Linking chromatin acylation mark-defined proteome and genome in living cells.

A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and ß-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation and established linkages for chromatin acylation mark-defined proteome, genome, and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr's gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the "metabolites-modification-regulation" axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.

SAR1B senses leucine levels to regulate mTORC1 signalling.

The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels1. Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B-GATOR2-mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer.

DeKinomics pulse-chases kinase functions in living cells.

Cellular context is crucial for understanding the complex and dynamic kinase functions in health and disease. Systematic dissection of kinase-mediated cellular processes requires rapid and precise stimulation ('pulse') of a kinase of interest, as well as global and in-depth characterization ('chase') of the perturbed proteome under living conditions. Here we developed an optogenetic 'pulse-chase' strategy, termed decaging kinase coupled proteomics (DeKinomics), for proteome-wide profiling of kinase-driven phosphorylation at second-timescale in living cells. We took advantage of the 'gain-of-function' feature of DeKinomics to identify direct kinase substrates and further portrayed the global phosphorylation of understudied receptor tyrosine kinases under native cellular settings. DeKinomics offered a general activation-based strategy to study kinase functions with high specificity and temporal resolution under living conditions.

Orange/far-red hybrid voltage indicators with reduced phototoxicity enable reliable long-term imaging in neurons and cardiomyocytes.

Hybrid voltage indicators (HVIs) are chemogenetic sensors that combines the superior photophysical properties of organic dyes and the genetic targetability of protein sensors to report transient membrane voltage changes. They exhibit boosted sensitivity in excitable cells such as neurons and cardiomyocytes. However, the voltage signals recorded during long-term imaging are severely diminished or distorted due to phototoxicity and photobleaching issues. To capture stable electrophysiological activities over a long time, we employ cyanine dyes conjugated with a cyclooctatetraene (COT) molecule as the fluorescence reporter of HVI. The resulting orange-emitting HVI-COT-Cy3 enables high-fidelity voltage imaging for up to 30 min in cultured primary neurons with a sensitivity of ~ -30% ΔF/F0 per action potential (AP). It also maximally preserves the signal of individual APs in cardiomyocytes. The far-red-emitting HVI-COT-Cy5 allows two-color voltage/calcium imaging with GCaMP6s in neurons and cardiomyocytes for 15 min. We leverage the HVI-COT series with reduced phototoxicity and photobleaching to evaluate the impact of drug candidates on the electrophysiology of excitable cells.

Time-resolved protein activation by proximal decaging in living systems.

A universal gain-of-function approach for selective and temporal control of protein activity in living systems is crucial to understanding dynamic cellular processes. Here we report development of a computationally aided and genetically encoded proximal decaging (hereafter, CAGE-prox) strategy that enables time-resolved activation of a broad range of proteins in living cells and mice. Temporal blockage of protein activity was computationally designed and realized by genetic incorporation of a photo-caged amino acid in proximity to the functional site of the protein, which can be rapidly removed upon decaging, resulting in protein re-activation. We demonstrate the wide applicability of our method on diverse protein families, which enabled orthogonal tuning of cell signalling and immune responses, temporal profiling of proteolytic substrates upon caspase activation as well as the development of protein-based pro-drug therapy. We envision that CAGE-prox will open opportunities for the gain-of-function study of proteins and dynamic biological processes with high precision and temporal resolution.

Cerebrovascular Complications Associated With Iatrogenic Fungal Meningitis Following Surgical Procedures in Mexico.

BACKGROUND: The current fungal meningitis outbreak caused by contaminated epidural anesthesia with Fusarium solani among patients who underwent surgical procedures in Matamoros, Mexico remains a cause of concern. Its association with an increased susceptibility for cerebrovascular complications (CVC) has not been reported. This single-center study describes 3 patients with a unique pattern of CVC attributed to fungal meningitis. METHODS: A retrospective case series of patients diagnosed with fungal meningitis following surgical procedures under contaminated epidural anesthesia who developed a unique pattern of CVC during their hospitalization. RESULTS: Three female patients (mean age, 35 years) with CVC due to iatrogenic fungal meningitis were included. Positive Fungitell ß-D-glucan assay in cerebrospinal fluid was documented in all cases, and F. solani was confirmed by polymerase chain reaction in case 3. All cases were complicated by severe vertebrobasilar circulation vasculopathy and arterial dissections with resultant subarachnoid hemorrhage and intraventricular hemorrhage, ultimately leading to patients' death. CONCLUSIONS: The death toll from the ongoing fungal meningitis outbreak keeps rising, underscoring the need for early recognition and aggressive treatment. We highlight the risk for vertebrobasilar circulation CVC among these patients. The angioinvasive nature of F. solani is yet to be clarified; however, a clear pattern has been observed. Public health awareness should be raised and a strong response should be pursued.

