ABSTRACT
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.
Subject(s)
Membrane Proteins , trans-Golgi Network , Humans , Membrane Proteins/metabolism , Protein Transport/physiology , trans-Golgi Network/metabolismABSTRACT
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.
Subject(s)
Disease Progression , Prostatic Neoplasms, Castration-Resistant , Signal Transduction , Animals , Humans , Male , Mice , Cell Line, Tumor , Cell Proliferation , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/pathology , Receptors, Androgen/metabolism , Signal Transduction/drug effects , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Transcription factors are among the most attractive therapeutic targets but are considered largely 'undruggable' in part due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration-resistant prostate cancer, is key for its activity as transcription factor, allowing it to translocate to the nucleus and partition into transcriptional condensates upon activation by androgens. On the basis of our understanding of the interactions stabilizing such condensates and of the structure that the domain adopts upon condensation, we optimized the structure of a small-molecule inhibitor previously identified by phenotypic screening. The optimized compounds had more affinity for their target, inhibited androgen-receptor-dependent transcriptional programs, and had an antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo. These results suggest that it is possible to rationally optimize, and potentially even to design, small molecules that target the activation domains of oncogenic transcription factors.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Prostatic Neoplasms , Male , Humans , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Receptors, Androgen/genetics , Receptors, Androgen/chemistry , Androgens/therapeutic use , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Protein Domains , Transcription Factors , Cell Line, TumorABSTRACT
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.
Subject(s)
Caenorhabditis elegans Proteins , Touch , Animals , Touch/physiology , Caenorhabditis elegans Proteins/genetics , Mechanoreceptors/physiology , Connectin , Mechanotransduction, Cellular/physiology , Caenorhabditis elegans/genetics , Neurons , Membrane Proteins/physiologyABSTRACT
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.
Subject(s)
Chromatin , Gene Expression Regulation , Cell Nucleus , MacrophagesABSTRACT
Multiciliated cells (MCCs) project dozens to hundreds of motile cilia from their apical surface to promote the movement of fluids or gametes in the mammalian brain, airway or reproductive organs. Differentiation of MCCs requires the sequential action of the Geminin family transcriptional activators, GEMC1 and MCIDAS, that both interact with E2F4/5-DP1. How these factors activate transcription and the extent to which they play redundant functions remains poorly understood. Here, we demonstrate that the transcriptional targets and proximal proteomes of GEMC1 and MCIDAS are highly similar. However, we identified distinct interactions with SWI/SNF subcomplexes; GEMC1 interacts primarily with the ARID1A containing BAF complex while MCIDAS interacts primarily with BRD9 containing ncBAF complexes. Treatment with a BRD9 inhibitor impaired MCIDAS-mediated activation of several target genes and compromised the MCC differentiation program in multiple cell based models. Our data suggest that the differential engagement of distinct SWI/SNF subcomplexes by GEMC1 and MCIDAS is required for MCC-specific transcriptional regulation and mediated by their distinct C-terminal domains.
Subject(s)
Gene Expression Regulation , Nuclear Proteins , Animals , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Cell Differentiation/genetics , MammalsABSTRACT
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.
Subject(s)
Cell Nucleolus , HMGB1 Protein , Humans , Arginine/genetics , Arginine/metabolism , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Cell Nucleolus/pathology , HMGB1 Protein/chemistry , HMGB1 Protein/genetics , HMGB1 Protein/metabolism , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/metabolism , Syndrome , Frameshift Mutation , Phase TransitionABSTRACT
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.
Subject(s)
Amyotrophic Lateral Sclerosis , Muscular Diseases , Humans , Amyotrophic Lateral Sclerosis/genetics , Aspartic Acid/genetics , Motor Neurons/metabolism , Muscular Diseases/pathology , MutationABSTRACT
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.
Subject(s)
Calcium , Receptors, Androgen , Mice , Animals , Receptors, Androgen/chemistry , Gain of Function Mutation , Cyclin-Dependent Kinases/genetics , Phosphoric Monoester Hydrolases/geneticsABSTRACT
The binding of intrinsically disordered proteins to globular ones can require the folding of motifs into α-helices. These interactions offer opportunities for therapeutic intervention but their modulation with small molecules is challenging because they bury large surfaces. Linear peptides that display the residues that are key for binding can be targeted to globular proteins when they form stable helices, which in most cases requires their chemical modification. Here we present rules to design peptides that fold into single α-helices by instead concatenating glutamine side chain to main chain hydrogen bonds recently discovered in polyglutamine helices. The resulting peptides are uncharged, contain only natural amino acids, and their sequences can be optimized to interact with specific targets. Our results provide design rules to obtain single α-helices for a wide range of applications in protein engineering and drug design.
