Mechanism of orphan subunit recognition during assembly quality control.

Cells contain numerous abundant molecular machines assembled from multiple subunits. Imbalances in subunit production and failed assembly generate orphan subunits that are eliminated by poorly defined pathways. Here, we determined how orphan subunits of the cytosolic chaperonin CCT are recognized. Several unassembled CCT subunits recruited the E3 ubiquitin ligase HERC2 using ZNRD2 as an adaptor. Both factors were necessary for orphan CCT subunit degradation in cells, sufficient for CCT subunit ubiquitination with purified factors, and necessary for optimal cell fitness. Domain mapping and structure prediction defined the molecular features of a minimal HERC2-ZNRD2-CCT module. The structural model, whose key elements were validated in cells using point mutants, shows why ZNRD2 selectively recognizes multiple orphaned CCT subunits without engaging assembled CCT. Our findings reveal how failures during CCT assembly are monitored and provide a paradigm for the molecular recognition of orphan subunits, the largest source of quality control substrates in cells.

Mechanism of ribosome-associated mRNA degradation during tubulin autoregulation.

Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.

TAF15 amyloid filaments in frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) causes frontotemporal dementia (FTD), the most common form of dementia after Alzheimer's disease, and is often also associated with motor disorders1. The pathological hallmarks of FTLD are neuronal inclusions of specific, abnormally assembled proteins2. In the majority of cases the inclusions contain amyloid filament assemblies of TAR DNA-binding protein 43 (TDP-43) or tau, with distinct filament structures characterizing different FTLD subtypes3,4. The presence of amyloid filaments and their identities and structures in the remaining approximately 10% of FTLD cases are unknown but are widely believed to be composed of the protein fused in sarcoma (FUS, also known as translocated in liposarcoma). As such, these cases are commonly referred to as FTLD-FUS. Here we used cryogenic electron microscopy (cryo-EM) to determine the structures of amyloid filaments extracted from the prefrontal and temporal cortices of four individuals with FTLD-FUS. Surprisingly, we found abundant amyloid filaments of the FUS homologue TATA-binding protein-associated factor 15 (TAF15, also known as TATA-binding protein-associated factor 2N) rather than of FUS itself. The filament fold is formed from residues 7-99 in the low-complexity domain (LCD) of TAF15 and was identical between individuals. Furthermore, we found TAF15 filaments with the same fold in the motor cortex and brainstem of two of the individuals, both showing upper and lower motor neuron pathology. The formation of TAF15 amyloid filaments with a characteristic fold in FTLD establishes TAF15 proteinopathy in neurodegenerative disease. The structure of TAF15 amyloid filaments provides a basis for the development of model systems of neurodegenerative disease, as well as for the design of diagnostic and therapeutic tools targeting TAF15 proteinopathy.

Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP type C.

Neurodegenerative diseases are characterized by the abnormal filamentous assembly of specific proteins in the central nervous system1. Human genetic studies have established a causal role for protein assembly in neurodegeneration2. However, the underlying molecular mechanisms remain largely unknown, which is limiting progress in developing clinical tools for these diseases. Recent advances in cryo-electron microscopy have enabled the structures of the protein filaments to be determined from the brains of patients1. All neurodegenerative diseases studied to date have been characterized by the self-assembly of proteins in homomeric amyloid filaments, including that of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) types A and B3,4. Here we used cryo-electron microscopy to determine filament structures from the brains of individuals with FTLD-TDP type C, one of the most common forms of sporadic FTLD-TDP. Unexpectedly, the structures revealed that a second protein, annexin A11 (ANXA11), co-assembles with TDP-43 in heteromeric amyloid filaments. The ordered filament fold is formed by TDP-43 residues G282/G284-N345 and ANXA11 residues L39-Y74 from their respective low-complexity domains. Regions of TDP-43 and ANXA11  that were previously implicated in protein-protein interactions form an extensive hydrophobic interface at the centre of the filament fold. Immunoblots of the filaments revealed that the majority of ANXA11 exists as an approximately 22 kDa N-terminal fragment lacking the annexin core domain. Immunohistochemistry of brain sections showed the colocalization of ANXA11 and TDP-43 in inclusions, redefining the histopathology of FTLD-TDP type C. This work establishes a central role for ANXA11 in FTLD-TDP type C. The unprecedented formation of heteromeric amyloid filaments in the human brain revises our understanding of amyloid assembly and may be of significance for the pathogenesis of neurodegenerative diseases.