Bioorthogonal Quinone Methide Decaging Enables Live-Cell Quantification of Tumor-Specific Immune Interactions.

Effective antitumor immunity hinges on the specific engagement between tumor and cytotoxic immune cells, especially cytotoxic T cells. Although investigating these intercellular interactions is crucial for characterizing immune responses and guiding immunotherapeutic applications, direct and quantitative detection of tumor-T cell interactions within a live-cell context remains challenging. We herein report a photocatalytic live-cell interaction labeling strategy (CAT-Cell) relying on the bioorthogonal decaging of quinone methide moieties for sensitive and selective investigation and quantification of tumor-T cell interactions. By developing quinone methide-derived probes optimized for capturing cell-cell interactions (CCIs), we demonstrated the capacity of CAT-Cell for detecting CCIs directed by various types of receptor-ligand pairs (e.g., CD40-CD40L, TCR-pMHC) and further quantified the strengths of tumor-T cell interactions that are crucial for evaluating the antitumor immune responses. We further applied CAT-Cell for ex vivo quantification of tumor-specific T cell interactions on splenocyte and solid tumor samples from mouse models. Finally, the broad compatibility and utility of CAT-Cell were demonstrated by integrating it with the antigen-specific targeting system as well as for tumor-natural killer cell interaction detection. By leveraging the bioorthogonal photocatalytic decaging chemistry on quinone methide, CAT-Cell provides a sensitive, tunable, universal, and noninvasive toolbox for unraveling and quantifying the crucial but delicate tumor-immune interactions under live-cell settings.

Increasing the efficiency and precision of prime editing with guide RNA pairs.

The recently reported prime editor (PE) can produce all types of base substitution, insertion and deletion, greatly expanding the scope of genome editing. However, improving the editing efficiency and precision of PE represents a major challenge. Here, we report an approach termed the homologous 3' extension mediated prime editor (HOPE). HOPE uses paired prime editing guide RNAs (pegRNAs) encoding the same edits in both sense and antisense DNA strands to achieve high editing efficiency in human embryonic kidney 293T cells as well as mismatch repair-deficient human colorectal carcinoma 116 cells. In addition, we found that HOPE shows greatly improved product purity compared to the original PE3 system. We envision that this enhanced tool could broaden both fundamental research and therapeutic applications of prime editing.

Decaging-to-labeling: Development and investigation of quinone methide warhead for protein labeling.

Biomolecule labeling in living systems is crucial for understanding biological processes and discovering therapeutic targets. A variety of labeling warheads have been developed for multiple biological applications, including proteomics, bioimaging, sequencing, and drug development. Quinone methides (QMs), a class of highly reactive Michael receptors, have recently emerged as prominent warheads for on-demand biomolecule labeling. Their highly flexible functionality and tunability allow for diverse biological applications, but remain poorly explored at present. In this regard, we designed, synthesized, and evaluated a series of new QM probes with a trifluoromethyl group at the benzyl position and substituents on the aromatic ring to manipulate their chemical properties for biomolecule labeling. The engineered QM warhead efficiently labeled proteins both in vitro and under living cell conditions, with significantly enhanced activity compared to previous QM warheads. We further analyzed the labeling efficacy with the assistance of density functional theory (DFT) calculations, which revealed that the QM generation process, rather than the reactivity of QM, contributes more predominantly to the labeling efficacy. Noteworthy, twelve nucleophilic residues on the BSA were labeled by the probe, including Cys, Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, Thr, Trp and Tyr. Given their high efficiency and tunability, these new QM warheads may hold great promise for a broad range of applications, especially spatiotemporal proteomic profiling for in-depth biological studies.

Spatiotemporally resolved subcellular phosphoproteomics.

