatively collapsed and hidden folded states when these are present in the vast conformational landscape accessible to disordered states of proteins. As a first application, we study the long-range tertiary interactions of acid-unfolded apomyoglobin from experimentally measured paramagnetic relaxation enhancement data.

Common pathophysiology for ANXA11 disorders caused by aspartate 40 variants.

OBJECTIVE: Mutations in ANXA11 cause amyotrophic lateral sclerosis (ALS) and have recently been identified as a cause of multisystem proteinopathy and adult-onset muscular dystrophy. These conditions are adult-onset diseases and result from the substitution of Aspartate 40 (Asp40) for an apolar residue in the intrinsically disordered domain (IDD) of ANXA11. Some ALS-related variants are known to affect ANXA11 IDD; however, the mechanism by which the myopathy occurs is unknown. METHODS: Genetic analysis was performed using WES-trio. For the study of variant pathogenicity, we used recombinant proteins, muscle biopsy, and fibroblasts. RESULTS: Here we describe an individual with severe and rapidly progressive childhood-onset oculopharyngeal muscular dystrophy who carries a new ANXA11 variant at position Asp40 (p.Asp40Ile; c.118_119delGAinsAT). p.Asp40Ile is predicted to enhance the aggregation propensity of ANXA11 to a greater extent than other changes affecting this residue. In vitro studies using recombinant ANXA11p.Asp40Ile showed abnormal phase separation and confirmed this variant is more aggregation-prone than the ALS-associated variant ANXA11p.Asp40Gly . The study of the patient's fibroblasts revealed defects in stress granules dynamics and clearance, and muscle histopathology showed a myopathic pattern with ANXA11 protein aggregates. Super-resolution imaging showed aggregates expressed as pearl strips or large complex structures in the sarcoplasm, and as layered subsarcolemmal chains probably reflecting ANXA11 multifunctionality. INTERPRETATION: We demonstrate common pathophysiology for disorders associated with ANXA11 Asp40 allelic variants. Clinical phenotypes may result from different deleterious impacts of variants upon ANXA11 stability against aggregation, and differential muscle or motor neuron dysfunction expressed as a temporal and tissue-specific continuum.

A MEC-2/stomatin condensate liquid-to-solid phase transition controls neuronal mechanotransduction during touch sensing.

A growing body of work suggests that the material properties of biomolecular condensates ensuing from liquid-liquid phase separation change with time. How this aging process is controlled and whether the condensates with distinct material properties can have different biological functions is currently unknown. Using Caenorhabditis elegans as a model, we show that MEC-2/stomatin undergoes a rigidity phase transition from fluid-like to solid-like condensates that facilitate transport and mechanotransduction, respectively. This switch is triggered by the interaction between the SH3 domain of UNC-89 (titin/obscurin) and MEC-2. We suggest that this rigidity phase transition has a physiological role in frequency-dependent force transmission in mechanosensitive neurons during body wall touch. Our data demonstrate a function for the liquid and solid phases of MEC-2/stomatin condensates in facilitating transport or mechanotransduction, and a previously unidentified role for titin homologues in neurons.

CEBPA phase separation links transcriptional activity and 3D chromatin hubs.

Cell identity is orchestrated through an interplay between transcription factor (TF) action and genome architecture. The mechanisms used by TFs to shape three-dimensional (3D) genome organization remain incompletely understood. Here we present evidence that the lineage-instructive TF CEBPA drives extensive chromatin compartment switching and promotes the formation of long-range chromatin hubs during induced B cell-to-macrophage transdifferentiation. Mechanistically, we find that the intrinsically disordered region (IDR) of CEBPA undergoes in vitro phase separation (PS) dependent on aromatic residues. Both overexpressing B cells and native CEBPA-expressing cell types such as primary granulocyte-macrophage progenitors, liver cells, and trophectoderm cells reveal nuclear CEBPA foci and long-range 3D chromatin hubs at CEBPA-bound regions. In short, we show that CEBPA can undergo PS through its IDR, which may underlie in vivo foci formation and suggest a potential role of PS in regulating CEBPA function.

Antagonistic effect of cyclin-dependent kinases and a calcium-dependent phosphatase on polyglutamine-expanded androgen receptor toxic gain of function.

Spinal and bulbar muscular atrophy is caused by polyglutamine (polyQ) expansions in androgen receptor (AR), generating gain-of-function toxicity that may involve phosphorylation. Using cellular and animal models, we investigated what kinases and phosphatases target polyQ-expanded AR, whether polyQ expansions modify AR phosphorylation, and how this contributes to neurodegeneration. Mass spectrometry showed that polyQ expansions preserve native phosphorylation and increase phosphorylation at conserved sites controlling AR stability and transactivation. In small-molecule screening, we identified that CDC25/CDK2 signaling could enhance AR phosphorylation, and the calcium-sensitive phosphatase calcineurin had opposite effects. Pharmacologic and genetic manipulation of these kinases and phosphatases modified polyQ-expanded AR function and toxicity in cells, flies, and mice. Ablation of CDK2 reduced AR phosphorylation in the brainstem and restored expression of Myc and other genes involved in DNA damage, senescence, and apoptosis, indicating that the cell cycle-regulated kinase plays more than a bystander role in SBMA-vulnerable postmitotic cells.