P66 is a bacterial mimic of CD47 that binds the anti-phagocytic receptor SIRPα and facilitates macrophage evasion by Borrelia burgdorferi.

Innate immunity, the first line of defense against pathogens, relies on efficient elimination of invading agents by phagocytes. In the co-evolution of host and pathogen, pathogens developed mechanisms to dampen and evade phagocytic clearance. Here, we report that bacterial pathogens can evade clearance by macrophages through mimicry at the mammalian anti-phagocytic "don't eat me" signaling axis between CD47 (ligand) and SIRPα (receptor). We identified a protein, P66, on the surface of Borrelia burgdorferi that, like CD47, is necessary and sufficient to bind the macrophage receptor SIRPα. Expression of the gene encoding the protein is required for bacteria to bind SIRPα or a high-affinity CD47 reagent. Genetic deletion of p66 increases phagocytosis by macrophages. Blockade of P66 during infection promotes clearance of the bacteria. This study demonstrates that mimicry of the mammalian anti-phagocytic protein CD47 by B. burgdorferi inhibits macrophage-mediated bacterial clearance. Such a mechanism has broad implications for understanding of host-pathogen interactions and expands the function of the established innate immune checkpoint receptor SIRPα. Moreover, this report reveals P66 as a novel therapeutic target in the treatment of Lyme Disease.

Longitudinal map of transcriptome changes in the Lyme pathogen Borrelia burgdorferi during tick-borne transmission.

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, adapts to vastly different environments as it cycles between tick vector and vertebrate host. During a tick bloodmeal, Bb alters its gene expression to prepare for vertebrate infection; however, the full range of transcriptional changes that occur over several days inside of the tick are technically challenging to capture. We developed an experimental approach to enrich Bb cells to longitudinally define their global transcriptomic landscape inside nymphal Ixodes scapularis ticks during a transmitting bloodmeal. We identified 192 Bb genes that substantially change expression over the course of the bloodmeal from 1 to 4 days after host attachment. The majority of upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 outer surface lipoproteins embedded in the unusual protein-rich coat of Bb. As these proteins may facilitate Bb interactions with the host, we utilized mass spectrometry to identify candidate tick proteins that physically associate with Bb. The Bb enrichment methodology along with the ex vivo Bb transcriptomes and candidate tick interacting proteins presented here provide a resource to facilitate investigations into key determinants of Bb priming and transmission during the tick stage of its unique transmission cycle.

EMC chaperone-CaV structure reveals an ion channel assembly intermediate.

Voltage-gated ion channels (VGICs) comprise multiple structural units, the assembly of which is required for function1,2. Structural understanding of how VGIC subunits assemble and whether chaperone proteins are required is lacking. High-voltage-activated calcium channels (CaVs)3,4 are paradigmatic multisubunit VGICs whose function and trafficking are powerfully shaped by interactions between pore-forming CaV1 or CaV2 CaVα1 (ref. 3), and the auxiliary CaVß5 and CaVα2δ subunits6,7. Here we present cryo-electron microscopy structures of human brain and cardiac CaV1.2 bound with CaVß3 to a chaperone-the endoplasmic reticulum membrane protein complex (EMC)8,9-and of the assembled CaV1.2-CaVß3-CaVα2δ-1 channel. These structures provide a view of an EMC-client complex and define EMC sites-the transmembrane (TM) and cytoplasmic (Cyto) docks; interaction between these sites and the client channel causes partial extraction of a pore subunit and splays open the CaVα2δ-interaction site. The structures identify the CaVα2δ-binding site for gabapentinoid anti-pain and anti-anxiety drugs6, show that EMC and CaVα2δ interactions with the channel are mutually exclusive, and indicate that EMC-to-CaVα2δ hand-off involves a divalent ion-dependent step and CaV1.2 element ordering. Disruption of the EMC-CaV complex compromises CaV function, suggesting that the EMC functions as a channel holdase that facilitates channel assembly. Together, the structures reveal a CaV assembly intermediate and EMC client-binding sites that could have wide-ranging implications for the biogenesis of VGICs and other membrane proteins.

MFGE8 links absorption of dietary fatty acids with catabolism of enterocyte lipid stores through HNF4γ-dependent transcription of CES enzymes.