TDP-43 forms amyloid filaments with a distinct fold in type A FTLD-TDP.

The abnormal assembly of TAR DNA-binding protein 43 (TDP-43) in neuronal and glial cells characterizes nearly all cases of amyotrophic lateral sclerosis (ALS) and around half of cases of frontotemporal lobar degeneration (FTLD)1,2. A causal role for TDP-43 assembly in neurodegeneration is evidenced by dominantly inherited missense mutations in TARDBP, the gene encoding TDP-43, that promote assembly and give rise to ALS and FTLD3-7. At least four types (A-D) of FTLD with TDP-43 pathology (FTLD-TDP) are defined by distinct brain distributions of assembled TDP-43 and are associated with different clinical presentations of frontotemporal dementia8. We previously showed, using cryo-electron microscopy, that TDP-43 assembles into amyloid filaments in ALS and type B FTLD-TDP9. However, the structures of assembled TDP-43 in FTLD without ALS remained unknown. Here we report the cryo-electron microscopy structures of assembled TDP-43 from the brains of three individuals with the most common type of FTLD-TDP, type A. TDP-43 formed amyloid filaments with a new fold that was the same across individuals, indicating that this fold may characterize type A FTLD-TDP. The fold resembles a chevron badge and is unlike the double-spiral-shaped fold of ALS and type B FTLD-TDP, establishing that distinct filament folds of TDP-43 characterize different neurodegenerative conditions. The structures, in combination with mass spectrometry, led to the identification of two new post-translational modifications of assembled TDP-43, citrullination and monomethylation of R293, and indicate that they may facilitate filament formation and observed structural variation in individual filaments. The structures of TDP-43 filaments from type A FTLD-TDP will guide mechanistic studies of TDP-43 assembly, as well as the development of diagnostic and therapeutic compounds for TDP-43 proteinopathies.

Macromolecular condensation buffers intracellular water potential.

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

Circadian regulation of macromolecular complex turnover and proteome renewal.

Although costly to maintain, protein homeostasis is indispensable for normal cellular function and long-term health. In mammalian cells and tissues, daily variation in global protein synthesis has been observed, but its utility and consequences for proteome integrity are not fully understood. Using several different pulse-labelling strategies, here we gain direct insight into the relationship between protein synthesis and abundance proteome-wide. We show that protein degradation varies in-phase with protein synthesis, facilitating rhythms in turnover rather than abundance. This results in daily consolidation of proteome renewal whilst minimising changes in composition. Coupled rhythms in synthesis and turnover are especially salient to the assembly of macromolecular protein complexes, particularly the ribosome, the most abundant species of complex in the cell. Daily turnover and proteasomal degradation rhythms render cells and mice more sensitive to proteotoxic stress at specific times of day, potentially contributing to daily rhythms in the efficacy of proteasomal inhibitors against cancer. Our findings suggest that circadian rhythms function to minimise the bioenergetic cost of protein homeostasis through temporal consolidation of protein turnover.

Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response.

Halofuginone (HF) is a phase 2 clinical compound that inhibits the glutamyl-prolyl-tRNA synthetase (EPRS) thereby inducing the integrated stress response (ISR). Here, we report that halofuginone indeed triggers the predicted canonical ISR adaptations, consisting of attenuation of protein synthesis and gene expression reprogramming. However, the former is surprisingly atypical and occurs to a similar magnitude in wild-type cells, cells lacking GCN2 and those incapable of phosphorylating eIF2α. Proline supplementation rescues the observed HF-induced changes indicating that they result from inhibition of EPRS. The failure of the GCN2-to-eIF2α pathway to elicit a measurable protective attenuation of translation initiation allows translation elongation defects to prevail upon HF treatment. Exploiting this vulnerability of the ISR, we show that cancer cells with increased proline dependency are more sensitive to halofuginone. This work reveals that the consequences of EPRS inhibition are more complex than anticipated and provides novel insights into ISR signaling, as well as a molecular framework to guide the targeted development of halofuginone as a therapeutic.

CRYPTOCHROMES promote daily protein homeostasis.

