Neuronal activity-driven O-GlcNAcylation promotes mitochondrial plasticity.

Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-linked N-acetyl glucosamine (O-GlcNAc) transferase regulates neuronal activity-driven mitochondrial bioenergetics in hippocampal and cortical neurons. We show that neuronal activity upregulates O-GlcNAcylation in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven glucose consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.

Post-translational modification-centric base editor screens to assess phosphorylation site functionality in high throughput.

Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here we describe the high-throughput, functional assessment of phosphorylation sites through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of PHLPP1, which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.

OGT prevents DNA demethylation and suppresses the expression of transposable elements in heterochromatin by restraining TET activity genome-wide.

The O- GlcNAc transferase OGT interacts robustly with all three mammalian TET methylcytosine dioxygenases. We show here that deletion of the Ogt gene in mouse embryonic stem cells (mESC) results in a widespread increase in the TET product 5-hydroxymethylcytosine (5hmC) in both euchromatic and heterochromatic compartments, with concomitant reduction of the TET substrate 5-methylcytosine (5mC) at the same genomic regions. mESC engineered to abolish the TET1-OGT interaction likewise displayed a genome-wide decrease of 5mC. DNA hypomethylation in OGT-deficient cells was accompanied by de-repression of transposable elements (TEs) predominantly located in heterochromatin, and this increase in TE expression was sometimes accompanied by increased cis -expression of genes and exons located 3' of the expressed TE. Thus, the TET-OGT interaction prevents DNA demethylation and TE expression in heterochromatin by restraining TET activity genome-wide. We suggest that OGT protects the genome against DNA hypomethylation and impaired heterochromatin integrity, preventing the aberrant increase in TE expression observed in cancer, autoimmune-inflammatory diseases, cellular senescence and ageing.

A biotin targeting chimera (BioTAC) system to map small molecule interactomes in situ.

Understanding how small molecules bind to specific protein complexes in living cells is critical to understanding their mechanism-of-action. Unbiased chemical biology strategies for direct readout of protein interactome remodelling by small molecules would provide advantages over target-focused approaches, including the ability to detect previously unknown ligand targets and complexes. However, there are few current methods for unbiased profiling of small molecule interactomes. To address this, we envisioned a technology that would combine the sensitivity and live-cell compatibility of proximity labelling coupled to mass spectrometry, with the specificity and unbiased nature of chemoproteomics. In this manuscript, we describe the BioTAC system, a small-molecule guided proximity labelling platform that can rapidly identify both direct and complexed small molecule binding proteins. We benchmark the system against µMap, photoaffinity labelling, affinity purification coupled to mass spectrometry and proximity labelling coupled to mass spectrometry datasets. We also apply the BioTAC system to provide interactome maps of Trametinib and analogues. The BioTAC system overcomes a limitation of current approaches and supports identification of both inhibitor bound and molecular glue bound complexes.

Proteome-wide base editor screens to assess phosphorylation site functionality in high-throughput.

Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe "signaling-to-transcription network" mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of the phosphatase PHLPP1 which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.

A Biotin Targeting Chimera (BioTAC) System to Map Small Molecule Interactomes in situ.

Unbiased chemical biology strategies for direct readout of protein interactome remodelling by small molecules provide advantages over target-focused approaches, including the ability to detect previously unknown targets, and the inclusion of chemical off-compete controls leading to high-confidence identifications. We describe the BioTAC system, a small-molecule guided proximity labelling platform, to rapidly identify both direct and complexed small molecule binding proteins. The BioTAC system overcomes a limitation of current approaches, and supports identification of both inhibitor bound and molecular glue bound complexes.

A regulatory circuit controlled by extranuclear and nuclear retinoic acid receptor α determines T cell activation and function.

Ligation of retinoic acid receptor alpha (RARα) by RA promotes varied transcriptional programs associated with immune activation and tolerance, but genetic deletion approaches suggest the impact of RARα on TCR signaling. Here, we examined whether RARα would exert roles beyond transcriptional regulation. Specific deletion of the nuclear isoform of RARα revealed an RARα isoform in the cytoplasm of T cells. Extranuclear RARα was rapidly phosphorylated upon TCR stimulation and recruited to the TCR signalosome. RA interfered with extranuclear RARα signaling, causing suboptimal TCR activation while enhancing FOXP3+ regulatory T cell conversion. TCR activation induced the expression of CRABP2, which translocates RA to the nucleus. Deletion of Crabp2 led to increased RA in the cytoplasm and interfered with signalosome-RARα, resulting in impaired anti-pathogen immunity and suppressed autoimmune disease. Our findings underscore the significance of subcellular RA/RARα signaling in T cells and identify extranuclear RARα as a component of the TCR signalosome and a determinant of immune responses.

