ABSTRACT
All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.
ABSTRACT
Allogeneic chimeric antigen receptor (CAR)-T cells hold great promise for expanding the accessibility of CAR-T therapy, whereas the risks of allograft rejection have hampered its application. Here, we genetically engineered healthy-donor-derived, CD19-targeting CAR-T cells using CRISPR-Cas9 to address the issue of immune rejection and treated one patient with refractory immune-mediated necrotizing myopathy and two patients with diffuse cutaneous systemic sclerosis with these cells. This study was registered at ClinicalTrials.gov (NCT05859997). The infused cells persisted for over 3 months, achieving complete B cell depletion within 2 weeks of treatment. During the 6-month follow-up, we observed deep remission without cytokine release syndrome or other serious adverse events in all three patients, primarily shown by the significant improvement in the clinical response index scores for the two diseases, respectively, and supported by the observations of reversal of inflammation and fibrosis. Our results demonstrate the high safety and promising immune modulatory effect of the off-the-shelf CAR-T cells in treating severe refractory autoimmune diseases.
ABSTRACT
Chimeric antigen receptor (CAR) T cell therapies have successfully treated hematological malignancies. Macrophages have also gained attention as an immunotherapy owing to their immunomodulatory capacity and ability to infiltrate solid tumors and phagocytize tumor cells. The first-generation CD3ζ-based CAR-macrophages could phagocytose tumor cells in an antigen-dependent manner. Here we engineered induced pluripotent stem cell-derived macrophages (iMACs) with toll-like receptor 4 intracellular toll/IL-1R (TIR) domain-containing CARs resulting in a markedly enhanced antitumor effect over first-generation CAR-macrophages. Moreover, the design of a tandem CD3ζ-TIR dual signaling CAR endows iMACs with both target engulfment capacity and antigen-dependent M1 polarization and M2 resistance in a nuclear factor kappa B (NF-κB)-dependent manner, as well as the capacity to modulate the tumor microenvironment. We also outline a mechanism of tumor cell elimination by CAR-induced efferocytosis against tumor cell apoptotic bodies. Taken together, we provide a second-generation CAR-iMAC with an ability for orthogonal phagocytosis and polarization and superior antitumor functions in treating solid tumors relative to first-generation CAR-macrophages.
Subject(s)
Neoplasms , Receptors, Chimeric Antigen , Humans , Receptors, Antigen, T-Cell , T-Lymphocytes , Cell Line, Tumor , Receptors, Chimeric Antigen/genetics , Immunotherapy, Adoptive/methods , Macrophages/pathology , Tumor MicroenvironmentABSTRACT
The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.
Subject(s)
Carcinogenesis/metabolism , Carcinoma, Hepatocellular/genetics , Cell Compartmentation/genetics , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/metabolism , Cyclic AMP/metabolism , HSP40 Heat-Shock Proteins/genetics , Liver Neoplasms/genetics , Signal Transduction , Animals , Carcinogenesis/drug effects , Carcinogenesis/genetics , Carcinoma, Hepatocellular/metabolism , Cell Compartmentation/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cyclic AMP/pharmacology , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/genetics , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoplasm/metabolism , Humans , Liver Neoplasms/metabolism , Mice , Oncogenes/genetics , Protein Domains , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins , Spectroscopy, Fourier Transform Infrared , Time-Lapse Imaging/methodsABSTRACT
Fibrosis can develop in most organs and causes organ failure. The most common type of lung fibrosis is known as idiopathic pulmonary fibrosis, in which fibrosis starts at the lung periphery and then progresses toward the lung center, eventually causing respiratory failure. Little is known about the mechanisms underlying the pathogenesis and periphery-to-center progression of the disease. Here we discovered that loss of Cdc42 function in alveolar stem cells (AT2 cells) causes periphery-to-center progressive lung fibrosis. We further show that Cdc42-null AT2 cells in both post-pneumonectomy and untreated aged mice cannot regenerate new alveoli, resulting in sustained exposure of AT2 cells to elevated mechanical tension. We demonstrate that elevated mechanical tension activates a TGF-ß signaling loop in AT2 cells, which drives the periphery-to-center progression of lung fibrosis. Our study establishes a direct mechanistic link between impaired alveolar regeneration, mechanical tension, and progressive lung fibrosis.
Subject(s)
Adult Stem Cells/metabolism , Idiopathic Pulmonary Fibrosis/etiology , Pulmonary Alveoli/metabolism , Adult Stem Cells/pathology , Aged , Alveolar Epithelial Cells/pathology , Animals , Biomechanical Phenomena/physiology , Female , Fibrosis/pathology , Humans , Idiopathic Pulmonary Fibrosis/metabolism , Idiopathic Pulmonary Fibrosis/pathology , Lung/pathology , Male , Mice , Middle Aged , Pulmonary Alveoli/pathology , Regeneration , Signal Transduction , Stem Cells/pathology , Stress, Mechanical , Stress, Physiological/physiology , Transforming Growth Factor beta/metabolism , cdc42 GTP-Binding Protein/genetics , cdc42 GTP-Binding Protein/metabolismABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 µg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.
Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Drug Evaluation, Preclinical/methods , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Vaccines, Inactivated/therapeutic use , Viral Vaccines/therapeutic use , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/genetics , COVID-19 , COVID-19 Vaccines , Chlorocebus aethiops , Coronavirus Infections/virology , Disease Models, Animal , Female , Guinea Pigs , Immunogenicity, Vaccine , Macaca fascicularis , Macaca mulatta , Male , Mice , Mice, Inbred BALB C , Phylogeny , Pneumonia, Viral/virology , Rabbits , Rats , Rats, Wistar , SARS-CoV-2 , Vaccines, Inactivated/adverse effects , Vero Cells , Viral Vaccines/adverse effectsABSTRACT
The ability to sense sour provides an important sensory signal to prevent the ingestion of unripe, spoiled, or fermented foods. Taste and somatosensory receptors in the oral cavity trigger aversive behaviors in response to acid stimuli. Here, we show that the ion channel Otopetrin-1, a proton-selective channel normally involved in the sensation of gravity in the vestibular system, is essential for sour sensing in the taste system. We demonstrate that knockout of Otop1 eliminates acid responses from sour-sensing taste receptor cells (TRCs). In addition, we show that mice engineered to express otopetrin-1 in sweet TRCs have sweet cells that also respond to sour stimuli. Next, we genetically identified the taste ganglion neurons mediating each of the five basic taste qualities and demonstrate that sour taste uses its own dedicated labeled line from TRCs in the tongue to finely tuned taste neurons in the brain to trigger aversive behaviors.
Subject(s)
Brain/physiology , Membrane Proteins/metabolism , Taste Buds/metabolism , Taste , Acids/pharmacology , Afferent Pathways/cytology , Afferent Pathways/metabolism , Afferent Pathways/physiology , Animals , Brain/cytology , Brain/metabolism , Female , Male , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Taste Buds/drug effects , Taste Buds/physiology , Taste PerceptionABSTRACT
While mutations affecting protein-coding regions have been examined across many cancers, structural variants at the genome-wide level are still poorly defined. Through integrative deep whole-genome and -transcriptome analysis of 101 castration-resistant prostate cancer metastases (109X tumor/38X normal coverage), we identified structural variants altering critical regulators of tumorigenesis and progression not detectable by exome approaches. Notably, we observed amplification of an intergenic enhancer region 624 kb upstream of the androgen receptor (AR) in 81% of patients, correlating with increased AR expression. Tandem duplication hotspots also occur near MYC, in lncRNAs associated with post-translational MYC regulation. Classes of structural variations were linked to distinct DNA repair deficiencies, suggesting their etiology, including associations of CDK12 mutation with tandem duplications, TP53 inactivation with inverted rearrangements and chromothripsis, and BRCA2 inactivation with deletions. Together, these observations provide a comprehensive view of how structural variations affect critical regulators in metastatic prostate cancer.
Subject(s)
Genomic Structural Variation/genetics , Prostatic Neoplasms/genetics , Aged , Aged, 80 and over , BRCA2 Protein/metabolism , Cyclin-Dependent Kinases/metabolism , DNA Copy Number Variations , Exome , Gene Expression Profiling/methods , Genomics/methods , Humans , Male , Middle Aged , Mutation , Neoplasm Metastasis/genetics , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Tandem Repeat Sequences/genetics , Tumor Suppressor Protein p53/metabolism , Whole Genome Sequencing/methodsABSTRACT
Spatiotemporal regulation of intracellular signaling molecules, such as the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures proper cellular function. Liquid-liquid phase separation (LLPS) of the ubiquitous PKA regulatory subunit RIα promotes cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα LLPS remain unclear. Here, we reveal that two separate dimerization interfaces, combined with the cAMP-induced unleashing of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence, drive RIα condensate formation in the cytosol of mammalian cells, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα pseudosubstrate region is critically involved in forming a non-canonical R:C complex, which recruits active PKA-C to RIα condensates to maintain low basal PKA activity in the cytosol. Our results suggest that RIα LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity.