Subject(s)
Glutamine , Peptides , Protein Conformation, alpha-Helical , Amino Acid Sequence , Protein Structure, Secondary , Peptides/chemistryABSTRACT
Intrinsically disordered proteins, which do not adopt well-defined structures under physiological conditions, are implicated in many human diseases. Small molecules that target the disordered transactivation domain of the androgen receptor have entered human trials for the treatment of castration-resistant prostate cancer (CRPC), but no structural or mechanistic rationale exists to explain their inhibition mechanisms or relative potencies. Here, we utilize all-atom molecular dynamics computer simulations to elucidate atomically detailed binding mechanisms of the compounds EPI-002 and EPI-7170 to the androgen receptor. Our simulations reveal that both compounds bind at the interface of two transiently helical regions and induce the formation of partially folded collapsed helical states. We find that EPI-7170 binds androgen receptor more tightly than EPI-002 and we identify a network of intermolecular interactions that drives higher affinity binding. Our results suggest strategies for developing more potent androgen receptor inhibitors and general strategies for disordered protein drug design.
Subject(s)
Prostatic Neoplasms, Castration-Resistant , Receptors, Androgen , Male , Humans , Receptors, Androgen/metabolism , Transcriptional Activation , Androgen Receptor Antagonists/pharmacology , Androgen Receptor Antagonists/therapeutic use , Benzhydryl Compounds/pharmacology , Prostatic Neoplasms, Castration-Resistant/drug therapyABSTRACT
The regular functioning of the nucleolus and nucleus-mitochondria crosstalk are considered unrelated processes, yet cytochrome c (Cc) migrates to the nucleus and even the nucleolus under stress conditions. Nucleolar liquid-liquid phase separation usually serves the cell as a fast, smart mechanism to control the spatial localization and trafficking of nuclear proteins. Actually, the alternative reading frame (ARF), a tumor suppressor protein sequestered by nucleophosmin (NPM) in the nucleoli, is shifted out from NPM upon DNA damage. DNA damage also triggers early translocation of respiratory Cc to nucleus before cytoplasmic caspase activation. Here, we show that Cc can bind to nucleolar NPM by triggering an extended-to-compact conformational change, driving ARF release. Such a NPM-Cc nucleolar interaction can be extended to a general mechanism for DNA damage in which the lysine-rich regions of Cc-rather than the canonical, arginine-rich stretches of membrane-less organelle components-controls the trafficking and availability of nucleolar proteins.
Subject(s)
Cytochromes c , Nucleophosmin , Arginine , Caspases , Lysine , Mitochondria/metabolism , Nuclear Proteins/metabolism , Tumor Suppressor ProteinsABSTRACT
Recombinant protein expression in E. coli often induces the expressed protein to accumulate in insoluble aggregates, named inclusion bodies (IBs), that represent easy to isolate, highly pure protein reservoirs. IBs can be solubilized by denaturing agents but this procedure requires, for complex globular proteins, a refolding step that can be challenging. However, the lack of cooperatively folded tertiary structure in intrinsically disordered proteins (IDP) makes them ideal candidates for this purification strategy. Given the wide abundance of IDPs, their relevance in many disease areas and the numerous IDP-associated biological functions, the interest in this class of proteins has increased substantially over the last decade. Here we present a broad and versatile method for the production and isolation of IDPs from inclusion bodies under denaturant conditions that overcomes the challenges associated with the propensity of these sequences to precipitate from solution and becoming proteolytically degraded.
Subject(s)
Intrinsically Disordered Proteins , Escherichia coli/genetics , Escherichia coli/metabolism , Inclusion Bodies/metabolism , Intrinsically Disordered Proteins/chemistry , Recombinant Proteins/chemistryABSTRACT
NAD metabolism is essential for all forms of life. Compartmental regulation of NAD+ consumption, especially between the nucleus and the mitochondria, is required for energy homeostasis. However, how compartmental regulation evolved remains unclear. In the present study, we investigated the evolution of the macrodomain-containing histone variant macroH2A1.1, an integral chromatin component that limits nuclear NAD+ consumption by inhibiting poly(ADP-ribose) polymerase 1 in vertebrate cells. We found that macroH2A originated in premetazoan protists. The crystal structure of the macroH2A macrodomain from the protist Capsaspora owczarzaki allowed us to identify highly conserved principles of ligand binding and pinpoint key residue substitutions, selected for during the evolution of the vertebrate stem lineage. Metabolic characterization of the Capsaspora lifecycle suggested that the metabolic function of macroH2A was associated with nonproliferative stages. Taken together, we provide insight into the evolution of a chromatin element involved in compartmental NAD regulation, relevant for understanding its metabolism and potential therapeutic applications.
