Multiple Signaling Roles of CD3ε and Its Application in CAR-T Cell Therapy.

A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3ε ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.

tRNA-m1A modification promotes T cell expansion via efficient MYC protein synthesis.

Naive T cells undergo radical changes during the transition from dormant to hyperactive states upon activation, which necessitates de novo protein production via transcription and translation. However, the mechanism whereby T cells globally promote translation remains largely unknown. Here, we show that on exit from quiescence, T cells upregulate transfer RNA (tRNA) m1A58 'writer' proteins TRMT61A and TRMT6, which confer m1A58 RNA modification on a specific subset of early expressed tRNAs. These m1A-modified early tRNAs enhance translation efficiency, enabling rapid and necessary synthesis of MYC and of a specific group of key functional proteins. The MYC protein then guides the exit of naive T cells from a quiescent state into a proliferative state and promotes rapid T cell expansion after activation. Conditional deletion of the Trmt61a gene in mouse CD4+ T cells causes MYC protein deficiency and cell cycle arrest, disrupts T cell expansion upon cognate antigen stimulation and alleviates colitis in a mouse adoptive transfer colitis model. Our study elucidates for the first time, to our knowledge, the in vivo physiological roles of tRNA-m1A58 modification in T cell-mediated pathogenesis and reveals a new mechanism of tRNA-m1A58-controlled T cell homeostasis and signal-dependent translational control of specific key proteins.

Poxviruses Evade Cytosolic Sensing through Disruption of an mTORC1-mTORC2 Regulatory Circuit.

Viruses employ elaborate strategies to coopt the cellular processes they require to replicate while simultaneously thwarting host antiviral responses. In many instances, how this is accomplished remains poorly understood. Here, we identify a protein, F17 encoded by cytoplasmically replicating poxviruses, that binds and sequesters Raptor and Rictor, regulators of mammalian target of rapamycin complexes mTORC1 and mTORC2, respectively. This disrupts mTORC1-mTORC2 crosstalk that coordinates host responses to poxvirus infection. During infection with poxvirus lacking F17, cGAS accumulates together with endoplasmic reticulum vesicles around the Golgi, where activated STING puncta form, leading to interferon-stimulated gene expression. By contrast, poxvirus expressing F17 dysregulates mTOR, which localizes to the Golgi and blocks these antiviral responses in part through mTOR-dependent cGAS degradation. Ancestral conservation of Raptor/Rictor across eukaryotes, along with expression of F17 across poxviruses, suggests that mTOR dysregulation forms a conserved poxvirus strategy to counter cytosolic sensing while maintaining the metabolic benefits of mTOR activity.

Prospectively Isolated Tetraspanin+ Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.

m6A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development.

N6-methyladenosine (m6A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m6A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m6A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m6A modified in their native state, characterized by low folding energies, and predominantly protein coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mavs or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m6A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.

Conserved class B GPCR activation by a biased intracellular agonist.

Class B G-protein-coupled receptors (GPCRs), including glucagon-like peptide 1 receptor (GLP1R) and parathyroid hormone 1 receptor (PTH1R), are important drug targets1-5. Injectable peptide drugs targeting these receptors have been developed, but orally available small-molecule drugs remain under development6,7. Here we report the high-resolution structure of human PTH1R in complex with the stimulatory G protein (Gs) and a small-molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the cytoplasmic interface of PTH1R with Gs. The PCO371-binding site is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. The residues that make up the PCO371-binding pocket are conserved in class B GPCRs, and a single alteration in PTH2R and two residue alterations in GLP1R convert these receptors to respond to PCO371. Functional assays reveal that PCO371 is a G-protein-biased agonist that is defective in promoting PTH1R-mediated arrestin signalling. Together, these results uncover a distinct binding site for designing small-molecule agonists for PTH1R and possibly other members of the class B GPCRs and define a receptor conformation that is specific only for G-protein activation but not arrestin signalling. These insights should facilitate the design of distinct types of class B GPCR small-molecule agonist for various therapeutic indications.

Plant phytochrome B is an asymmetric dimer with unique signalling potential.