Activation mechanism and activity of globupain, a thermostable C11 protease from the Arctic Mid-Ocean Ridge hydrothermal system.

Deep-sea hydrothermal vents offer unique habitats for heat tolerant enzymes with potential new enzymatic properties. Here, we present the novel C11 protease globupain, which was prospected from a metagenome-assembled genome of uncultivated Archaeoglobales sampled from the Soria Moria hydrothermal vent system located on the Arctic Mid-Ocean Ridge. Sequence comparisons against the MEROPS-MPRO database showed that globupain has the highest sequence identity to C11-like proteases present in human gut and intestinal bacteria. Successful recombinant expression in Escherichia coli of the wild-type zymogen and 13 mutant substitution variants allowed assessment of residues involved in maturation and activity of the enzyme. For activation, globupain required the addition of DTT and Ca2+. When activated, the 52kDa proenzyme was processed at K137 and K144 into a 12kDa light- and 32kDa heavy chain heterodimer. A structurally conserved H132/C185 catalytic dyad was responsible for the proteolytic activity, and the enzyme demonstrated the ability to activate in-trans. Globupain exhibited caseinolytic activity and showed a strong preference for arginine in the P1 position, with Boc-QAR-aminomethylcoumarin (AMC) as the best substrate out of a total of 17 fluorogenic AMC substrates tested. Globupain was thermostable (Tm activated enzyme = 94.51°C ± 0.09°C) with optimal activity at 75°C and pH 7.1. Characterization of globupain has expanded our knowledge of the catalytic properties and activation mechanisms of temperature tolerant marine C11 proteases. The unique combination of features such as elevated thermostability, activity at relatively low pH values, and ability to operate under high reducing conditions makes globupain a potential intriguing candidate for use in diverse industrial and biotechnology sectors.

Development of subunit selective substrates for Trichomonas vaginalis proteasome.

The protozoan parasite, Trichomonas vaginalis (Tv) causes trichomoniasis, the most common, non-viral, sexually transmitted infection in the world. Only two closely related drugs are approved for its treatment. The accelerating emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health. There is an urgent need for novel effective anti-parasitic compounds. The proteasome is a critical enzyme for T. vaginalis survival and was validated as a drug target to treat trichomoniasis. However, to develop potent inhibitors of the T. vaginalis proteasome, it is essential that we understand which subunits should be targeted. Previously, we identified two fluorogenic substrates that were cleaved by T. vaginalis proteasome, however after isolating the enzyme complex and performing an in-depth substrate specificity study, we have now designed three fluorogenic reporter substrates that are each specific for one catalytic subunit. We screened a library of peptide epoxyketone inhibitors against the live parasite and evaluated which subunits are targeted by the top hits. Together we show that targeting of the ß5 subunit of T. vaginalis is sufficient to kill the parasite, however, targeting of ß5 plus either ß1 or ß2 results in improved potency.

CALR-mutated cells are vulnerable to combined inhibition of the proteasome and the endoplasmic reticulum stress response.

Cancer is driven by somatic mutations that provide a fitness advantage. While targeted therapies often focus on the mutated gene or its direct downstream effectors, imbalances brought on by cell-state alterations may also confer unique vulnerabilities. In myeloproliferative neoplasms (MPN), somatic mutations in the calreticulin (CALR) gene are disease-initiating through aberrant binding of mutant CALR to the thrombopoietin receptor MPL and ligand-independent activation of JAK-STAT signaling. Despite these mechanistic insights into the pathogenesis of CALR-mutant MPN, there are currently no mutant CALR-selective therapies available. Here, we identified differential upregulation of unfolded proteins, the proteasome and the ER stress response in CALR-mutant hematopoietic stem cells (HSCs) and megakaryocyte progenitors. We further found that combined pharmacological inhibition of the proteasome and IRE1-XBP1 axis of the ER stress response preferentially targets Calr-mutated HSCs and megakaryocytic-lineage cells over wild-type cells in vivo, resulting in an amelioration of the MPN phenotype. In serial transplantation assays following combined proteasome/IRE1 inhibition for six weeks, we did not find preferential depletion of Calr-mutant long-term HSCs. Together, these findings leverage altered proteostasis in Calr-mutant MPN to identify combinatorial dependencies that may be targeted for therapeutic benefit and suggest that eradicating disease-propagating Calr-mutant LT-HSCs may require more sustained treatment.

