EMBO Journal最新文献

Chromosome segregation relies on kinetochores that assemble on specialized centromeric chromatin containing a histone H3 variant. In budding yeast, a single centromeric nucleosome containing Cse4 assembles at a sequence-defined 125 bp centromere. Yeast centromeric sequences are poor templates for nucleosome formation in vitro, suggesting the existence of mechanisms that specifically stabilize Cse4 nucleosomes in vivo. The extended Cse4 N-terminal tail binds to the chaperone Scm3, and a short essential region called END within the N-terminal tail binds the inner kinetochore complex Okp1/Ame1. To address the roles of these interactions, we utilized single-molecule fluorescence assays to monitor Cse4 during kinetochore assembly. We found that Okp1/Ame1 and Scm3 independently stabilize Cse4 at centromeres via their END interaction. Scm3 and Cse4 stability at the centromere are enhanced by Ipl1/Aurora B phosphorylation of the Cse4 END, identifying a previously unknown role for Ipl1 in ensuring Cse4 stability. Strikingly, a phosphomimetic mutation in the Cse4 END restores Cse4 recruitment in mutants defective in Okp1/Ame1 binding. Together, these data suggest that a key function of the essential Cse4 N-terminus is to ensure Cse4 localization at centromeres.

The carboxyl terminus of Hsc70-interacting protein (CHIP) is pivotal for managing misfolded and aggregated proteins via chaperone networks and degradation pathways. In a preclinical rodent model of CHIP-related ataxia, we observed that CHIP mutations lead to increased levels of phosphodiesterase 9A (PDE9A), whose role in this context remains poorly understood. Here, we investigated the molecular mechanisms underlying the role of PDE9A in CHIP-related ataxia and demonstrated that CHIP binds to PDE9A, facilitating its polyubiquitination and autophagic degradation. Conversely, dysfunctional CHIP disrupts this process, resulting in PDE9A accumulation, increased cGMP hydrolysis, and impaired PKG phosphorylation of CHIP at serine 19. This cascade further amplifies PDE9A accumulation, ultimately disrupting mitophagy and triggering neuronal apoptosis. Elevated PKA levels inhibit PDE9A degradation, further exacerbating this neuronal dysfunction. Notably, pharmacological inhibition of PDE9A via Bay 73-6691 or virus-mediated CHIP expression restored the balance of cGMP/cAMP signalling. These interventions protect against cerebellar neuropathologies, particularly Purkinje neuron mitophagy dysfunction. Thus, PDE9A upregulation considerably exacerbates ataxia associated with CHIP mutations, and targeting the interaction between PDE9A and CHIP is an innovative therapeutic strategy for CHIP-related ataxia.

Mitochondrial metabolism requires the chaperoned import of disulfide-stabilized proteins via CHCHD4/MIA40 and its enigmatic interaction with oxidoreductase Apoptosis-inducing factor (AIF). By crystallizing human CHCHD4's AIF-interaction domain with an activated AIF dimer, we uncover how NADH allosterically configures AIF to anchor CHCHD4's β-hairpin and histidine-helix motifs to the inner mitochondrial membrane. The structure further reveals a similarity between the AIF-interaction domain and recognition sequences of CHCHD4 substrates. NMR and X-ray scattering (SAXS) solution measurements, mutational analyses, and biochemistry show that the substrate-mimicking AIF-interaction domain shields CHCHD4's redox-sensitive active site. Disrupting this shield critically activates CHCHD4 substrate affinity and chaperone activity. Regulatory-domain sequestration by NADH-activated AIF directly stimulates chaperone binding and folding, revealing how AIF mediates CHCHD4 mitochondrial import. These results establish AIF as an integral component of the metazoan disulfide relay and point to NADH-activated dimeric AIF as an organizational import center for CHCHD4 and its substrates. Importantly, AIF regulation of CHCHD4 directly links AIF's cellular NAD(H) sensing to CHCHD4 chaperone function, suggesting a mechanism to balance tissue-specific oxidative phosphorylation (OXPHOS) capacity with NADH availability.

Pericytes are essential for capillary stability and homeostasis, with impaired pericyte function linked to diseases like pulmonary arterial hypertension. Investigating pericyte biology has been challenging due to the lack of specific markers, making it difficult to distinguish pericytes from other stromal cells. Using bioinformatic analysis and RNAscope, we identified Higd1b as a unique gene marker for pericytes and subsequently generated a knock-in mouse line, Higd1b-CreERT2, that accurately labels pericytes in the lung and heart. Single-cell RNA sequencing revealed two distinct Higd1b+ pericyte subtypes: while Type 1 pericytes support capillary homeostasis, Type 2 pericytes accumulate in arterioles, and co-express smooth muscle markers and higher levels of vimentin under hypoxic conditions. Lastly, healthy human lung pericytes with upregulation of vimentin exhibited increased adhesion, migration, and higher expression levels of the smooth muscle marker SM22 in vitro. These findings highlight the specialization of pulmonary pericytes and their contribution to vascular remodeling during hypoxia-induced pulmonary hypertension.