Many aspects of plant photoperception are mediated by the phytochrome (Phy) family of bilin-containing photoreceptors that reversibly interconvert between inactive Pr and active Pfr conformers1,2. Despite extensive biochemical studies, full understanding of plant Phy signalling has remained unclear due to the absence of relevant 3D models. Here we report a cryo-electron microscopy structure of Arabidopsis PhyB in the Pr state that reveals a topologically complex dimeric organization that is substantially distinct from its prokaryotic relatives. Instead of an anticipated parallel architecture, the C-terminal histidine-kinase-related domains (HKRDs) associate head-to-head, whereas the N-terminal photosensory regions associate head-to-tail to form a parallelogram-shaped platform with near two-fold symmetry. The platform is internally linked by the second of two internal Per/Arnt/Sim domains that binds to the photosensory module of the opposing protomer and a preceding 'modulator' loop that assembles tightly with the photosensory module of its own protomer. Both connections accelerate the thermal reversion of Pfr back to Pr, consistent with an inverse relationship between dimer assembly and Pfr stability. Lopsided contacts between the HKRDs and the platform create profound asymmetry to PhyB that might imbue distinct signalling potentials to the protomers. We propose that this unique structural dynamism creates an extensive photostate-sensitive surface for conformation-dependent interactions between plant Phy photoreceptors and their signalling partners.

Molecular Basis for Hormone Recognition and Activation of Corticotropin-Releasing Factor Receptors.

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.

Haplotype-resolved 3D chromatin architecture of the hybrid pig.

In diploid mammals, allele-specific three-dimensional (3D) genome architecture may lead to imbalanced gene expression. Through ultradeep in situ Hi-C sequencing of three representative somatic tissues (liver, skeletal muscle, and brain) from hybrid pigs generated by reciprocal crosses of phenotypically and physiologically divergent Berkshire and Tibetan pigs, we uncover extensive chromatin reorganization between homologous chromosomes across multiple scales. Haplotype-based interrogation of multi-omic data revealed the tissue dependence of 3D chromatin conformation, suggesting that parent-of-origin-specific conformation may drive gene imprinting. We quantify the effects of genetic variations and histone modifications on allelic differences of long-range promoter-enhancer contacts, which likely contribute to the phenotypic differences between the parental pig breeds. We also observe the fine structure of somatically paired homologous chromosomes in the pig genome, which has a functional implication genome-wide. This work illustrates how allele-specific chromatin architecture facilitates concomitant shifts in allele-biased gene expression, as well as the possible consequential phenotypic changes in mammals.

MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche.

Intestinal stromal cells are known to modulate the propagation and differentiation of intestinal stem cells1,2. However, the precise cellular and molecular mechanisms by which this diverse stromal cell population maintains tissue homeostasis and repair are poorly understood. Here we describe a subset of intestinal stromal cells, named MAP3K2-regulated intestinal stromal cells (MRISCs), and show that they are the primary cellular source of the WNT agonist R-spondin 1 following intestinal injury in mice. MRISCs, which are epigenetically and transcriptomically distinct from subsets of intestinal stromal cells that have previously been reported3-6, are strategically localized at the bases of colon crypts, and function to maintain LGR5+ intestinal stem cells and protect against acute intestinal damage through enhanced R-spondin 1 production. Mechanistically, this MAP3K2 specific function is mediated by a previously unknown reactive oxygen species (ROS)-MAP3K2-ERK5-KLF2 axis to enhance production of R-spondin 1. Our results identify MRISCs as a key component of an intestinal stem cell niche that specifically depends on MAP3K2 to augment WNT signalling for the regeneration of damaged intestine.

Structural basis for activation of somatostatin receptor 5 by cyclic neuropeptide agonists.

Somatostatin receptor 5 (SSTR5) is an important G protein-coupled receptor and drug target for neuroendocrine tumors and pituitary disorders. This study presents two high-resolution cryogenicelectron microscope structures of the SSTR5-Gi complexes bound to the cyclic neuropeptide agonists, cortistatin-17 (CST17) and octreotide, with resolutions of 2.7 Å and 2.9 Å, respectively. The structures reveal that binding of these peptides causes rearrangement of a "hydrophobic lock", consisting of residues from transmembrane helices TM3 and TM6. This rearrangement triggers outward movement of TM6, enabling Gαi protein engagement and receptor activation. In addition to hydrophobic interactions, CST17 forms conserved polar contacts similar to somatostatin-14 binding to SSTR2, while further structural and functional analysis shows that extracellular loops differently recognize CST17 and octreotide. These insights elucidate agonist selectivity and activation mechanisms of SSTR5, providing valuable guidance for structure-based drug development targeting this therapeutically relevant receptor.

Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action.

Ago2 differentially regulates oncogenic and tumor-suppressive miRNAs in cancer cells. This discrepancy suggests a secondary event regulating Ago2/miRNA action in a context-dependent manner. We show here that a positive charge of Ago2 K212, that is preserved by SIR2-mediated Ago2 deacetylation in cancer cells, is responsible for the direct interaction between Ago2 and Caveolin-1 (CAV1). Through this interaction, CAV1 sequesters Ago2 on the plasma membranes and regulates miRNA-mediated translational repression in a compartment-dependent manner. Ago2/CAV1 interaction plays a role in miRNA-mediated mRNA suppression and in miRNA release via extracellular vesicles (EVs) from tumors into the circulation, which can be used as a biomarker of tumor progression. Increased Ago2/CAV1 interaction with tumor progression promotes aggressive cancer behaviors, including metastasis. Ago2/CAV1 interaction acts as a secondary event in miRNA-mediated suppression and increases the complexity of miRNA actions in cancer.

UBE2M Is a Stress-Inducible Dual E2 for Neddylation and Ubiquitylation that Promotes Targeted Degradation of UBE2F.

UBE2M and UBE2F are two family members of neddylation E2 conjugating enzyme that, together with E3s, activate CRLs (Cullin-RING Ligases) by catalyzing cullin neddylation. However, whether and how two E2s cross-talk with each other are largely unknown. Here, we report that UBE2M is a stress-inducible gene subjected to cis-transactivation by HIF-1 and AP1, and MLN4924, a small molecule inhibitor of E1 NEDD8-activating enzyme (NAE), upregulates UBE2M via blocking degradation of HIF-1α and c-JUN. UBE2M is a dual E2 for targeted ubiquitylation and degradation of UBE2F, acting as a neddylation E2 to activate CUL3-Keap1 E3 under physiological conditions but as a ubiquitylation E2 for Parkin-DJ-1 E3 under stressed conditions. UBE2M-induced UBE2F degradation leads to CRL5 inactivation and subsequent NOXA accumulation to suppress the growth of lung cancer cells. Collectively, our study establishes a negative regulatory axis between two neddylation E2s with UBE2M ubiquitylating UBE2F, and two CRLs with CRL3 inactivating CRL5.

The giant flexoelectric effect in a luffa plant-based sponge for green devices and energy harvesters.

Soft materials that can produce electrical energy under mechanical stimulus or deform significantly via moderate electrical fields are important for applications ranging from soft robotics to biomedical science. Piezoelectricity, the property that would ostensibly promise such a realization, is notably absent from typical soft matter. Flexoelectricity is an alternative form of electromechanical coupling that universally exists in all dielectrics and can generate electricity under nonuniform deformation such as flexure and conversely, a deformation under inhomogeneous electrical fields. The flexoelectric coupling effect is, however, rather modest for most materials and thus remains a critical bottleneck. In this work, we argue that a significant emergent flexoelectric response can be obtained by leveraging a hierarchical porous structure found in biological materials. We experimentally illustrate our thesis for a natural dry luffa vegetable-based sponge and demonstrate an extraordinarily large mass- and deformability-specific electromechanical response with the highest-density-specific equivalent piezoelectric coefficient known for any material (50 times that of polyvinylidene fluoride and more than 10 times that of lead zirconate titanate). Finally, we demonstrate the application of the fabricated natural sponge as green, biodegradable flexible smart devices in the context of sensing (e.g., for speech, touch pressure) and electrical energy harvesting.

Human FAM111A inhibits vaccinia virus replication by degrading viral protein I3 and is antagonized by poxvirus host range factor SPI-1.