Retron reverse transcriptase termination and phage defense are dependent on host RNase H1.

Retrons are bacterial retroelements that produce single-stranded, reverse-transcribed DNA (RT-DNA) that is a critical part of a newly discovered phage defense system. Short retron RT-DNAs are produced from larger, structured RNAs via a unique 2'-5' initiation and a mechanism for precise termination that is not yet understood. Interestingly, retron reverse transcriptases (RTs) typically lack an RNase H domain and, therefore, depend on endogenous RNase H1 to remove RNA templates from RT-DNA. We find evidence for an expanded role of RNase H1 in the mechanism of RT-DNA termination, beyond the mere removal of RNA from RT-DNA:RNA hybrids. We show that endogenous RNase H1 determines the termination point of the retron RT-DNA, with differing effects across retron subtypes, and that these effects can be recapitulated using a reduced, in vitro system. We exclude mechanisms of termination that rely on steric effects of RNase H1 or RNA secondary structure and, instead, propose a model in which the tertiary structure of the single-stranded RT-DNA and remaining RNA template results in termination. Finally, we show that this mechanism affects cellular function, as retron-based phage defense is weaker in the absence of RNase H1.

Mechanisms encoding STAT functional diversity for context-specific inflammatory responses.

Cells integrate complex cytokine cues and other inflammatory stimuli through activation of the signal transducers and activators of transcription (STAT) family of transcription factors to drive the appropriate anti-microbial, inflammatory, and resolving functions. Here, we discuss recent progress in our understanding of mechanisms supporting STAT functional diversity. Signaling component availability and the strength of receptor and STAT interactions emerge as important determinants of immune function. The resultant dynamics of STAT activation, together with stimulus-specific variation in STAT post-translationally modified states, will impact downstream binding partners to support transcription of distinct gene subsets. Understanding how context-dependent STAT function is encoded to dictate cytokine specificity, crosstalk, and control of inflammation will guide therapeutic efforts to selectively perturb STAT-regulated responses.

Congenital anemia reveals distinct targeting mechanisms for master transcription factor GATA1.

Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.

Identification of RIOK2 as a master regulator of human blood cell development.

Anemia is a major comorbidity in aging, chronic kidney and inflammatory diseases, and hematologic malignancies. However, the transcriptomic networks governing hematopoietic differentiation in blood cell development remain incompletely defined. Here we report that the atypical kinase RIOK2 (right open reading frame kinase 2) is a master transcription factor (TF) that not only drives erythroid differentiation, but also simultaneously suppresses megakaryopoiesis and myelopoiesis in primary human stem and progenitor cells. Our study reveals the previously uncharacterized winged helix-turn-helix DNA-binding domain and two transactivation domains of RIOK2 that are critical to regulate key hematopoietic TFs GATA1, GATA2, SPI1, RUNX3 and KLF1. This establishes RIOK2 as an integral component of the transcriptional regulatory network governing human hematopoietic differentiation. Importantly, RIOK2 mRNA expression significantly correlates with these TFs and other hematopoietic genes in myelodysplastic syndromes, acute myeloid leukemia and chronic kidney disease. Further investigation of RIOK2-mediated transcriptional pathways should yield therapeutic approaches to correct defective hematopoiesis in hematologic disorders.

Novel Antibodies for the Simple and Efficient Enrichment of Native O-GlcNAc Modified Peptides.