The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock-controlled protein expression, driven by daily cycles of CRYPTOCHROME-dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild-type and CRY-deficient fibroblasts under constant conditions. In CRY-deficient cells, we found that temporal variation in protein, phosphopeptide, and K+ abundance was at least as great as wild-type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY-deficient cells. This proteome imbalance in CRY-deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide-ranging phenotypes of CRY-deficient mice. Rather than generating large-scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post-translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation.

Architecture of the biofilm-associated archaic Chaperone-Usher pilus CupE from Pseudomonas aeruginosa.

Chaperone-Usher Pathway (CUP) pili are major adhesins in Gram-negative bacteria, mediating bacterial adherence to biotic and abiotic surfaces. While classical CUP pili have been extensively characterized, little is known about so-called archaic CUP pili, which are phylogenetically widespread and promote biofilm formation by several human pathogens. In this study, we present the electron cryomicroscopy structure of the archaic CupE pilus from the opportunistic human pathogen Pseudomonas aeruginosa. We show that CupE1 subunits within the pilus are arranged in a zigzag architecture, containing an N-terminal donor ß-strand extending from each subunit into the next, where it is anchored by hydrophobic interactions, with comparatively weaker interactions at the rest of the inter-subunit interface. Imaging CupE pili on the surface of P. aeruginosa cells using electron cryotomography shows that CupE pili adopt variable curvatures in response to their environment, which might facilitate their role in promoting cellular attachment. Finally, bioinformatic analysis shows the widespread abundance of cupE genes in isolates of P. aeruginosa and the co-occurrence of cupE with other cup clusters, suggesting interdependence of cup pili in regulating bacterial adherence within biofilms. Taken together, our study provides insights into the architecture of archaic CUP pili, providing a structural basis for understanding their role in promoting cellular adhesion and biofilm formation in P. aeruginosa.

Selective inhibition of protein secretion by abrogating receptor-coat interactions during ER export.

Protein secretion is an essential process that drives cell growth, movement, and communication. Protein traffic within the secretory pathway occurs via transport intermediates that bud from one compartment and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human proprotein convertase subtilisin/kexin type 9 (PCSK9) is a determinant of cholesterol metabolism whose secretion is mediated by a specific cargo adaptor protein, SEC24A. We map a series of protein-protein interactions between PCSK9, its endoplasmic reticulum (ER) export receptor SURF4, and SEC24A that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-phenylbutyrate (4-PBA), a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small-molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.

Investigating the Regulation of Ribosomal Protein S6 Kinase 1 by CoAlation.

Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a "dual anchor" mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors.

Cryo-EM structures of amyloid-ß filaments with the Arctic mutation (E22G) from human and mouse brains.

The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-ß (Aß)], causes dominantly inherited Alzheimer's disease. Here, we report the high-resolution cryo-EM structures of Aß filaments from the frontal cortex of a previously described case (AßPParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12-V40 (human Arctic fold A) and E11-G37 (human Arctic fold B). They have a substructure (residues F20-G37) in common with the folds of type I and type II Aß42. When compared to the structures of wild-type Aß42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant Aß molecules and promote filament formation. A minority of Aß42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Aß filaments with the Arctic mutation from mouse knock-in line AppNL-G-F. Most filaments are made of two identical mutant protofilaments that extend from D1 to G37 (AppNL-G-F murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The AppNL-G-F murine Arctic fold differs from the human Arctic folds, but shares some substructure.

New SNCA mutation and structures of α-synuclein filaments from juvenile-onset synucleinopathy.

A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant α-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of α-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new α-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36-100 of wild-type α-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type α-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant α-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation.

Akt and SGK protein kinases are required for efficient feeding by macropinocytosis.

Macropinocytosis is an actin-driven process of large-scale and non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum In Dictyostelium, macropinocytic cups are organized by 'macropinocytic patches' in the plasma membrane. These contain activated Ras, Rac and phospholipid PIP3, and direct actin polymerization to their periphery. We show that a Dictyostelium Akt (PkbA) and an SGK (PkbR1) protein kinase act downstream of PIP3 and, together, are nearly essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Through phosphoproteomics, we identify a RhoGAP, GacG, as a PkbA and PkbR1 target, and show that it is required for efficient macropinocytosis and expansion of macropinocytic patches. The function of Akt and SGK in cell feeding through control of macropinosome size has implications for cancer cell biology.