Enterocytes modulate the extent of postprandial lipemia by storing dietary fats in cytoplasmic lipid droplets (cLDs). We have previously shown that the integrin ligand MFGE8 links absorption of dietary fats with activation of triglyceride (TG) hydrolases that catabolize cLDs for chylomicron production. Here, we identify CES1D as the key hydrolase downstream of the MFGE8-αvß5 integrin pathway that regulates catabolism of diet-derived cLDs. Mfge8 knockout (KO) enterocytes have reduced CES1D transcript and protein levels and reduced protein levels of the transcription factor HNF4γ. Both Ces1d and Hnf4γ KO mice have decreased enterocyte TG hydrolase activity coupled with retention of TG in cLDs. Mechanistically, MFGE8-dependent fatty acid uptake through CD36 stabilizes HNF4γ protein level; HNF4γ then increases Ces1d transcription. Our work identifies a regulatory network that regulates the severity of postprandial lipemia by linking dietary fat absorption with protein stabilization of a transcription factor that increases expression of hydrolases responsible for catabolizing diet-derived cLDs.

Secreted fungal virulence effector triggers allergic inflammation via TLR4.

Invasive fungal pathogens are major causes of human mortality and morbidity1,2. Although numerous secreted effector proteins that reprogram innate immunity to promote virulence have been identified in pathogenic bacteria, so far, there are no examples of analogous secreted effector proteins produced by human fungal pathogens. Cryptococcus neoformans, the most common cause of fungal meningitis and a major pathogen in AIDS, induces a pathogenic type 2 response characterized by pulmonary eosinophilia and alternatively activated macrophages3-8. Here, we identify CPL1 as an effector protein secreted by C. neoformans that drives alternative activation (also known as M2 polarization) of macrophages to enable pulmonary infection in mice. We observed that CPL1-enhanced macrophage polarization requires Toll-like receptor 4, which is best known as a receptor for bacterial endotoxin but is also a poorly understood mediator of allergen-induced type 2 responses9-12. We show that this effect is caused by CPL1 itself and not by contaminating lipopolysaccharide. CPL1 is essential for virulence, drives polarization of interstitial macrophages in vivo, and requires type 2 cytokine signalling for its effect on infectivity. Notably, C. neoformans associates selectively with polarized interstitial macrophages during infection, suggesting a mechanism by which C. neoformans generates its own intracellular replication niche within the host. This work identifies a circuit whereby a secreted effector protein produced by a human fungal pathogen reprograms innate immunity, revealing an unexpected role for Toll-like receptor 4 in promoting the pathogenesis of infectious disease.

Proteomic characterization of phagocytic primary human monocyte-derived macrophages.

Macrophages play a vital role in the innate immune system, identifying and destroying unwanted cells. However, it has been difficult to attain a comprehensive understanding of macrophage protein abundance due to technical limitations. In addition, it remains unclear how changes in proteome composition are linked to phagocytic activity. In this study we developed methods to derive human macrophages and prepare them for mass spectrometry analysis in order to more-deeply understand the proteomic consequences of macrophage stimulation. Interferon gamma (IF-g), an immune stimulating cytokine, was used to induce macrophage activation, increasing phagocytosis of cancer cells by 2-fold. These conditions were used to perform comparative shotgun proteomics between resting macrophages and stimulated macrophages with increased phagocytic activity. Our analysis revealed that macrophages bias their protein production toward biological processes associated with phagocytosis and antigen processing in response to stimulation. We confirmed our findings by antibody-based western blotting experiments, validating both previously reported and novel proteins of interest. In addition to whole protein changes, we evaluated active protein synthesis by treating cells with the methionine surrogate probe homopropargylglycine (HPG). We saw increased rates of HPG incorporation during phagocytosis-inducing stimulation, suggesting protein synthesis rates are altered by stimulation. Together our findings provide the most comprehensive proteomic insight to date into primary human macrophages. We anticipate that this data can be used as a launchpoint to generate new hypotheses about innate immune function.

Improved Electrophile Design for Exquisite Covalent Molecule Selectivity.

