Arabinosyl (ara) nucleoside analogues such as vidarabine (araA) and cytarabine (araC) are antimetabolites known to inhibit DNA polymerases in isolation. However, their effects on DNA synthesis catalysed by the multi-protein replisome remain unknown. Here, we uncover a central role for the lagging-strand DNA polymerase, Pol δ, in tolerating these drugs. Upon DNA synthesis inhibition by ara nucleotides, Pol δ replaces the canonical leading-strand DNA polymerase, Pol ε, to synthesise and proofread nascent leading strands in vitro and in vivo, with these Pol δ activities limiting helicase-polymerase uncoupling and intra-S checkpoint activation. Removal of ara analogues from nascent strands by DNA polymerase proofreading is likely the primary resistance mechanism against these drugs since a template-embedded analogue can be bypassed by the replisome. This study defines the initial response of the eukaryotic replisome to araA and araC and may have implications for how resistance arises against them, and other nucleoside analogue chemotherapeutics, in the clinic.

Humoral immune responses rely on germinal center (GC) reactions to generate long-lasting antibody responses with affinity maturation. CD137 (4-1BB) agonists are known to exert inhibitory effects on B-cell responses; however, the underlying mechanisms remain unclear. Here, we show that agonistic anti-CD137 monoclonal antibodies target multiple CD137-expressing cell types, including activated CD8[+] T-cells, regulatory T-cells, follicular regulatory T-cells, and follicular dendritic cells. Anti-CD137 antibodies strongly disrupt GC reactions upon vaccination, preventing the development of anti-drug antibodies, and suppress ectopic B-cell responses within tumor-associated tertiary lymphoid structures in mice. Mechanistic analyses demonstrate that GC disruption occurs independently of IFN-γ, IL-12, and CD8[+] T-cells, but is critically dependent on Foxp3[+] T-cells. Combined transcriptomic profiling, immune phenotyping, and functional GC suppression assays reveal that anti-CD137 treatment increases the abundance and immunosuppressive activity of regulatory and follicular regulatory T-cells in vaccinated mice. Our results uncover the cellular basis of CD137-mediated suppression of humoral immunity and provide insight into rational combinatorial immunotherapeutic strategies incorporating CD137 agonists under active clinical development.

Aging is a major contributor to the increasing incidence of acute kidney injury (AKI) and chronic kidney disease (CKD), however the molecular events explaining aging-related nephropathies remains elusive. Here, we found that the RAS signaling adapter protein Erbin (ErbB2-interacting protein) is abundantly expressed in renal tubular cells, but its levels are decreased in murine AKI models and human CKD patient samples. Tubule-specific deletion of Erbin rendered mice highly susceptible to acute kidney injury, and exacerbated renal fibrosis. In contrast, restoring Erbin protected against AKI and CKD. Moreover, tubular Erbin loss in the naturally-aged kidney aggravates aging-associated nephropathies. Transcriptome sequencing revealed a causal association between Erbin loss and cell-cycle alterations in tubular epithelial cells. Depleting Erbin increased the proportion of tubular cells in G2/M phase and accelerated cellular senescence in vitro and in vivo. Mechanistically, Erbin protects tubular cells from senescence by stabilizing YB-1, which maintains CDK1 and cyclin B1 expression, and facilitates cell-cycle progression. Thus, restoring Erbin represents a potential strategy for chronic kidney disease and aging nephropathies.

Photosynthetic eukaryotes have undergone evolutionary shifts from aquatic to terrestrial habitats, accompanied by changes in genome organization and gene regulation. Yet, the evolution of transfer RNA (tRNA) gene repertoires has received limited attention despite their central role in translation. Here, we review how tRNA gene content, structure, and genomic organization diversified across photosynthetic lineages, mainly Archaeplastida, and how changes relate to evolutionary transitions. We show that tRNA gene repertoires are shaped by ecological transitions, genome architecture, and translational demands. We highlight terrestrialization as a shift in tRNA evolution, marked by loss of selenocysteine and its dedicated tRNA, and changes in intron prevalence and structure. Copy number variation correlates with codon usage and amino acid composition, and in angiosperms, nuclear tRNA genes display reinforced cis-regulatory elements consistent with increased translational demands. We show that plant tRNA genes exhibit evenly dispersed arrangements, except in some algae enriched in clustered configurations. Together, these observations support a model in which tRNA gene repertoires are drivers of genome evolution, integrating translational demand, genomic organization, and ecological adaptation across photosynthetic lineages.