Zoonotic poxviruses such as mpox virus (MPXV) continue to threaten public health safety since the eradication of smallpox. Vaccinia virus (VACV), the prototypic poxvirus used as the vaccine strain for smallpox eradication, is the best-characterized member of the poxvirus family. VACV encodes a serine protease inhibitor 1 (SPI-1) conserved in all orthopoxviruses, which has been recognized as a host range factor for modified VACV Ankara (MVA), an approved smallpox vaccine and a promising vaccine vector. FAM111A (family with sequence similarity 111 member A), a nuclear protein that regulates host DNA replication, was shown to restrict the replication of a VACV SPI-1 deletion mutant (VACV-ΔSPI-1) in human cells. Nevertheless, the detailed antiviral mechanisms of FAM111A were unresolved. Here, we show that FAM111A is a potent restriction factor for VACV-ΔSPI-1 and MVA. Deletion of FAM111A rescued the replication of MVA and VACV-ΔSPI-1 and overexpression of FAM111A significantly reduced viral DNA replication and virus titers but did not affect viral early gene expression. The antiviral effect of FAM111A necessitated its trypsin-like protease domain and DNA-binding domain but not the PCNA-interacting motif. We further identified that FAM111A translocated into the cytoplasm upon VACV infection by degrading the nuclear pore complex via its protease activity, interacted with VACV DNA-binding protein I3, and promoted I3 degradation through autophagy. Moreover, SPI-1 from VACV, MPXV, or lumpy skin disease virus was able to antagonize FAM111A by prohibiting its nuclear export. Our findings reveal the detailed mechanism by which FAM111A inhibits VACV and provide explanations for the immune evasive function of VACV SPI-1.

MSI-XGNN: an explainable GNN computational framework integrating transcription- and methylation-level biomarkers for microsatellite instability detection.

Microsatellite instability (MSI) is a hypermutator phenotype caused by DNA mismatch repair deficiency. MSI has been reported in various human cancers, particularly colorectal, gastric and endometrial cancers. MSI is a promising biomarker for cancer prognosis and immune checkpoint blockade immunotherapy. Several computational methods have been developed for MSI detection using DNA- or RNA-based approaches based on next-generation sequencing. Epigenetic mechanisms, such as DNA methylation, regulate gene expression and play critical roles in the development and progression of cancer. We here developed MSI-XGNN, a new computational framework for predicting MSI status using bulk RNA-sequencing and DNA methylation data. MSI-XGNN is an explainable deep learning model that combines a graph neural network (GNN) model to extract features from the gene-methylation probe network with a CatBoost model to classify MSI status. MSI-XGNN, which requires tumor-only samples, exhibited comparable performance with two well-known methods that require tumor-normal paired sequencing data, MSIsensor and MANTIS and better performance than several other tools. MSI-XGNN also showed good generalizability on independent validation datasets. MSI-XGNN identified six MSI markers consisting of four methylation probes (EPM2AIP1|MLH1:cg14598950, EPM2AIP1|MLH1:cg27331401, LNP1:cg05428436 and TSC22D2:cg15048832) and two genes (RPL22L1 and MSH4) constituting the optimal feature subset. All six markers were significantly associated with beneficial tumor microenvironment characteristics for immunotherapy, such as tumor mutation burden, neoantigens and immune checkpoint molecules such as programmed cell death-1 and cytotoxic T-lymphocyte antigen-4. Overall, our study provides a powerful and explainable deep learning model for predicting MSI status and identifying MSI markers that can potentially be used for clinical MSI evaluation.

INPP5K controls the dynamic structure and signaling of wild-type and mutated, leukemia-associated IL-7 receptors.

