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One of the hallmarks of Alzheimer’s disease and several other neurodegenerative disorders is the aggregation of tau protein into fibrillar structures. Building on recent reports that tau readily undergoes liquid–liquid phase separation (LLPS), here we explored the relationship between disease-related mutations, LLPS, and tau fibrillation. Our data demonstrate that, in contrast to previous suggestions, pathogenic mutations within the pseudorepeat region do not affect tau441’s propensity to form liquid droplets. LLPS does, however, greatly accelerate formation of fibrillar aggregates, and this effect is especially dramatic for tau441 variants with disease-related mutations. Most important, this study also reveals a previously unrecognized mechanism by which LLPS can regulate the rate of fibrillation in mixtures containing tau isoforms with different aggregation propensities. This regulation results from unique properties of proteins under LLPS conditions, where total concentration of all tau variants in the condensed phase is constant. Therefore, the presence of increasing proportions of the slowly aggregating tau isoform gradually lowers the concentration of the isoform with high aggregation propensity, reducing the rate of its fibrillation. This regulatory mechanism may be of direct relevance to phenotypic variability of tauopathies, as the ratios of fast and slowly aggregating tau isoforms in brain varies substantially in different diseases.

Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein 1 , 2 , to trigger target cell pyroptosis 3 . GSDMB and the charter gasdermin family member GSDMD 4 , 5 have been inconsistently reported to be degraded by the Shigella flexneri ubiquitin-ligase virulence factor IpaH7.8 (refs. 6 , 7 ). Whether and how IpaH7.8 targets both gasdermins is undefined, and the pyroptosis function of GSDMB has even been questioned recently 6 , 8 . Here we report the crystal structure of the IpaH7.8–GSDMB complex, which shows how IpaH7.8 recognizes the GSDMB pore-forming domain. We clarify that IpaH7.8 targets human (but not mouse) GSDMD through a similar mechanism. The structure of full-length GSDMB suggests stronger autoinhibition than in other gasdermins 9 , 10 . GSDMB has multiple splicing isoforms that are equally targeted by IpaH7.8 but exhibit contrasting pyroptotic activities. Presence of exon 6 in the isoforms dictates the pore-forming, pyroptotic activity in GSDMB. We determine the cryo-electron microscopy structure of the 27-fold-symmetric GSDMB pore and depict conformational changes that drive pore formation. The structure uncovers an essential role for exon-6-derived elements in pore assembly, explaining pyroptosis deficiency in the non-canonical splicing isoform used in recent studies 6 , 8 . Different cancer cell lines have markedly different isoform compositions, correlating with the onset and extent of pyroptosis following GZMA stimulation. Our study illustrates fine regulation of GSDMB pore-forming activity by pathogenic bacteria and mRNA splicing and defines the underlying structural mechanisms. The cryo-EM structure of the GSDMB pore reveals mechanisms by which GSDMB pore-forming activity is regulated by pathogenic bacteria and mRNA splicing.

Dipeptidyl peptidase-4 (DPP4) modulates inflammation by enzymatic cleavage of immunoregulatory peptides and through its soluble form (sDPP4) that directly engages immune cells. Here we examine whether reduction of DPP4 activity alters inflammation. Prolonged DPP4 inhibition increases plasma levels of sDPP4, and induces sDPP4 expression in lymphocyte-enriched organs in mice. Bone marrow transplantation experiments identify hematopoietic cells as the predominant source of plasma sDPP4 following catalytic DPP4 inhibition. Surprisingly, systemic DPP4 inhibition increases plasma levels of inflammatory markers in regular chow-fed but not in high fat-fed mice. Plasma levels of sDPP4 and biomarkers of inflammation are lower in metformin-treated subjects with type 2 diabetes (T2D) and cardiovascular disease, yet exhibit considerable inter-individual variation. Sitagliptin therapy for 12 months reduces DPP4 activity yet does not increase markers of inflammation or levels of sDPP4. Collectively our findings dissociate levels of DPP4 enzyme activity, sDPP4 and biomarkers of inflammation in mice and humans. DPP4 inhibitors are used for the treatment of diabetes, but the impact of DPP4 activity and soluble DPP4 on development of diabetes-associated inflammation remains uncertain. Here the authors study whether DPP4 inhibition controls sDPP4 and inflammatory biomarkers, and demonstrate that DPP4 inhibition is dissociated from changes in inflammation in mice and humans.