Covalent inhibitors are viable therapeutics. However, off-target reactivity challenges the field. Chemists have attempted to solve this issue by varying the reactivity attributes of electrophilic warheads. Here, we report the development of an approach to increase the selectivity of covalent molecules that is independent of warhead reactivity features and can be used in concert with existing methods. Using the scaffold of the Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib for our proof-of-concept, we reasoned that increasing the steric bulk of fumarate-based electrophiles on Ibrutinib should improve selectivity via the steric exclusion of off-targets but retain rates of cysteine reactivity comparable to that of an acrylamide. Using chemical proteomic techniques, we demonstrate that elaboration of the electrophile to a tert-butyl (t-Bu) fumarate ester decreases time-dependent off-target reactivity and abolishes time-independent off-target reactivity. While an alkyne-bearing probe analogue of Ibrutinib has 247 protein targets, our t-Bu fumarate probe analogue has only 7. Of these 7 targets, BTK is the only time-independent target. The t-Bu inhibitor itself is also more selective for BTK, reducing off-targets by 70%. We investigated the consequences of treatment with Ibrutinib and our t-Bu analogue and discovered that only 8 proteins are downregulated in response to treatment with the t-Bu analogue compared to 107 with Ibrutinib. Of these 8 proteins, 7 are also downregulated by Ibrutinib and a majority of these targets are associated with BTK biology. Taken together, these findings reveal an opportunity to increase cysteine-reactive covalent inhibitor selectivity through electrophilic structure optimization.

Refinement of Covalent EGFR Inhibitor AZD9291 to Eliminate Off-target Activity.

Non-small-cell lung cancer (NSCLC) is a major disease that accounts for 85% of all lung cancer cases which claimed around 1.8 billion lives worldwide in 2020. Tyrosine kinase inhibitors (TKIs) that target EGFR have been used for the treatment of NSCLC, but often develop drug resistance, and the covalent inhibitor AZD9291 has been developed to tackle the problem of drug resistance mediated by the T790M EGFR mutation; however, there is a side effect of hyperglycemia that may be due to off-target activity. This study examines analogues of AZD9291 by chemical proteomics, identifying analogues that maintain T790M-EGFR engagement while showing reduced cross-reactivity with off-targets.

Proteomic analysis of young and old mouse hematopoietic stem cells and their progenitors reveals post-transcriptional regulation in stem cells.

The balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for a healthy blood supply; imbalances underlie hematological diseases. The importance of HSCs and their progenitors have led to their extensive characterization at genomic and transcriptomic levels. However, the proteomics of hematopoiesis remains incompletely understood. Here we report a proteomics resource from mass spectrometry of mouse young adult and old adult mouse HSCs, multipotent progenitors and oligopotent progenitors; 12 cell types in total. We validated differential protein levels, including confirmation that Dnmt3a protein levels are undetected in young adult mouse HSCs until forced into cycle. Additionally, through integrating proteomics and RNA-sequencing datasets, we identified a subset of genes with apparent post-transcriptional repression in young adult mouse HSCs. In summary, we report proteomic coverage of young and old mouse HSCs and progenitors, with broader implications for understanding mechanisms for stem cell maintenance, niche interactions and fate determination.

O-Acetylated Chemical Reporters of Glycosylation Can Display Metabolism-Dependent Background Labeling of Proteins but Are Generally Reliable Tools for the Identification of Glycoproteins.

Monosaccharide analogs bearing bioorthogonal functionalities, or metabolic chemical reporters (MCRs) of glycosylation, have been used for approximately two decades for the visualization and identification of different glycoproteins. More recently, proteomics analyses have shown that per-O-acetylated MCRs can directly and chemically react with cysteine residues in lysates and potentially cells, drawing into question the physiological relevance of the labeling. Here, we report robust metabolism-dependent labeling by Ac42AzMan but not the structurally similar Ac44AzGal. However, the levels of background chemical-labeling of cell lysates by both reporters are low and identical. We then characterized Ac42AzMan labeling and found that the vast majority of the labeling occurs on intracellular proteins but that this MCR is not converted to previously characterized reporters of intracellular O-GlcNAc modification. Additionally, we used isotope targeted glycoproteomics (IsoTaG) proteomics to show that essentially all of the Ac42AzMan labeling is on cysteine residues. Given the implications this result has for the identification of intracellular O-GlcNAc modifications using MCRs, we then performed a meta-analysis of the potential O-GlcNAcylated proteins identified by different techniques. We found that many of the proteins identified by MCRs have also been found by other methods. Finally, we randomly selected four proteins that had only been identified as O-GlcNAcylated by MCRs and showed that half of them were indeed modified. Together, these data indicate that the selective metabolism of certain MCRs is responsible for S-glycosylation of proteins in the cytosol and nucleus. However, these results also show that MCRs are still good tools for unbiased identification of glycosylated proteins, as long as complementary methods are employed for confirmation.