Subject(s)
Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , Phase Separation , Animals , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/genetics , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/chemistry , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/metabolism , Signal Transduction , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Mammals/metabolismABSTRACT
Cell signaling is a complex process. The faithful transduction of information into specific cellular actions depends on the synergistic effects of many regulatory molecules, nurtured by their strict spatiotemporal regulation. Over the years, we have gained copious insights into the subcellular architecture supporting this spatiotemporal control, including the roles of membrane-bound organelles and various signaling nanodomains. Recently, liquid-liquid phase separation (LLPS) has been recognized as another potentially ubiquitous framework for organizing signaling molecules with high specificity and precise spatiotemporal control in cells. Here, we review the pervasive role of LLPS in signal transduction, highlighting several key pathways that intersect with LLPS, including examples in which LLPS is controlled by signaling events. We also examine how LLPS orchestrates signaling by compartmentalizing signaling molecules, amplifying signals non-linearly, and moderating signaling dynamics. We focus on the specific molecules that drive LLPS and highlight the known functional and pathological consequences of LLPS in each pathway.
Subject(s)
Cell Compartmentation , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Space/metabolism , Intrinsically Disordered Proteins/metabolism , Organelles/metabolism , Signal Transduction , Animals , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intrinsically Disordered Proteins/chemistry , Time FactorsABSTRACT
p53 is critical for tumor suppression but also elicits detrimental effects when aberrantly overexpressed. Thus, multiple regulators, including RNA-binding protein RBM38, are found to tightly control p53 expression. Interestingly, RBM38 is unique in that it can either suppress or enhance p53 mRNA translation via altered interaction with eIF4E potentially mediated by serine-195 (S195) in RBM38. Thus, multiple RBM38/eIF4E knock-in (KI) cell lines were generated to investigate the significance of eIF4E-RBM38 interaction in controlling p53 activity. We showed that KI of RBM38-S195D or -Y192C enhances, whereas KI of RBM38-S195K/R/L weakens, the binding of eIF4E to p53 mRNA and subsequently p53 expression. We also showed that KI of eIF4E-D202K weakens the interaction of eIF4E with RBM38 and thereby enhances p53 expression, suggesting that D202 in eIF4E interacts with S195 in RBM38. Moreover, we generated an Rbm38 S193D KI mouse model in which human-equivalent serine-193 is substituted with aspartic acid. We showed that S193D KI enhances p53-dependent cellular senescence and that S193D KI mice have a shortened life span and are prone to spontaneous tumors, chronic inflammation, and liver steatosis. Together, we provide in vivo evidence that the RBM38-eIF4E loop can be explored to fine-tune p53 expression for therapeutic development.
Subject(s)
Eukaryotic Initiation Factor-4E/metabolism , Gene Expression Regulation, Neoplastic/genetics , RNA-Binding Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Carcinogenesis/genetics , Cell Line , Cellular Senescence/genetics , Eukaryotic Initiation Factor-4E/genetics , Fatty Liver/genetics , Gene Knock-In Techniques , Inflammation/genetics , Longevity/genetics , Mice , Protein Binding/genetics , RNA-Binding Proteins/genetics , Tumor Suppressor Protein p53/geneticsABSTRACT
G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)-a master regulator of cell proliferation and survival1. However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood2-4. Here we investigated how spatially organized ß2-adrenergic receptor (ß2AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors5, we show that ß2AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gαs and requires ligand-stimulated ß2AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gαs, RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal ß2AR and Gαs signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis.
Subject(s)
Adrenergic Agents , Endosomes , Extracellular Signal-Regulated MAP Kinases , Receptors, Adrenergic, beta-2 , Adrenergic Agents/metabolism , Adrenergic Agents/pharmacology , Cell Proliferation , Endosomes/drug effects , Endosomes/enzymology , Endosomes/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Genes, myc , GTP-Binding Protein alpha Subunits, Gs/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Phosphorylation/drug effects , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
The cell wall shapes plant cell morphogenesis and affects the plasticity of organ growth. However, the way in which cell wall establishment is regulated by ethylene remains largely elusive. Here, by analyzing cell wall patterns, cell wall composition and gene expression in rice (Oryza sativa, L.) roots, we found that ethylene induces cell wall thickening and the expression of cell wall synthesis-related genes, including CELLULOSE SYNTHASE-LIKE C1, 2, 7, 9, 10 (OsCSLC1, 2, 7, 9, 10) and CELLULOSE SYNTHASE A3, 4, 7, 9 (OsCESA3, 4, 7, 9). Overexpression and mutant analyses revealed that OsCSLC2 and its homologs function in ethylene-mediated induction of xyloglucan biosynthesis mainly in the cell wall of root epidermal cells. Moreover, OsCESA-catalyzed cellulose deposition in the cell wall was enhanced by ethylene. OsCSLC-mediated xyloglucan biosynthesis likely plays an important role in restricting cell wall extension and cell elongation during the ethylene response in rice roots. Genetically, OsCSLC2 acts downstream of ETHYLENE-INSENSITIVE3-LIKE1 (OsEIL1)-mediated ethylene signaling, and OsCSLC1, 2, 7, 9 are directly activated by OsEIL1. Furthermore, the auxin signaling pathway is synergistically involved in these regulatory processes. These findings link plant hormone signaling with cell wall establishment, broadening our understanding of root growth plasticity in rice and other crops.