The downstream signaling of the interleukin-7 (IL-7) receptor (IL-7R) plays important physiological and pathological roles, including the differentiation of lymphoid cells and proliferation of acute lymphoblastic leukemia cells. Gain-of-function mutations in the IL-7Rα chain, the specific component of the receptor for IL-7, result in constitutive, IL-7-independent signaling and trigger acute lymphoblastic leukemia. Here, we show that the loss of the phosphoinositide 5-phosphatase INPP5K is associated with increased levels of the INPP5K substrate phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) and causes an altered dynamic structure of the IL-7 receptor. We discovered that the IL-7Rα chain contains a very conserved positively charged polybasic amino acid sequence in its cytoplasmic juxtamembrane region; this region establish stronger ionic interactions with negatively charged PtdIns(4,5)P2 in the absence of INPP5K, freezing the IL-7Rα chain structure. This dynamic structural alteration causes defects in IL-7R signaling, culminating in decreased expressions of EBF1 and PAX5 transcription factors, in microdomain formation, cytoskeletal reorganization, and bone marrow B-cell differentiation. Similar alterations after the reduced INPP5K expression also affected mutated, constitutively activated IL-7Rα chains that trigger leukemia development, leading to reduced cell proliferation. Altogether, our results indicate that the lipid 5-phosphatase INPP5K hydrolyzes PtdIns(4,5)P2, allowing the requisite conformational changes of the IL-7Rα chain for optimal signaling.

Phytochrome interacting factor regulates stomatal aperture by coordinating red light and abscisic acid.

Stomata are crucial valves coordinating the fixation of carbon dioxide by photosynthesis and water loss through leaf transpiration. Phytochrome interacting factors (PIFs) are negative regulators of red light responses that belong to the basic helix-loop-helix family of transcription factors. Here, we show that the rice (Oryza sativa) PIF family gene OsPIL15 acts as a negative regulator of stomatal aperture to control transpiration in rice. OsPIL15 reduces stomatal aperture by activating rice ABSCISIC ACID INSENSITIVE 5 (OsABI5), which encodes a critical positive regulator of ABSCISIC ACID (ABA) signaling in rice. Moreover, OsPIL15 interacts with the NIGT1/HRS1/HHO family transcription factor rice HRS1 HOMOLOG 3 (OsHHO3) to possibly enhance the regulation of stomatal aperture. Notably, we discovered that the maize (Zea mays) PIF family genes ZmPIF1 and ZmPIF3, which are homologous to OsPIL15, are also involved in the regulation of stomatal aperture in maize, indicating that PIF-mediated regulation of stomatal aperture may be conserved in the plant lineage. Our findings explain the molecular mechanism by which PIFs play a role in red-light-mediated stomatal opening, and demonstrate that PIFs regulate stomatal aperture by coordinating the red light and ABA signaling pathways.

Structural basis for heparan sulfate co-polymerase action by the EXT1-2 complex.

Heparan sulfate (HS) proteoglycans are extended (-GlcAß1,4GlcNAcα1,4-)n co-polymers containing decorations of sulfation and epimerization that are linked to cell surface and extracellular matrix proteins. In mammals, HS repeat units are extended by an obligate heterocomplex of two exostosin family members, EXT1 and EXT2, where each protein monomer contains distinct GT47 (GT-B fold) and GT64 (GT-A fold) glycosyltransferase domains. In this study, we generated human EXT1-EXT2 (EXT1-2) as a functional heterocomplex and determined its structure in the presence of bound donor and acceptor substrates. Structural data and enzyme activity of catalytic site mutants demonstrate that only two of the four glycosyltransferase domains are major contributors to co-polymer syntheses: the EXT1 GT-B fold ß1,4GlcA transferase domain and the EXT2 GT-A fold α1,4GlcNAc transferase domain. The two catalytic sites are over 90 Å apart, indicating that HS is synthesized by a dissociative process that involves a single catalytic site on each monomer.

Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing.

Paraspeckles are subnuclear RNA-protein structures that are implicated in important processes including cellular stress response, differentiation, and cancer progression. However, it is unclear how paraspeckles impart their physiological effect at the molecular level. Through biochemical analyses, we show that paraspeckles interact with the SWI/SNF chromatin-remodeling complex. This is specifically mediated by the direct interaction of the long-non-coding RNA NEAT1 of the paraspeckles with ARID1B of the cBAF-type SWI/SNF complex. Strikingly, ARID1B depletion, in addition to resulting in loss of interaction with the SWI/SNF complex, decreases the binding of paraspeckle proteins to chromatin modifiers, transcription factors, and histones. Functionally, the loss of ARID1B and NEAT1 influences the transcription and the alternative splicing of a common set of genes. Our findings reveal that dynamic granules such as the paraspeckles may leverage the specificity of epigenetic modifiers to impart their regulatory effect, thus providing a molecular basis for their function.