Metabolic Labeling for the Visualization and Identification of Potentially O-GlcNAc-Modified Proteins.

O-GlcNAcylation is a posttranslational modification involving the addition of the single monosaccharide N-acetylglucosamine (GlcNAc) onto serine and threonine residues of intracellular proteins. Though O-GlcNAc is found on ∼1000 proteins in mammals, its specific function on individual substrates remains largely a mystery. To overcome this shortcoming, work has been put toward developing metabolic chemical reporters (MCRs) to label O-GlcNAcylated proteins for subsequent biochemical analysis. Typically, these MCRs are GlcNAc or GalNAc analogs functionalized with azide or alkyne handles. These unnatural sugar moieties can be metabolically incorporated directly on to protein substrates. The protocols outlined in this article describe how to use MCRs as tools for visualizing and identifying potentially O-GlcNAc modified proteins via in-gel fluorescence, Western blotting, and mass spectrometry. Taken together, MCR labeling provides a powerful tool to discover where and when substrates are O-GlcNAc modified. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Treatment of cells and CuAAC Basic Protocol 2: In-gel fluorescence of labeled cell lysates (1 mg scale) Basic Protocol 3: Enrichment of labeled proteins, trypsinolysis, and collection of peptides for proteomics Basic Protocol 4: Proteomic identification of labeled proteins.

CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy.

Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called 'don't eat me' signals-including CD471, programmed cell death ligand 1 (PD-L1)2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)3. Monoclonal antibodies that antagonize the interaction of 'don't eat me' signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers4,5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown 'don't eat me' signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24-Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy.

The Proteome-Wide Potential for Reversible Covalency at Cysteine.

Reversible covalency, achieved with, for instance, highly electron-deficient olefins, offers a compelling strategy to design chemical probes and drugs that benefit from the sustained target engagement afforded by irreversible compounds, while avoiding permanent protein modification. Reversible covalency has mainly been evaluated for cysteine residues in individual kinases and the broader potential for this strategy to engage cysteines across the proteome remains unexplored. Herein, we describe a mass-spectrometry-based platform that integrates gel filtration with activity-based protein profiling to assess cysteine residues across the human proteome for both irreversible and reversible interactions with small-molecule electrophiles. Using this method, we identify numerous cysteine residues from diverse protein classes that are reversibly engaged by cyanoacrylamide fragment electrophiles, revealing the broad potential for reversible covalency as a strategy for chemical-probe discovery.

Dimethyl Fumarate Disrupts Human Innate Immune Signaling by Targeting the IRAK4-MyD88 Complex.

Dimethyl fumarate (DMF) is a prescribed treatment for multiple sclerosis and has also been used to treat psoriasis. The electrophilicity of DMF suggests that its immunosuppressive activity is related to the covalent modification of cysteine residues in the human proteome. Nonetheless, our understanding of the proteins modified by DMF in human immune cells and the functional consequences of these reactions remains incomplete. In this study, we report that DMF inhibits human plasmacytoid dendritic cell function through a mechanism of action that is independent of the major electrophile sensor NRF2. Using chemical proteomics, we instead identify cysteine 13 of the innate immune kinase IRAK4 as a principal cellular target of DMF. We show that DMF blocks IRAK4-MyD88 interactions and IRAK4-mediated cytokine production in a cysteine 13-dependent manner. Our studies thus identify a proteomic hotspot for DMF action that constitutes a druggable protein-protein interface crucial for initiating innate immune responses.

Azide- and Alkyne-Bearing Metabolic Chemical Reporters of Glycosylation Show Structure-Dependent Feedback Inhibition of the Hexosamine Biosynthetic Pathway.

Metabolic chemical reporters (MCRs) of protein glycosylation are analogues of natural monosaccharides that bear reactive groups, like azides and alkynes. When they are added to living cells and organisms, these small molecules are biosynthetically transformed into nucleotide donor sugars and then used by glycosyltransferases to modify proteins. Subsequent installation of tags by bioorthogonal chemistries can then enable the visualization and enrichment of these glycoproteins. Although this two-step procedure is powerful, the use of MCRs has the potential to change the endogenous production of the natural repertoire of donor sugars. A major route for the generation of these glycosyltransferase substrates is the hexosamine biosynthetic pathway (HBP), which results in uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Interestingly, the rate-determining enzyme of the HBP, glutamine fructose-6-phosphate amidotransferase (GFAT), is feedback inhibited by UDP-GlcNAc. This raises the possibility that a build-up of UDP-MCRs would block the biosynthesis of UDP-GlcNAc, resulting in off target effects. Here, we directly test this possibility with recombinant human GFAT and a small panel of synthetic UDP-MCRs. We find that MCRs with larger substitutions at the N-acetyl position do not inhibit GFAT, whereas those with modifications of the 2- or 6-hydroxy group do. These results further illuminate the considerations that should be applied to the use of MCRs.