Subject(s)
Energy Metabolism/physiology , Histones/genetics , Histones/metabolism , NAD/metabolism , Cell Nucleus/metabolism , Chromatin/metabolism , DNA Repair/genetics , Eukaryota/metabolism , Humans , Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitorsABSTRACT
The phase equilibria of intrinsically disordered proteins are exquisitely sensitive to changes in solution conditions and this can be used to investigate the driving forces of phase separation in vitro as well as the biological roles of phase transitions in live cells. Here we investigate how using D2 O as co-solvent in an aqueous buffer changes the phase equilibrium of a fragment of the activation domain of the androgen receptor, a transcription factor that plays a role in the development of the male phenotype and is a therapeutic target for castration resistant prostate cancer. We show how replacing even small fractions of H2 O with D2 O increases the propensity of this fragment to undergo liquid-liquid phase separation, likely reflecting a stabilization of the hydrophobic interactions that drive condensation. Our results indicate that it is necessary to take this effect into consideration when studying phase separation phenomena with biophysical methods that require using D2 O as a co-solvent. In addition, they suggest that additions of D2 O may be used to enhance phase separation phenomena in cells, facilitating their observation.
Subject(s)
Deuterium Oxide/chemistry , Intrinsically Disordered Proteins/chemistry , Receptors, Androgen/chemistry , Humans , Protein DomainsABSTRACT
Intrinsically disordered domains represent attractive therapeutic targets because they play key roles in cancer, as well as in neurodegenerative and infectious diseases. They are, however, considered undruggable because they do not form stable binding pockets for small molecules and, therefore, have not been prioritized in drug discovery. Under physiological solution conditions many biomedically relevant intrinsically disordered proteins undergo phase separation processes leading to the formation of mesoscopic highly dynamic assemblies, generally known as biomolecular condensates that define environments that can be quite different from the solutions surrounding them. In what follows, we review key recent findings in this area and show how biomolecular condensation can offer opportunities for modulating the activities of intrinsically disordered targets.
Subject(s)
Intrinsically Disordered Proteins/metabolism , Pharmaceutical Preparations/chemistry , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Drug Discovery , Humans , Phase Transition/drug effects , Protein Binding , Protein Multimerization/drug effects , Pyrimidines/pharmacology , Structure-Activity Relationship , Thermodynamics , Transition TemperatureABSTRACT
Biomolecular condensates are mesoscopic biomolecular assemblies devoid of long range order that contribute to important cellular functions. They form reversibly, are stabilized by numerous but relatively weak intermolecular interactions, and their formation can be regulated by various cellular signals including changes in local concentration, post-translational modifications, energy-consuming processes, and biomolecular interactions. Condensates formed by liquid-liquid phase separation are initially liquid but are metastable relative to hydrogels or irreversible solids that have been associated with protein aggregation diseases and are stabilized by stronger, more permanent interactions. As a consequence of this, a series of cellular mechanisms are available to regulate not only biomolecular condensation but also the physical properties of the condensates.
Subject(s)
Signal Transduction , Protein Processing, Post-Translational , ProteinsABSTRACT
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.
Subject(s)
Nanoparticles/chemistry , Peptide Fragments/chemistry , alpha-Synuclein/chemistry , Adhesiveness , Adsorption , Amino Acid Sequence , Protein Domains , Surface PropertiesABSTRACT
Synucleinopathies are a group of disorders characterized by the accumulation of α-Synuclein amyloid inclusions in the brain. Preventing α-Synuclein aggregation is challenging because of the disordered nature of the protein and the stochastic nature of fibrillogenesis, but, at the same time, it is a promising approach for therapeutic intervention in these pathologies. A high-throughput screening initiative allowed us to discover ZPDm, the smallest active molecule in a library of more than 14.000 compounds. Although the ZPDm structure is highly related to that of the previously described ZPD-2 aggregation inhibitor, we show here that their mechanisms of action are entirely different. ZPDm inhibits the aggregation of wild-type, A30P, and H50Q α-Synuclein variants in vitro and interferes with α-Synuclein seeded aggregation in protein misfolding cyclic amplification assays. However, ZPDm distinctive feature is its strong potency to dismantle preformed α-Synuclein amyloid fibrils. Studies in a Caenorhabditis elegans model of Parkinson's Disease, prove that these in vitro properties are translated into a significant reduction in the accumulation of α-Synuclein inclusions in ZPDm treated animals. Together with previous data, the present work illustrates how different chemical groups on top of a common molecular scaffold can result in divergent but complementary anti-amyloid activities.
ABSTRACT
High solvent exposure of certain sequences located in intrinsically disordered regions (IDRs) may eventually lead to aggregation, as is the case for some low-complexity regions (LCRs) and short linear motifs (SLiMs). In particular, polyglutamine (polyQ) tracts are LCRs of variable length highly enriched in glutamine residues. They are common in transcription factors, and their length can have an impact on transcriptional activity. In nine proteins, polyQ tract expansions beyond specific thresholds cause nine neurodegenerative diseases, and aggregates formed by the protein harboring the polyQ tract can be detected in affected individuals. A structural characterization of polyQ proteins in their monomeric form is key to understand how their expansion can affect their aggregation propensity. In this regard, nuclear magnetic resonance (NMR) spectroscopy can provide high-resolution structural information. Here, we present a protocol to prepare monomeric samples of isotope-enriched short helical polyQ peptides based on the sequence of the androgen receptor (AR) suitable for NMR characterization and suggest different ways to adapt it for the production and monomerization of other relatively short IDR sequences and SLiMs.