Polyglucosans are glycogen molecules with overlong chains, which are hyperphosphorylated in the neurodegenerative Lafora disease (LD). Brain polyglucosan bodies (PBs) cause fatal neurodegenerative diseases including Lafora disease and adult polyglucosan body disease (ABPD), for which treatments, biomarkers, and good understanding of their pathogenesis are currently missing. Mutations in the genes for the phosphatase laforin or the E3 ubiquitin ligase malin can cause LD. By depleting PTG, an activator of the glycogen chain-elongating enzyme glycogen synthase (GYS1), in laforin- and malin-deficient LD mice, we show that abnormal glycogen chain lengths and not hyperphosphorylation underlie polyglucosan formation, and that polyglucosan bodies induce neuroinflammation. We provide evidence indicating that a small pool of overactive GYS1 contributes to glycogen insolubility in LD and APBD. In contrast to previous findings, metabolomics experiments using in situ-fixed brains reveal only modest metabolic changes in laforin-deficient mice. These changes are not replicated in malin-deficient or APBD mice, and are not normalized in rescued LD mice. Finally, we identify a pool of metabolically volatile malto-oligoglucans as a polyglucosan body- and neuroinflammation-associated brain energy source, and promising candidate biomarkers for LD and APBD, including malto-oligoglucans and the neurodegeneration marker CHI3L1/YKL40.

ABCB1 is a broad-spectrum efflux pump central to cellular drug handling and multidrug resistance in humans. However, how it is able to recognize and transport a wide range of diverse substrates remains poorly understood. Here we present cryo-EM structures of lipid-embedded human ABCB1 in conformationally distinct apo-, substrate-bound, inhibitor-bound, and nucleotide-trapped states at 3.4-3.9 Å resolution, in the absence of stabilizing antibodies or mutations. The substrate-binding site is located within one half of the molecule and, in the apo state, is obstructed by the transmembrane helix (TM) 4. Substrate and inhibitor binding are distinguished by major TM rearrangements and their ligand binding chemistry, with TM4 playing a central role in all conformational transitions. Furthermore, our data identify secondary structure-breaking residues that impart localized TM flexibility and asymmetry between the two transmembrane domains. The resulting structural changes and lipid interactions that are induced by substrate and inhibitor binding can predict substrate-binding profiles and may direct ABCB1 inhibitor design.

Dysregulation of RNA processing has in recent years emerged as a significant contributor to neurodegeneration. The diverse mechanisms and molecular functions underlying RNA processing underscore the essential role of RNA regulation in maintaining neuronal health and function. RNA molecules are bound by RNA-binding proteins (RBPs), and interactions between RNAs and RBPs are commonly affected in neurodegeneration. In this review, we highlight recent progress in understanding dysregulated RNA-processing pathways and the causes of RBP dysfunction across various neurodegenerative diseases. We discuss both established and emerging mechanisms of RNA-mediated neuropathogenesis in this rapidly evolving field. Furthermore, we explore the development of potential RNA-targeting therapeutic approaches for the treatment of neurodegenerative diseases.

Cancer cells rely on invasive growth to survive in a hostile microenvironment; this growth is characterised by interconnected processes such as epithelial-to-mesenchymal transition and migration. A master regulator of these events is the MET oncogene, which is overexpressed in the majority of cancers; however, since mutations in the MET oncogene are seen only rarely in cancers and are relatively infrequent, the mechanisms that cause this widespread MET overexpression remain obscure. Here, we show that the 5' untranslated region (5'UTR) of MET mRNA harbours two functional stress-responsive elements, conferring translational regulation by the integrated stress response (ISR), regulated by phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) at serine 52. ISR activation by serum starvation, leucine deprivation, hypoxia, irradiation, thapsigargin or gemcitabine is followed by MET protein overexpression. We mechanistically link MET translation to the ISR by (i) mutation of the two uORFs within the MET 5'UTR, (ii) CRISPR/Cas9-mediated mutation of eIF2α (S52A), or (iii) the application of ISR pathway inhibitors. All of these interventions reduce stress-induced MET overexpression. Finally, we show that blocking stress-induced MET translation blunts MET-dependent invasive growth. These findings indicate that upregulation of the MET oncogene is a functional requirement linking integrated stress response to cancer progression.

The ESCRT machinery mediates membrane remodeling in numerous processes in cells including cell division and nuclear membrane reformation. The identification of ESCRT homologs in Asgard archaea, currently considered the closest prokaryotic relative of eukaryotes, implies a role for ESCRTs in the membrane remodeling processes that occurred during eukaryogenesis. Yet, the function of these distant ESCRT homologs is mostly unresolved. Here we show that Asgard ESCRT-III proteins of the Lokiarcheota self-assemble into helical filaments, a hallmark of the ESCRT system. We determined the cryo-EM structure of the filaments at 3.6 Å resolution and found that they share features of bacterial and eukaryotic ESCRT-III assemblies. Markedly, Asgard ESCRT-III filaments bound and deformed eukaryotic-like membrane vesicles. Oligonucleotides facilitated the assembly of ESCRT-III filaments and tuned the extent of membrane remodeling. The ability of Asgard archaeal ESCRTs to remodel eukaryotic-like membranes, which are fundamentally different from archaeal membranes, and the structural properties of these proteins places them at the junction between prokaryotes and eukaryotes.

The complement system and neutrophils constitute the two main pillars of the host innate immune defense against infection by bacterial pathogens. Here, we identify T-Mac, a novel virulence factor of the periodontal pathogen Treponema denticola that allows bacteria to evade both defense systems. We show that T-Mac is expressed as a pre-protein that is cleaved into two functional units. The N-terminal fragment has two immunoglobulin-like domains and binds with high affinity to the major neutrophil chemokine receptors FPR1 and CXCR1, blocking N-formyl-Met-Leu-Phe- and IL-8-induced neutrophil chemotaxis and activation. The C-terminal fragment functions as a cysteine protease with a unique proteolytic activity and structure, which degrades several components of the complement system, such as C3 and C3b. Murine infection studies further reveal a critical T-Mac role in tissue damage and inflammation caused by bacterial infection. Collectively, these results disclose a novel innate immunity-evasion strategy, and open avenues for investigating the role of cysteine proteases and immunoglobulin-like domains of gram-positive and -negative bacterial pathogens.