Perforin-2 is a pore-forming protein localised to the endocytic compartments of dendritic cells and macrophages. It is reported to perform two distinct functions during immune responses: attacking intravacuolar pathogens, and forming pores in endocytic compartments to enable cytosolic delivery of antigens during cross-presentation. The molecular mechanisms that regulate perforin-2 remain unknown. Here, we address how cross-presenting dendritic cells control pore formation in phagosomes while maintaining the integrity of their endocytic compartments. We demonstrate that perforin-2 undergoes extensive proteolytic processing involving multiple endocytic proteases. Although the transmembrane anchor has been proposed to protect host membranes by orienting pores towards bacterial targets, we find that endocytic escape is mediated by full-length, membrane-anchored perforin-2 rather than by the proteolytically released ectodomain. Moreover, we show that perforin-2-mediated antigen translocation does not require low pH, explaining how perforin-2 can form pores in cross-presenting dendritic cells which do not acidify their phagosomes. Our findings point to a critical role of the transmembrane anchor in perforin-2 biology and suggest that perforin-2 employs distinct mechanisms of pore formation during anti-bacterial defence and cross-presentation.

Tau protein aggregates adopt distinct conformations across tauopathies, yet the protein interactions engaged by disease-specific polymorphs remain poorly characterized. Here, we demonstrate that conformationally distinct tau polymorphs associate with disease-specific interaction networks across Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and dementia with Lewy bodies (DLB). Interactome profiling of tau aggregates from PBS- and sarkosyl-soluble brain fractions identified 493 high-confidence interactors exhibiting remarkable disease specificity. As an exploratory feature discovery machine learning classification discriminated against diseases using as few as four to six protein features. AD tau selectively engaged glycolytic enzymes, TCA cycle components, and glutamate/GABA cycling machinery, with the astrocytic transporter SLC1A2 showing 27-fold enrichment. PSP tau exhibited extensive interactor depletion alongside selective proteasome enrichment, whereas DLB tau associated with neurogenesis modulators while depleting neuroinflammatory mediators. Interaction patterns were corroborated by parallel reaction monitoring mass spectrometry and proximity ligation assays and corresponded to disease-specific post-translational modification profiles. These findings show that tau polymorph conformations are associated with disease-specific interaction networks, providing molecular insight into tauopathy heterogeneity.

During each cell cycle, cells must not only duplicate the DNA sequence but also preserve the three-dimensional genome architecture required for gene regulation and DNA repair. After replication, this requires coordination between cohesin-mediated loop extrusion and sister-chromatid cohesion for homology-directed DNA repair. Because extrusion promotes sister resolution whereas cohesion can impede extrusion, how both activities coexist on replicated chromosomes is unclear. Here we show that cohesin function is partitioned within the nested hierarchy of topologically associating domains (TADs): cohesive cohesin accumulates at high-level boundaries to form "cohesion zones," while loop-extruding cohesin occupies boundaries across all hierarchical levels. Our data support a model in which this segregation emerges from dynamic interplay between cohesin pools and semi-permeable CTCF barriers, which act in cis and in trans to constrain sister-sister misalignment. Thus, a CTCF-defined boundary framework enables sister tethering while preserving dynamic loop folding, allowing replicated genomes to simultaneously support gene regulation and faithful DNA repair.

Target-directed microRNA degradation (TDMD) is an emerging post-transcriptional mechanism that controls miRNA turnover, yet its role in human cancers remains largely unexplored. Here, we combine CRISPRi-mediated ZSWIM8 depletion, miRNA-seq, and AGO2-eCLIP to define the TDMD landscape across breast cancer subtypes. We identify 19 high-confidence TDMD substrates, including miR-29b-3p and miR-33a/b-5p, and show that TDMD shapes miRNA target occupancy and target repression. Integration with single-cell transcriptomics reveals that TDMD of miR-29b-3p triggered by NREP transcript is associated with transcriptional plasticity along the epithelial-mesenchymal axis and marks a stem-like subpopulation of malignat cells with tumor-initiating potential in triple-negative breast cancer. Unexpectedly, we also uncover a non-canonical TDMD mechanism, independent of ubiquitin ligase ZSWIM8 and the proteasome, which includes SERPINE1-triggered miR-30c-5p degradation, conferring paclitaxel resistance and enhancing sphere-forming potential. These findings establish target-directed microRNA degradation as a functional layer of miRNA regulation in cancer, linking miRNA decay to cellular state transitions and therapeutic response. Our results broaden the mechanistic spectrum of TDMD and provide a framework to investigate how regulated miRNA decay contributes to aggressive breast cancer phenotypes.