The post-translational modification of proteins regulates many biological processes. Their dysfunction relates to diseases. Ubiquitination is one of the post-translational modifications that target lysine residue and regulate many cellular processes. Three enzymes are required for achieving the ubiquitination reaction: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E3s play a pivotal role in selecting substrates. Many structural studies have been conducted to reveal the molecular mechanism of the ubiquitination reaction. Recently, the structure of PCAF_N, a newly categorized E3 ligase, was reported. We present a review of the recent progress toward the structural understanding of E3 ligases.

In a previous Phase 1/2b malaria vaccine trial testing the 3D7 isoform of the malaria vaccine candidate Merozoite surface protein 2 (MSP2), parasite densities in children were reduced by 62%. However, breakthrough parasitemias were disproportionately of the alternate dimorphic form of MSP2, the FC27 genotype. We therefore undertook a dose-escalating, double-blinded, placebo-controlled Phase 1 trial in healthy, malaria-naïve adults of MSP2-C1, a vaccine containing recombinant forms of the two families of msp2 alleles, 3D7 and FC27 (EcMSP2-3D7 and EcMSP2-FC27), formulated in equal amounts with Montanide® ISA 720 as a water-in-oil emulsion. The trial was designed to include three dose cohorts (10, 40, and 80 µg), each with twelve subjects receiving the vaccine and three control subjects receiving Montanide® ISA 720 adjuvant emulsion alone, in a schedule of three doses at 12-week intervals. Due to unexpected local reactogenicity and concern regarding vaccine stability, the trial was terminated after the second immunisation of the cohort receiving the 40 µg dose; no subjects received the 80 µg dose. Immunization induced significant IgG responses to both isoforms of MSP2 in the 10 µg and 40 µg dose cohorts, with antibody levels by ELISA higher in the 40 µg cohort. Vaccine-induced antibodies recognised native protein by Western blots of parasite protein extracts and by immunofluorescence microscopy. Although the induced anti-MSP2 antibodies did not directly inhibit parasite growth in vitro, IgG from the majority of individuals tested caused significant antibody-dependent cellular inhibition (ADCI) of parasite growth. As the majority of subjects vaccinated with MSP2-C1 developed an antibody responses to both forms of MSP2, and that these antibodies mediated ADCI provide further support for MSP2 as a malaria vaccine candidate. However, in view of the reactogenicity of this formulation, further clinical development of MSP2-C1 will require formulation of MSP2 in an alternative adjuvant. Australian New Zealand Clinical Trials Registry 12607000552482.

In the absence of DHX9, circular RNAs accumulate and transcription and translation are dysregulated—effects that are exacerbated by concomitant depletion of the RNA-editing enzyme ADAR. DHX9 suppresses Alu-derived defects In the human genome, there are more than a million copies of the Alu transposable element. Movement of Alu elements is a common source of mutations, but as insertions usually occur in non-coding regions, they are often without discernible effect. Alu elements located near one another in an inverted orientation will form secondary structures that may affect various nuclear processes. Asifa Akhtar and colleagues find that the RNA helicase, DHX9, binds transcribed ‘IRAlus’ (inverted repeat Alu elements). In the absence of DHX9, circular RNAs accumulate, and transcription and translation are dysregulated. These effects are further exacerbated by co-depletion of DHX9 and ADAR p150, an interferon-inducible RNA modification enzyme. The authors conclude that these proteins protect against transposon insertion, which can have deleterious effects on gene expression. Transposable elements are viewed as ‘selfish genetic elements’, yet they contribute to gene regulation and genome evolution in diverse ways 1 . More than half of the human genome consists of transposable elements 2 . Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome 2 . Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability 3 . Alu elements are the main targets of the RNA-editing enzyme ADAR 4 and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC 5 , but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant 6 nuclear RNA helicase 7 , binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.

Splicing varies across brain regions, but the single-cell resolution of regional variation is unclear. We present a single-cell investigation of differential isoform expression (DIE) between brain regions using single-cell long-read sequencing in mouse hippocampus and prefrontal cortex in 45 cell types at postnatal day 7 ( www.isoformAtlas.com ). Isoform tests for DIE show better performance than exon tests. We detect hundreds of DIE events traceable to cell types, often corresponding to functionally distinct protein isoforms. Mostly, one cell type is responsible for brain-region specific DIE. However, for fewer genes, multiple cell types influence DIE. Thus, regional identity can, although rarely, override cell-type specificity. Cell types indigenous to one anatomic structure display distinctive DIE, e.g. the choroid plexus epithelium manifests distinct transcription-start-site usage. Spatial transcriptomics and long-read sequencing yield a spatially resolved splicing map. Our methods quantify isoform expression with cell-type and spatial resolution and it contributes to further our understanding of how the brain integrates molecular and cellular complexity. Alternative RNA splicing varies across the brain. Its mapping at single cell resolution is unclear. Here, the authors provide a spatial and single-cell splicing atlas reporting brain region- and cell type-specific expression of different isoforms in the postnatal mouse brain.