Catalysts from synthetic genetic polymers.

The emergence of catalysis in early genetic polymers such as RNA is considered a key transition in the origin of life, pre-dating the appearance of protein enzymes. DNA also demonstrates the capacity to fold into three-dimensional structures and form catalysts in vitro. However, to what degree these natural biopolymers comprise functionally privileged chemical scaffolds for folding or the evolution of catalysis is not known. The ability of synthetic genetic polymers (XNAs) with alternative backbone chemistries not found in nature to fold into defined structures and bind ligands raises the possibility that these too might be capable of forming catalysts (XNAzymes). Here we report the discovery of such XNAzymes, elaborated in four different chemistries (arabino nucleic acids, ANA; 2'-fluoroarabino nucleic acids, FANA; hexitol nucleic acids, HNA; and cyclohexene nucleic acids, CeNA) directly from random XNA oligomer pools, exhibiting in trans RNA endonuclease and ligase activities. We also describe an XNA-XNA ligase metalloenzyme in the FANA framework, establishing catalysis in an entirely synthetic system and enabling the synthesis of FANA oligomers and an active RNA endonuclease FANAzyme from its constituent parts. These results extend catalysis beyond biopolymers and establish technologies for the discovery of catalysts in a wide range of polymer scaffolds not found in nature. Evolution of catalysis independent of any natural polymer has implications for the definition of chemical boundary conditions for the emergence of life on Earth and elsewhere in the Universe.

Protein CoAlation and antioxidant function of coenzyme A in prokaryotic cells.

In all living organisms, coenzyme A (CoA) is an essential cofactor with a unique design allowing it to function as an acyl group carrier and a carbonyl-activating group in diverse biochemical reactions. It is synthesized in a highly conserved process in prokaryotes and eukaryotes that requires pantothenic acid (vitamin B5), cysteine and ATP. CoA and its thioester derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. A novel unconventional function of CoA in redox regulation has been recently discovered in mammalian cells and termed protein CoAlation. Here, we report for the first time that protein CoAlation occurs at a background level in exponentially growing bacteria and is strongly induced in response to oxidizing agents and metabolic stress. Over 12% of Staphylococcus aureus gene products were shown to be CoAlated in response to diamide-induced stress. In vitro CoAlation of S. aureus glyceraldehyde-3-phosphate dehydrogenase was found to inhibit its enzymatic activity and to protect the catalytic cysteine 151 from overoxidation by hydrogen peroxide. These findings suggest that in exponentially growing bacteria, CoA functions to generate metabolically active thioesters, while it also has the potential to act as a low-molecular-weight antioxidant in response to oxidative and metabolic stress.

Protein CoAlation: a redox-regulated protein modification by coenzyme A in mammalian cells.

Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry, we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation). We show that protein CoAlation is a reversible post-translational modification that is induced in mammalian cells and tissues by oxidising agents and metabolic stress. Many key cellular enzymes were found to be CoAlated in vitro and in vivo in ways that modified their activities. Our study reveals that protein CoAlation is a widespread post-translational modification which may play an important role in redox regulation under physiological and pathophysiological conditions.

Nanostructures from Synthetic Genetic Polymers.

Nanoscale objects of increasing complexity can be constructed from DNA or RNA. However, the scope of potential applications could be enhanced by expanding beyond the moderate chemical diversity of natural nucleic acids. Here, we explore the construction of nano-objects made entirely from alternative building blocks: synthetic genetic polymers not found in nature, also called xeno nucleic acids (XNAs). Specifically, we describe assembly of 70 kDa tetrahedra elaborated in four different XNA chemistries (2'-fluro-2'-deoxy-ribofuranose nucleic acid (2'F-RNA), 2'-fluoroarabino nucleic acids (FANA), hexitol nucleic acids (HNA), and cyclohexene nucleic acids (CeNA)), as well as mixed designs, and a ∼600 kDa all-FANA octahedron, visualised by electron microscopy. Our results extend the chemical scope for programmable nanostructure assembly, with implications for the design of nano-objects and materials with an expanded range of structural and physicochemical properties, including enhanced biostability.