Protein folding in the endoplasmic reticulum (ER) relies on N-linked glycosylation and glycan remodeling to guide quality control. Major histocompatibility complex class I (MHC-I) molecules, essential for adaptive immunity, undergo a specialized maturation pathway involving the peptide-loading complex (PLC), the editor TAPBPR, the UDP-glucose:glycoprotein glucosyltransferase, and the lectin chaperone calreticulin. However, how glycan-dependent mechanisms coordinate MHC-I transfer between these factors has remained unclear. Using a fully reconstituted system, we show that retrograde transfer of peptide-receptive MHC-I from TAPBPR to tapasin requires calreticulin recognition of monoglucosylated MHC-I glycans. While calreticulin's C-terminal acidic helix is dispensable for releasing reglucosylated MHC-I from TAPBPR, it is essential for productive docking of MHC-I onto tapasin. These findings reveal a glycan-surveillance mechanism that enables retrieval of suboptimally loaded MHC-I molecules missed by the initial quality control at the PLC. Our work defines a glycan-dependent chaperone network, finely tuned by a combination of low-micromolar interactions between the constituents, that ensures efficient MHC-I maturation and illustrates fundamental principles of ER protein quality control.

VCP/p97 is an AAA+ ATPase that, together with its cofactors UFD1-NPL4 (p97-UN), unfolds ubiquitylated substrates to maintain cellular homeostasis. The human p97-UN complex associates with additional cofactors, but how these cofactors modulate p97-UN activity is not fully understood. Here, we screen cofactors and identify FAF2 to potently enhance substrate unfolding by p97-UN. Using biochemical and structural approaches, we show how FAF2 engages p97-UN and polyubiquitin to promote unfolding. We define a conserved activation motif in FAF2 that contacts both UFD1 and the ubiquitin proximal to the initiator, thereby stabilizing and supporting the unfolding of the initiator ubiquitin in a UFD1-dependent manner. We leverage the features of the FAF2 activation motif to engineer de novo proteins that potently enhance unfolding, providing a rational strategy to boost p97 activity. Our findings reveal how cofactors can provide additional adaptive control, fine-tuning human p97 activity to unfold challenging substrates and those modified with short ubiquitin chains.

Neoepitope-based therapies hold great promise for cancer immunotherapy because they target tumor-specific mutations and elicit potent anti-tumor T-cell responses. However, their clinical implementation remains limited by the complexity of neoepitope discovery and uncertainty regarding presentation by tumor cells. A potential alternative is the generation of immunogenic neoepitopes directly within cancer cells through programmable RNA editing. Here, we develop Short Precise-Encodable ADAR Recruiting (SPEAR) gRNAs that harness endogenous ADAR1 to direct precise adenosine-to-inosine (A-to-I) editing at selected transcript sites. Using these gRNAs, we demonstrate the generation of immunogenic neoepitopes through RNA editing at the transcript level, termed editopes. In a proof-of-concept model based on the melanoma antigen MART-1, SPEAR-mediated RNA editing restored antigen-specific T cell recognition and enabled tumor control in vivo. Finally, we developed a computational pipeline to identify candidate tumor-selective neoepitopes across multiple cancer types amenable to guided RNA editing. Our findings establish programmable RNA editing as a strategy for engineering immunogenic editopes and provide a framework for neoepitope-directed cancer immunotherapy.