ABSTRACT
The phylogeny and divergence timing of the Neoavian radiation remain controversial despite recent progress. We analyzed the genomes of 124 species across all Neoavian orders, using data from 25,460 loci spanning four DNA classes, including 5,756 coding sequences, 12,449 conserved nonexonic elements, 4,871 introns, and 2,384 intergenic segments. We conducted a comprehensive sensitivity analysis to account for the heterogeneity across different DNA classes, leading to an optimal tree of Neoaves with high resolution. This phylogeny features a novel Neoavian dichotomy comprising two monophyletic clades: a previously recognized Telluraves (land birds) and a newly circumscribed Aquaterraves (waterbirds and relatives). Molecular dating analyses with 20 fossil calibrations indicate that the diversification of modern birds began in the Late Cretaceous and underwent a constant and steady radiation across the KPg boundary, concurrent with the rise of angiosperms as well as other major Cenozoic animal groups including placental and multituberculate mammals. The KPg catastrophe had a limited impact on avian evolution compared to the Paleocene-Eocene Thermal Maximum, which triggered a rapid diversification of seabirds. Our findings suggest that the evolution of modern birds followed a slow process of gradualism rather than a rapid process of punctuated equilibrium, with limited interruption by the KPg catastrophe. This study places bird evolution into a new context within vertebrates, with ramifications for the evolution of the Earth's biota.
Subject(s)
Fossils , Magnoliopsida , Pregnancy , Female , Animals , Magnoliopsida/genetics , Placenta , Phylogeny , Birds/genetics , Mammals/genetics , DNA, Mitochondrial/genetics , Biological EvolutionABSTRACT
Real-time visualization of a wide range of biochemical processes in living systems is being made possible through the development and application of genetically encoded fluorescent reporters. These versatile biosensors have proven themselves tailor-made to the study of signal transduction, and in this review, we discuss some of the unique insights that they continue to provide regarding the spatial organization and dynamic regulation of intracellular signaling networks. In addition, we explore the more recent push to expand the scope of biological phenomena that can be monitored using these reporters, while also considering the potential to integrate this highly adaptable technology with a number of emerging techniques that may significantly broaden our view of how networks of biochemical processes shape larger biological phenomena.
Subject(s)
Biosensing Techniques/methods , Fluorescence Resonance Energy Transfer/methods , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Signal Transduction/physiology , Animals , Calcium/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Green Fluorescent Proteins/chemistry , Intracellular Membranes/chemistry , Models, MolecularABSTRACT
Stimulatory immune receptor NKG2D binds diverse ligands to elicit differential anti-tumor and anti-virus immune responses. Two conflicting degeneracy recognition models based on static crystal structures and in-solution binding affinities have been considered for almost two decades. Whether and how NKG2D recognizes and discriminates diverse ligands still remain unclear. Using live-cell-based single-molecule biomechanical assay, we characterized the in situ binding kinetics of NKG2D interacting with different ligands in the absence or presence of mechanical force. We found that mechanical force application selectively prolonged NKG2D interaction lifetimes with the ligands MICA and MICB, but not with ULBPs, and that force-strengthened binding is much more pronounced for MICA than for other ligands. We also integrated steered molecular dynamics simulations and mutagenesis to reveal force-induced rotational conformational changes of MICA, involving formation of additional hydrogen bonds on its binding interface with NKG2D, impeding MICA dissociation under force. We further provided a kinetic triggering model to reveal that force-dependent affinity determines NKG2D ligand discrimination and its downstream NK cell activation. Together, our results demonstrate that NKG2D has a discrimination power to recognize different ligands, which depends on selective mechanical force-induced ligand conformational changes.
Subject(s)
NK Cell Lectin-Like Receptor Subfamily K/chemistry , Binding Sites , Cells, Cultured , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/metabolism , Humans , K562 Cells , Ligands , Mechanical Phenomena , Molecular Dynamics Simulation , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Protein Binding , Single Molecule ImagingABSTRACT
Dynamic metabolism is exhibited by early mammalian embryos to support changing cell fates during development. It is widely acknowledged that metabolic pathways not only satisfy cellular energetic demands, but also play pivotal roles in the process of cell signalling, gene regulation, cell proliferation and differentiation. Recently, various new technological advances have been made in metabolomics and computational analysis, deepening our understanding of the crucial role of dynamic metabolism during early mammalian embryogenesis. In this Review, we summarize recent studies on oocyte and embryo metabolism and its regulation, with a particular focus on its association with key developmental events such as fertilization, zygote genome activation and cell fate determination. In addition, we discuss the mechanisms of certain metabolites that, in addition to serving as energy sources, contribute to epigenetic modifications.