The RAS–RAF pathway is one of the most commonly dysregulated in human cancers 1 – 3 . Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation 4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer 5 and active RAF dimer 5 – 8 bound to 14-3-3 9 , 10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF 11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS 11 – 13 , but the structure of SHOC2 and the architecture of the presumptive SHOC2–PP1C–RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2–PP1C–MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway. Cryo-electron microscopy structure, molecular dynamics and biochemical analyses of the SHOC2–PP1C–MRAS complex demonstrate the dependence of the complex formation on RAS–GTP and identify the determinants of RAS isoform preference for SHOC2–PP1C and specificity of the complex for RAF dephosphorylation.

The ordered assembly of tau protein into abnormal filamentous inclusions underlies many human neurodegenerative diseases 1 . Tau assemblies seem to spread through specific neural networks in each disease 2 , with short filaments having the greatest seeding activity 3 . The abundance of tau inclusions strongly correlates with disease symptoms 4 . Six tau isoforms are expressed in the normal adult human brain—three isoforms with four microtubule-binding repeats each (4R tau) and three isoforms that lack the second repeat (3R tau) 1 . In various diseases, tau filaments can be composed of either 3R or 4R tau, or of both. Tau filaments have distinct cellular and neuroanatomical distributions 5 , with morphological and biochemical differences suggesting that they may be able to adopt disease-specific molecular conformations 6 , 7 . Such conformers may give rise to different neuropathological phenotypes 8 , 9 , reminiscent of prion strains 10 . However, the underlying structures are not known. Using electron cryo-microscopy, we recently reported the structures of tau filaments from patients with Alzheimer’s disease, which contain both 3R and 4R tau 11 . Here we determine the structures of tau filaments from patients with Pick’s disease, a neurodegenerative disorder characterized by frontotemporal dementia. The filaments consist of residues Lys254–Phe378 of 3R tau, which are folded differently from the tau filaments in Alzheimer’s disease, establishing the existence of conformers of assembled tau. The observed tau fold in the filaments of patients with Pick’s disease explains the selective incorporation of 3R tau in Pick bodies, and the differences in phosphorylation relative to the tau filaments of Alzheimer’s disease. Our findings show how tau can adopt distinct folds in the human brain in different diseases, an essential step for understanding the formation and propagation of molecular conformers. The structures of tau filaments from patients with the neurodegenerative disorder Pick’s disease show that the filament fold is different from that of the tau filaments found in Alzheimer’s disease.

Fast chemical communication in the nervous system is mediated by neurotransmitter-gated ion channels. The prototypical member of this class of cell surface receptors is the cation-selective nicotinic acetylcholine receptor. As with most ligand-gated ion channels, nicotinic receptors assemble as oligomers of subunits, usually as hetero-oligomers and often with variable stoichiometries 1 . This intrinsic heterogeneity in protein composition provides fine tunability in channel properties, which is essential to brain function, but frustrates structural and biophysical characterization. The α4β2 subtype of the nicotinic acetylcholine receptor is the most abundant isoform in the human brain and is the principal target in nicotine addiction. This pentameric ligand-gated ion channel assembles in two stoichiometries of α- and β-subunits (2α:3β and 3α:2β). Both assemblies are functional and have distinct biophysical properties, and an imbalance in the ratio of assemblies is linked to both nicotine addiction 2 , 3 and congenital epilepsy 4 , 5 . Here we leverage cryo-electron microscopy to obtain structures of both receptor assemblies from a single sample. Antibody fragments specific to β2 were used to ‘break’ symmetry during particle alignment and to obtain high-resolution reconstructions of receptors of both stoichiometries in complex with nicotine. The results reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the two different stoichiometries of this receptor. Cryo-electron microscopy structures of two stoichiometries of heteromeric acetylcholine receptors in complex with nicotine reveal principles of subunit assembly and the structural basis of the distinctive biophysical and pharmacological properties of the different stoichiometries.