Proteome-wide profiling of protein phosphorylation has been widely used to reveal the underlying mechanism of diverse cellular signaling events. Yet, characterizing subcellular phosphoproteome with high spatial-temporal resolution has remained challenging. Herein, we developed a subcellular-specific uncaging-assisted biotinylation and mapping of phosphoproteome (SubMAPP) strategy to monitor the phosphorylation dynamics of subcellular proteome in living cells and animals. Our method capitalizes on the genetically encoded bioorthogonal decaging strategy, which enables the rapid activation of subcellular localized proximity labeling biotin ligase through either light illumination or small-molecule triggers. By further adopting an integrated orthogonal pull-down strategy with quantitative mass spectrometry, SubMAPP allowed for the investigation of subcellular phosphoproteome dynamics, revealing the altered phosphorylation patterns of endoplasmic reticulum (ER) luminal proteins under ER stress. Finally, we further expanded the scope of the SubMAPP strategy to primary neuron culture and living mice.

Optical Control of Protein Functions via Genetically Encoded Photocaged Aspartic Acids.

Site-specific protein decaging by light has become an effective approach for in situ manipulation of protein activities in a gain-of-function fashion. Although successful decaging of amino acid side chains of Lys, Tyr, Cys, and Glu has been demonstrated, this strategy has not been extended to aspartic acid (Asp), an essential amino acid residue with a range of protein functions and protein-protein interactions. We herein reported a genetically encoded photocaged Asp and applied it to the photocontrolled manipulation of a panel of proteins including firefly luciferase, kinases (e.g., BRAF), and GTPase (e.g., KRAS) as well as mimicking the in situ phosphorylation event on kinases. As a new member of the increasingly expanded amino acid-decaging toolbox, photocaged Asp may find broad applications for gain-of-function study of diverse proteins as well as biological processes in living cells.

A Dual-Mechanism Targeted Bioorthogonal Prodrug Therapy.

Bioorthogonal prodrug therapies offer an intriguing two-component system that features enhanced circulating stability and controlled activation on demand. Current strategies often deliver either the prodrug or its complementary activator to the tumor with a monomechanism targeted mechanism, which cannot achieve the desired antitumor efficacy and safety profile. The orchestration of two distinct and orthogonal mechanisms should overcome the hierarchical heterogeneity of solid tumors to improve the delivery efficiency of both components simultaneously for bio-orthogonal prodrug therapies. We herein developed a dual-mechanism targeted bioorthogonal prodrug therapy by integrating two orthogonal, receptor-independent tumor-targeting strategies. We first employed the endogenous albumin transport system to generate the in situ albumin-bound, bioorthogonal-caged doxorubicin prodrug with extended plasma circulation and selective accumulation at the tumor site. We then employed enzyme-instructed self-assembly (EISA) to specifically enrich the bioorthogonal activators within tumor cells. As each targeted delivery mode induced an intrinsic pharmacokinetic profile, further optimization of the administration sequence according to their pharmacokinetics allowed the spatiotemporally controlled prodrug activation on-target and on-demand. Taken together, by orchestrating two discrete and receptor-independent targeting strategies, we developed an all-small-molecule based bioorthogonal prodrug system for dual-mechanism targeted anticancer therapies to maximize therapeutic efficacy and minimize adverse drug reactions for chemotherapeutic agents.

Near Infrared Light-Triggered Photocatalytic Decaging for Remote-Controlled Spatiotemporal Activation in Living Mice.

Spatiotemporal manipulation of biological processes in living animals using noninvasive, remote-controlled stimuli is a captivating but challenging endeavor. Herein, we present the development of a biocompatible photocatalytic technology termed CAT-NIR, which uses external near infrared light (NIR, 740 nm) to trigger decaging reactions in living mice. The Os(II) terpyridine complex was identified as an efficient NIR photocatalyst for promoting deboronative hydroxylation reactions via superoxide generation in the presence of NIR light, resulting in the deprotection of phenol groups and the release of bioactive molecules under living conditions. The validation of the CAT-NIR system was demonstrated through the NIR-triggered rescue of fluorophores, prodrugs as well as biomolecules ranging from amino acids, peptides to proteins. Furthermore, by combining genetic code expansion and computer-aided screening, CAT-NIR could regulate affibody binding to the cell surface receptor HER2, providing a selective cell tagging technology through external NIR light. In particular, the tissue-penetrating ability of NIR light allowed for facile prodrug activation in living mice, enabling noninvasive, remote-controlled rescue of drug molecules. Given its broad adaptability, this CAT-NIR system may open new opportunities for manipulating the functions of bioactive molecules in living animals using external NIR light with spatiotemporal resolution.