Antibodies against posttranslational modifications (PTMs) such as lysine acetylation, ubiquitin remnants, or phosphotyrosine have resulted in significant advances in our understanding of the fundamental roles of these PTMs in biology. However, the roles of a number of PTMs remain largely unexplored due to the lack of robust enrichment reagents. The addition of N-acetylglucosamine to serine and threonine residues (O-GlcNAc) by the O-GlcNAc transferase (OGT) is a PTM implicated in numerous biological processes and disease states but with limited techniques for its study. Here, we evaluate a new mixture of anti-O-GlcNAc monoclonal antibodies for the immunoprecipitation of native O-GlcNAcylated peptides from cells and tissues. The anti-O-GlcNAc antibodies display good sensitivity and high specificity toward O-GlcNAc-modified peptides and do not recognize O-GalNAc or GlcNAc in extended glycans. Applying this antibody-based enrichment strategy to synaptosomes from mouse brain tissue samples, we identified over 1300 unique O-GlcNAc-modified peptides and over 1000 sites using just a fraction of sample preparation and instrument time required in other landmark investigations of O-GlcNAcylation. Our rapid and robust method greatly simplifies the analysis of O-GlcNAc signaling and will help to elucidate the role of this challenging PTM in health and disease.

Spatiotemporally-resolved mapping of RNA binding proteins via functional proximity labeling reveals a mitochondrial mRNA anchor promoting stress recovery.

Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, we show that it is possible to map specific protein subclasses within distinct compartments of living cells. In particular, we develop a method to enrich subcompartment-specific RNA binding proteins (RBPs) by combining peroxidase-catalyzed PL with organic-aqueous phase separation of crosslinked protein-RNA complexes ("APEX-PS"). We use APEX-PS to generate datasets of nuclear, nucleolar, and outer mitochondrial membrane (OMM) RBPs, which can be mined for novel functions. For example, we find that the OMM RBP SYNJ2BP retains specific nuclear-encoded mitochondrial mRNAs at the OMM during translation stress, facilitating their local translation and import of protein products into the mitochondrion during stress recovery. Functional PL in general, and APEX-PS in particular, represent versatile approaches for the discovery of proteins with novel function in specific subcellular compartments.

Blockade of IL-22 signaling reverses erythroid dysfunction in stress-induced anemias.

Patients with myelodysplastic syndromes (MDSs) display severe anemia but the mechanisms underlying this phenotype are incompletely understood. Right open-reading-frame kinase 2 (RIOK2) encodes a protein kinase located at 5q15, a region frequently lost in patients with MDS del(5q). Here we show that hematopoietic cell-specific haploinsufficient deletion of Riok2 (Riok2f/+Vav1cre) led to reduced erythroid precursor frequency leading to anemia. Proteomic analysis of Riok2f/+Vav1cre erythroid precursors suggested immune system activation, and transcriptomic analysis revealed an increase in p53-dependent interleukin (IL)-22 in Riok2f/+Vav1cre CD4+ T cells (TH22). Further, we discovered that the IL-22 receptor, IL-22RA1, was unexpectedly present on erythroid precursors. Blockade of IL-22 signaling alleviated anemia not only in Riok2f/+Vav1cre mice but also in wild-type mice. Serum concentrations of IL-22 were increased in the subset of patients with del(5q) MDS as well as patients with anemia secondary to chronic kidney disease. This work reveals a possible therapeutic opportunity for reversing many stress-induced anemias by targeting IL-22 signaling.

Proximity labeling in mammalian cells with TurboID and split-TurboID.

This protocol describes the use of TurboID and split-TurboID in proximity labeling applications for mapping protein-protein interactions and subcellular proteomes in live mammalian cells. TurboID is an engineered biotin ligase that uses ATP to convert biotin into biotin-AMP, a reactive intermediate that covalently labels proximal proteins. Optimized using directed evolution, TurboID has substantially higher activity than previously described biotin ligase-related proximity labeling methods, such as BioID, enabling higher temporal resolution and broader application in vivo. Split-TurboID consists of two inactive fragments of TurboID that can be reconstituted through protein-protein interactions or organelle-organelle interactions, which can facilitate greater targeting specificity than full-length enzymes alone. Proteins biotinylated by TurboID or split-TurboID are then enriched with streptavidin beads and identified by mass spectrometry. Here, we describe fusion construct design and characterization (variable timing), proteomic sample preparation (5-7 d), mass spectrometric data acquisition (2 d), and proteomic data analysis (1 week).

RNA-protein interaction mapping via MS2- or Cas13-based APEX targeting.

RNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. We used two approaches to target the engineered peroxidase APEX2 to specific cellular RNAs for RNA-centered proximity biotinylation of protein interaction partners. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured candidate binding partners for hTR, including more than a dozen proteins not previously linked to hTR. We validated the interaction between hTR and the N6-methyladenosine (m6A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m6A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA-protein interactions in living cells.