Transient receptor potential vanilloid 3 (TRPV3) is a non-selective cation channel highly expressed in the skin and intestine. While its roles in itch and skin inflammation are established, the physiological role of TRPV3 in the intestine remains relatively poorly understood. Topical application of a TRPV3 inhibitor, KM-001, has entered phase I clinical trials for the treatment of pruritus; however, its poor metabolic stability limits broader therapeutic application. Here, we show that the KM-001-derivative Colivin has comparable TRPV3-blocking activity, but enhanced metabolic stability and increased oral bioavailability. Cryo-EM and site-directed mutagenesis analyses established that Colivin binds to the vanilloid binding pocket of TRPV3, stabilizing one of two distinct non-conducting conformations. Oral administration of Colivin effectively suppressed DSS-induced ulcerative colitis (UC) in wild-type, but not Trpv3-deficient mice. Collectively, our findings establish TRPV3 inhibition as a novel therapeutic strategy for UC, offer key structural insights into the inhibition mechanism of TRPV3, and provide critical structural insights for the rational design of potent and selective TRPV3 inhibitors.

Macrophages can adopt diverse functional states in response to environmental cues, a process that is fundamentally controlled at the level of transcriptional regulation. In this review, we outline a hierarchical framework of transcription factor activity that underpins macrophage identity and activation. Firstly, lineage-determining transcription factors establish the cell-type-specific chromatin landscape during development. Upon tissue seeding, local signals shape the activity of additional transcription factors that refine enhancer landscapes and drive tissue-specific macrophage phenotypes. Macrophages further adopt their functional states when exposed to potential threats to tissue homeostasis, such as bacterial ligands or inflammatory cytokines. In response, signal-dependent transcription factors are activated and initiate signal-appropriate transcriptional programs. The specificity and durability of these responses are determined by secondary transcription factors that modulate the magnitude, timing, and maintenance of stimulus-induced transcriptional programs. Collectively, macrophage responses emerge from the activity of interconnected layers of cell-type-, tissue-, and signal-dependent transcription factors, forming complex regulatory networks that inflict stimulus-specific outcomes. Dysregulation of these networks can transcriptionally rewire macrophages toward disease-associated states. Delineating transcriptional regulators that distinguish signal responses may enable more targeted disease interventions.

Multicellular cyanobacteria have evolved sophisticated cell-cell communication machinery to exchange, synchronize, and coordinate the efforts of individual cells. Analogous to gap junctions that have traditionally been regarded as a eukaryotic feature, multicellular cyanobacteria coordinate their cell-cell communication via septal junctions (SJs). However, the signals that regulate cell-cell communication and septal-junction assembly are largely unknown. Lately, calcium signaling has been implicated in regulating cell junctions in eukaryotes. We recently discovered a Ca[2+]-binding protein, CSE, which is exclusively found in multicellular cyanobacteria. Here, we investigate CSE as a potential link between calcium signaling and cell-cell communication. We solve the NMR structure of CSE in its Ca[2+]-bound state and revealed that CSE acts as Ca[2+]-buffer protein. Using cryo-electron tomography, we find that Δcse mutant cells display significantly fewer septal junctions as well as SJ precursors known as nanopores. This indicates that CSE is not only essential for Ca[2+] homeostasis, but also mediates cell-cell communication via regulating SJs and nanopores formation, and establishes Ca[2+] signaling and CSE as key players regulating cyanobacterial multicellularity. Furthermore, these findings highlight calcium signaling as a conserved principle for regulating cellular junctions in organisms that diverged a billion years ago.

Prolonged translational arrests caused by defective mRNAs activate the ribosome-associated protein quality control (RQC) pathway, which marks harmful incomplete proteins for degradation. Multipass transmembrane proteins have increased propensity to be targeted by the RQC, raising the question of whether problems in transmembrane domain insertion and assembly can also cause RQC-eliciting translational arrests. Here, we investigated RQC-mediated quality control of CFTR, a large transmembrane protein mutated in cystic fibrosis. Reporter assays showed that although a fraction of nascent CFTR expressed in HEK293 cells arrested during translation and activated the RQC, multiple interventions compromising CFTR folding and membrane insertion did not exacerbate this response. CFTR translation abortion was also largely unaffected by regulators of translation kinetics such as codon usage, the ribosome collision sensor GCN1, and the SRP ER targeting complex. We propose that the RQC can be triggered by the inherent difficulties in synthesizing transmembrane segments, resulting from their inappropriate interaction with the protein synthesis machinery. Our study uncovers and characterizes a novel physiological role for the RQC in dealing with elongation-arrested transmembrane proteins.