Controlled Genetic Encoding of Unnatural Amino Acids in a Protein Nanopore.

Conventional protein engineering methods for modifying protein nanopores are typically limited to 20 natural amino acids, which restrict the diversity of the nanopores in structure and function. To enrich the chemical environment inside the nanopore, we employed the genetic code expansion (GCE) technique to site-specifically incorporate the unnatural amino acid (UAA) into the sensing region of aerolysin nanopores. This approach leveraged the efficient pyrrolysine-based aminoacyl-tRNA synthetase-tRNA pair for a high yield of pore-forming protein. Both molecular dynamics (MD) simulations and single-molecule sensing experiments demonstrated that the conformation of UAA residues provided a favorable geometric orientation for the interactions of target molecules and the pore. This rationally designed chemical environment enabled the direct discrimination of multiple peptides containing hydrophobic amino acids. Our work provides a new framework for endowing nanopores with unique sensing properties that are difficult to achieve using classical protein engineering approaches.

Bioorthogonally Activatable Base Editing for On-Demand Pyroptosis.

Pyroptosis is an inflammatory cell death form triggered by protease-mediated truncation and release of the N-terminal pore-forming domain of the gasdermin (GSDM) family proteins in various cell types. We report a Bioorthogonally ACtivatable Base editor (BaseBAC) for in situ and on-demand initiation of cell-type-specific pyroptosis. We first made the enzymatic activity of a cytosine base editor (CBE) switchable by establishing a bioorthogonal blockage on the PAM-interacting residue to control its DNA-binding ability. The resulting BaseBAC allowed in situ control of base editing on the GSDME gene that switched to the truncated expression of its N-terminal domain to activate pyroptosis. BaseBAC offers a general method for on-demand awakening of functional domains of self-inhibiting proteins and the corresponding cellular processes with high specificity in living systems.

Antigen-Specific T Cell Detection via Photocatalytic Proximity Cell Labeling (PhoXCELL).

Quantitative detection and characterization of antigen-specific T cells are crucial to our understanding of immune responses as well as the development of new immunotherapies. Herein, we report a spatiotemporally resolved method for the detection and quantification of cell-cell interactions via Photocatalytic proXimity CELl Labeling (PhoXCELL). The biocompatible photosensitizer dibromofluorescein (DBF) was leveraged and optimized as a nongenetic alternative of enzymatic approaches for efficient generation of singlet oxygen upon photoirradiation (520 nm) on the cell surface, which allowed the subsequent labeling of nearby oxidized proteins with primary aliphatic amine-based probes. We demonstrated that DBF-functionalized dendritic cells (DCs) could spatiotemporally label interacting T cells in immune synapses via rapid photoirradiation with quantitatively discriminated interaction strength, which revealed distinct gene signatures for T cells that strongly interact with antigen-pulsed DCs. Furthermore, we employed PhoXCELL to simultaneously detect tumor antigen-specific CD8+ as well as CD4+ T cells from tumor-infiltrating lymphocytes and draining lymph nodes in murine tumor models, enabling PhoXCELL as a powerful platform to identify antigen-specific T cells in T cell receptor (TCR)-relevant personal immunotherapy.

Covalently Engineered Protein Minibinders with Enhanced Neutralization Efficacy against Escaping SARS-CoV-2 Variants.

The rapid emergence and spread of escaping mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly challenged our efforts in fighting against the COVID-19 pandemic. A broadly neutralizing reagent against these concerning variants is thus highly desirable for the prophylactic and therapeutic treatments of SARS-CoV-2 infection. We herein report a covalent engineering strategy on protein minibinders for potent neutralization of the escaping variants such as B.1.617.2 (Delta), B.1.617.1 (Kappa), and B.1.1.529 (Omicron) through in situ cross-linking with the spike receptor binding domain (RBD). The resulting covalent minibinder (GlueBinder) exhibited enhanced blockage of RBD-human angiotensin-converting enzyme 2 (huACE2) interaction and more potent neutralization effect against the Delta variant than its noncovalent counterpart as demonstrated on authentic virus. By leveraging the covalent chemistry against escaping mutations, our strategy may be generally applicable for restoring and enhancing the potency of neutralizing antibodies to SARS-CoV-2 and other rapidly evolving viral targets.

