EMBO Journal最新文献

Breast cancer is a leading cause of mortality worldwide. Pharmacological inhibitors of cyclin-dependent kinases (CDK) 4 and 6 (CDK4/6i) inhibit breast cancer growth by inducing a senescent-like state. However, the long-term treatment efficacy remains limited by the development of drug resistance, so clearance of senescent-like cancer cells may extend the durability of treatment. However, we show here that while CDK4/6i-treated breast cancer cells exhibit various senescence-associated phenotypes, they remain insensitive to common senolytic compounds. By searching for novel vulnerabilities, we identify a significantly increased lysosomal mass and altered lysosomal structure across various breast cancer cell types upon exposure to CDK4/6i in preclinical systems and clinical specimens. We demonstrate that these CDK4/6i-induced lysosomal alterations render breast cancer cells sensitive to lysosomotropic agents, such as L-leucyl-L-leucine methyl ester (LLOMe) and salinomycin. Importantly, sequential treatment with CDK4/6i and lysosomotropic agents effectively reduces the growth of both hormone receptor-positive (HR⁺) and subsets of triple-negative breast cancer (TNBC) cells in vivo. This sequential therapeutic strategy offers a promising approach to eliminate CDK4/6i-induced senescent(-like) cells, potentially reducing tumor recurrence and enhancing the overall efficacy of breast cancer therapy.

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.

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.

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.

Genomic DNA is assembled into chromatin by histones, and extruded into loops by cohesin. These mechanisms control important genomic functions, but whether histones and cohesin cooperate in genome regulation is poorly understood. Here we identify Phf2, a member of the Jumonji-C family of histone demethylases, as a cohesin-interacting protein. Phf2 binds to H3K4me3 nucleosomes at active transcription start sites (TSSs), but also co-localizes with cohesin. Cohesin depletion reduces Phf2 binding at sites lacking H3K4me3, and depletion of Wapl and CTCF re-positions Phf2 together with cohesin in the genome, resulting in the accumulation of both proteins in chromosomal regions called vermicelli and cohesin islands. Conversely, Phf2 depletion reduces cohesin binding at TSSs lacking CTCF and decreases the number of short cohesin loops, while increasing the length of heterochromatic B compartments. These results suggest that Phf2 is an 'epigenetic reader', which is translocated through the genome by cohesin-mediated DNA loop extrusion, and which recruits cohesin to active TSSs and limits the size of B compartments. These findings reveal an unexpected degree of cooperativity between epigenetic and architectural mechanisms of eukaryotic genome regulation.

During mitosis, the condensin I and II complexes compact chromatin into chromosomes. Loss of the chromokinesin, KIF4A, results in reduced condensin I association with chromosomes, but the molecular mechanism behind this phenotype is unknown. In this study, we reveal that KIF4A binds directly to the human condensin I HAWK subunit, NCAPG, via a conserved disordered short linear motif (SLiM) located in its C-terminal tail. KIF4A competes for NCAPG binding to an overlapping site with SLiMs at the N-terminus of NCAPH and the C-terminus of NCAPD2, which mediate two auto-inhibitory interactions within condensin I. Consistently, the KIF4A SLiM peptide alone is sufficient to stimulate ATPase and DNA loop extrusion activities of condensin I. We identify similar SLiMs in the known yeast condensin interactors, Sgo1 and Lrs4, which bind yeast condensin subunit, Ycg1, the equivalent HAWK to NCAPG. Our findings, together with previous work on condensin II and cohesin, demonstrate that SLiM binding to the NCAPG-equivalent HAWK subunit is a conserved mechanism of regulation in SMC complexes.

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.

The cellular concentrations of splicing factors (SFs) are critical for controlling alternative splicing. Most serine and arginine-enriched (SR) protein SFs regulate their own concentration via a homeostatic feedback mechanism that involves regulation of inclusion of non-coding 'poison exons' (PEs) that target transcripts for nonsense-mediated decay. The importance of SR protein PE splicing during animal development is largely unknown despite PE ultra-conservation across animal genomes. To address this, we used mouse genetics to disrupt an ultra-conserved PE in the Tra2b gene encoding the SR protein Tra2β. Focussing on germ cell development, we found that Tra2b PE deletion causes azoospermia due to catastrophic cell death during meiotic prophase. Failure to proceed through meiosis was associated with increased Tra2b expression sufficient to drive aberrant Tra2β protein hyper-responsive splice patterns. Although critical for meiotic prophase, Tra2b PE deletion spared earlier mitotically active germ cells, even though these still required Tra2b gene function. Our data indicate that PE splicing control prevents the accumulation of toxic levels of Tra2β protein that are incompatible with meiotic prophase. This unexpected connection with male fertility helps explain Tra2b PE ultra-conservation and indicates the importance of evaluating PE function in animal models.

Chloride (Cl^-) ions cause major damage to crops in saline soils. Understanding the key factors that influence Cl^- uptake and translocation will aid the breeding of more salt-tolerant crops. Here, using genome-wide association study and transcriptomic analysis, we identified a NITRATE TRANSPORTER 1 (NRT1)/PEPTIDE TRANSPORTER family (NPF) protein, GmNPF7.5, as the dominant gene locus influencing Cl^- homeostasis in soybean (Glycine max). A natural SNP variation resulted in two haplotypes (GmNPF7.5^HapA and GmNPF7.5^HapB), which was associated with Cl^- content. GmNPF7.5^HapA mediated Cl^- or nitrate (NO₃^-) uptake in a pH-dependent manner and exhibited higher permeability for Cl^- over NO₃^-. The suppression of GmNPF7.5^HapA expression decreased Cl^- accumulation and salt damage in plants, whereas its overexpression showed the opposite effects. The elite haplotype GmNPF7.5^HapB diminished Cl^- transport activity independently from NO₃^- permeability, thus enhancing soybean salt tolerance. Furthermore, the protein kinase GmPI4Kγ4 could phosphorylate GmNPF7.5, which repressed Cl^- uptake without affecting NO₃^- permeability. Our findings define a regulatory mechanism for Cl^- control under NaCl stress, providing a strategy for the improvement of salt tolerance in soybean plants.