The New Chemical Reporter 6-Alkynyl-6-deoxy-GlcNAc Reveals O-GlcNAc Modification of the Apoptotic Caspases That Can Block the Cleavage/Activation of Caspase-8.

O-GlcNAc modification (O-GlcNAcylation) is required for survival in mammalian cells. Genetic and biochemical experiments have found that increased modification inhibits apoptosis in tissues and cell culture and that lowering O-GlcNAcylation induces cell death. However, the molecular mechanisms by which O-GlcNAcylation might inhibit apoptosis are still being elucidated. Here, we first synthesize a new metabolic chemical reporter, 6-Alkynyl-6-deoxy-GlcNAc (6AlkGlcNAc), for the identification of O-GlcNAc-modified proteins. Subsequent characterization of 6AlkGlcNAc shows that this probe is selectively incorporated into O-GlcNAcylated proteins over cell-surface glycoproteins. Using this probe, we discover that the apoptotic caspases are O-GlcNAcylated, which we confirmed using other techniques, raising the possibility that the modification affects their biochemistry. We then demonstrate that changes in the global levels of O-GlcNAcylation result in a converse change in the kinetics of caspase-8 activation during apoptosis. Finally, we show that caspase-8 is modified at residues that can block its cleavage/activation. Our results provide the first evidence that the caspases may be directly affected by O-GlcNAcylation as a potential antiapoptotic mechanism.

Metabolic Chemical Reporters of Glycans Exhibit Cell-Type-Selective Metabolism and Glycoprotein Labeling.

Since the pioneering work by Reutter and co-workers that demonstrated structural flexibility in the carbohydrate biosynthesis and glycosylation pathways, many different labs have used unnatural monosaccharide analogues to perform glycan engineering on the surface of living cells. A subset of these unnatural monosaccharides contain bioorthogonal groups that enable the selective installation of visualization or enrichment tags. These metabolic chemical reporters (MCRs) have proven to be powerful for the unbiased identification of glycoproteins; however, they do have certain limitations. For example, they are incorporated substoichiometrically into glycans, and most MCRs are not selective for one class (e.g., O-GlcNAcylation) of glycoprotein. Here, we explore the relationship between the biosynthesis of MCR donor sugars in cells and the labeling levels of four different N-acetylglucosamine- and N-acetylgalactosamine-based MCRs. We found that the buildup of the different donor sugars correlated well with the overall labeling levels but less so with intracellular labeling of proteins by O-GlcNAcylation.

The Small Molecule 2-Azido-2-deoxy-glucose Is a Metabolic Chemical Reporter of O-GlcNAc Modifications in Mammalian Cells, Revealing an Unexpected Promiscuity of O-GlcNAc Transferase.

Glycans can be directly labeled using unnatural monosaccharide analogs, termed metabolic chemical reporters (MCRs). These compounds enable the secondary visualization and identification of glycoproteins by taking advantage of bioorthogonal reactions. Most widely used MCRs have azides or alkynes at the 2-N-acetyl position but are not selective for one class of glycoprotein over others. To address this limitation, we are exploring additional MCRs that have bioorthogonal functionality at other positions. Here, we report the characterization of 2-azido-2-deoxy-glucose (2AzGlc). We find that 2AzGlc selectively labels intracellular O-GlcNAc modifications, which further supports a somewhat unexpected, structural flexibility in this pathway. In contrast to the endogenous modification N-acetyl-glucosamine (GlcNAc), we find that 2AzGlc is not dynamically removed from protein substrates and that treatment with higher concentrations of per-acetylated 2AzGlc is toxic to cells. Finally, we demonstrate that this toxicity is an inherent property of the small-molecule, as removal of the 6-acetyl-group renders the corresponding reporter nontoxic but still results in protein labeling.