Selective Endothelial Hyperactivation of Oncogenic KRAS Induces Brain Arteriovenous Malformations in Mice.

OBJECTIVE: Brain arteriovenous malformations (bAVMs) are a leading cause of hemorrhagic stroke and neurological deficits in children and young adults, however, no pharmacological intervention is available to treat these patients. Although more than 95% of bAVMs are sporadic without family history, the pathogenesis of sporadic bAVMs is largely unknown, which may account for the lack of therapeutic options. KRAS mutations are frequently observed in cancer, and a recent unprecedented finding of these mutations in human sporadic bAVMs offers a new direction in the bAVM research. Using a novel adeno-associated virus targeting brain endothelium (AAV-BR1), the current study tested if endothelial KRASG12V mutation induces sporadic bAVMs in mice. METHODS: Five-week-old mice were systemically injected with either AAV-BR1-GFP or -KRASG12V . At 8 weeks after the AAV injection, bAVM formation and characteristics were addressed by histological and molecular analyses. The effect of MEK/ERK inhibition on KRASG12V -induced bAVMs was determined by treatment of trametinib, a US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor. RESULTS: The viral-mediated KRASG12V overexpression induced bAVMs, which were composed of a tangled nidus mirroring the distinctive morphology of human bAVMs. The bAVMs were accompanied by focal angiogenesis, intracerebral hemorrhages, altered vascular constituents, neuroinflammation, and impaired sensory/cognitive/motor functions. Finally, we confirmed that bAVM growth was inhibited by trametinib treatment. INTERPRETATION: Our innovative approach using AAV-BR1 confirms that KRAS mutations promote bAVM development via the MEK/ERK pathway, and provides a novel preclinical mouse model of bAVMs which will be useful to develop a therapeutic strategy for patients with bAVM. ANN NEUROL 2021;89:926-941.

Intellectual disability-associated gene ftsj1 is responsible for 2'-O-methylation of specific tRNAs.

tRNA modifications at the anti-codon loop are critical for accurate decoding. FTSJ1 was hypothesized to be a human tRNA 2'-O-methyltransferase. tRNAPhe (GAA) from intellectual disability patients with mutations in ftsj1 lacks 2'-O-methylation at C32 and G34 (Cm32 and Gm34). However, the catalytic activity, RNA substrates, and pathogenic mechanism of FTSJ1 remain unknown, owing, in part, to the difficulty in reconstituting enzymatic activity in vitro. Here, we identify an interacting protein of FTSJ1, WDR6. For the first time, we reconstitute the 2'-O-methylation activity of the FTSJ1-WDR6 complex in vitro, which occurs at position 34 of specific tRNAs with m1 G37 as a prerequisite. We find that modifications at positions 32, 34, and 37 are interdependent and occur in a hierarchical order in vivo. We also show that the translation efficiency of the UUU codon, but not the UUC codon decoded by tRNAPhe (GAA), is reduced in ftsj1 knockout cells. Bioinformatics analysis reveals that almost 40% of the high TTT-biased genes are related to brain/nervous functions. Our data potentially enhance our understanding of the relationship between FTSJ1 and nervous system development.

Lysine acetylation activates 6-phosphogluconate dehydrogenase to promote tumor growth.

Although the oxidative pentose phosphate pathway is important for tumor growth, how 6-phosphogluconate dehydrogenase (6PGD) in this pathway is upregulated in human cancers is unknown. We found that 6PGD is commonly activated in EGF-stimulated cells and human cancer cells by lysine acetylation. Acetylation at K76 and K294 of 6PGD promotes NADP(+) binding to 6PGD and formation of active 6PGD dimers, respectively. Moreover, we identified DLAT and ACAT2 as upstream acetyltransferases of K76 and K294, respectively, and HDAC4 as the deacetylase of both sites. Expressing acetyl-deficient mutants of 6PGD in cancer cells significantly attenuated cell proliferation and tumor growth. This is due in part to reduced levels of 6PGD products ribulose-5-phosphate and NADPH, which led to reduced RNA and lipid biosynthesis as well as elevated ROS. Furthermore, 6PGD activity is upregulated with increased lysine acetylation in primary leukemia cells from human patients, providing mechanistic insights into 6PGD upregulation in cancer cells.