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.

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.

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.

Metabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible Inhibitors.

Electrophilic small molecules are an important class of chemical probes and drugs that produce biological effects by irreversibly modifying proteins. Examples of electrophilic drugs include covalent kinase inhibitors that are used to treat cancer and the multiple sclerosis drug dimethyl fumarate. Optimized covalent drugs typically inactivate their protein targets rapidly in cells, but ensuing time-dependent, off-target protein modification can erode selectivity and diminish the utility of reactive small molecules as chemical probes and therapeutics. Here, we describe an approach to confer kinetic selectivity to electrophilic drugs. We show that an analogue of the covalent Bruton's tyrosine kinase (BTK) inhibitor Ibrutinib bearing a fumarate ester electrophile is vulnerable to enzymatic metabolism on a time-scale that preserves rapid and sustained BTK inhibition, while thwarting more slowly accumulating off-target reactivity in cell and animal models. These findings demonstrate that metabolically labile electrophilic groups can endow covalent drugs with kinetic selectivity to enable perturbation of proteins and biochemical pathways with greater precision.

Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells.

Dimethyl fumarate (DMF) is an electrophilic drug that is used to treat autoimmune conditions, including multiple sclerosis and psoriasis. The mechanism of action of DMF is unclear but may involve the covalent modification of proteins or DMF serving as a prodrug that is converted to monomethyl fumarate (MMF). We found that DMF, but not MMF, blocked the activation of primary human and mouse T cells. Using a quantitative, site-specific chemical proteomic platform, we determined the DMF sensitivity of >2400 cysteine residues in human T cells. Cysteines sensitive to DMF, but not MMF, were identified in several proteins with established biochemical or genetic links to T cell function, including protein kinase Cθ (PKCθ). DMF blocked the association of PKCθ with the costimulatory receptor CD28 by perturbing a CXXC motif in the C2 domain of this kinase. Mutation of these DMF-sensitive cysteines also impaired PKCθ-CD28 interactions and T cell activation, designating the C2 domain of PKCθ as a key functional, electrophile-sensing module important for T cell biology.

Chemoproteomics reveals Toll-like receptor fatty acylation.

BACKGROUND: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells. RESULTS: A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands. CONCLUSIONS: This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. S-palmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.

Changes in metabolic chemical reporter structure yield a selective probe of O-GlcNAc modification.

Metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain bioorthogonal functionalities and enable the direct visualization and identification of glycoproteins from living cells. Each MCR was initially thought to report on specific types of glycosylation. We and others have demonstrated that several MCRs are metabolically transformed and enter multiple glycosylation pathways. Therefore, the development of selective MCRs remains a key unmet goal. We demonstrate here that 6-azido-6-deoxy-N-acetyl-glucosamine (6AzGlcNAc) is a specific MCR for O-GlcNAcylated proteins. Biochemical analysis and comparative proteomics with 6AzGlcNAc, N-azidoacetyl-glucosamine (GlcNAz), and N-azidoacetyl-galactosamine (GalNAz) revealed that 6AzGlcNAc exclusively labels intracellular proteins, while GlcNAz and GalNAz are incorporated into a combination of intracellular and extracellular/lumenal glycoproteins. Notably, 6AzGlcNAc cannot be biosynthetically transformed into the corresponding UDP sugar-donor by the canonical salvage-pathway that requires phosphorylation at the 6-hydroxyl. In vitro experiments showed that 6AzGlcNAc can bypass this roadblock through direct phosphorylation of its 1-hydroxyl by the enzyme phosphoacetylglucosamine mutase (AGM1). Taken together, 6AzGlcNAc enables the specific analysis of O-GlcNAcylated proteins, and these results suggest that specific MCRs for other types of glycosylation can be developed. Additionally, our data demonstrate that cells are equipped with a somewhat unappreciated metabolic flexibility with important implications for the biosynthesis of natural and unnatural carbohydrates.

Identification of O-GlcNAc modification targets in mouse retinal pericytes: implication of p53 in pathogenesis of diabetic retinopathy.

Hyperglycemia is the primary cause of the majority of diabetes complications, including diabetic retinopathy (DR). Hyperglycemic conditions have a detrimental effect on many tissues and cell types, especially the retinal vascular cells including early loss of pericytes (PC). However, the mechanisms behind this selective sensitivity of retinal PC to hyperglycemia are undefined. The O-linked ß-N-acetylglucosamine (O-GlcNAc) modification is elevated under hyperglycemic condition, and thus, may present an important molecular modification impacting the hyperglycemia-driven complications of diabetes. We have recently demonstrated that the level of O-GlcNAc modification in response to high glucose is variable in various retinal vascular cells. Retinal PC responded with the highest increase in O-GlcNAc modification compared to retinal endothelial cells and astrocytes. Here we show that these differences translated into functional changes, with an increase in apoptosis of retinal PC, not just under high glucose but also under treatment with O-GlcNAc modification inducers, PUGNAc and Thiamet-G. To gain insight into the molecular mechanisms involved, we have used click-It chemistry and LC-MS analysis and identified 431 target proteins of O-GlcNAc modification in retinal PC using an alkynyl-modified GlcNAc analog (GlcNAlk). Among the O-GlcNAc target proteins identified here 115 of them were not previously reported to be target of O-GlcNAc modification. We have identified at least 34 of these proteins with important roles in various aspects of cell death processes. Our results indicated that increased O-GlcNAc modification of p53 was associated with an increase in its protein levels in retinal PC. Together our results suggest that post-translational O-GlcNAc modification of p53 and its increased levels may contribute to selective early loss of PC during diabetes. Thus, modulation of O-GlcNAc modification may provide a novel treatment strategy to prevent the initiation and progression of DR.

An alkyne-aspirin chemical reporter for the detection of aspirin-dependent protein modification in living cells.

Aspirin (acetylsalicylic acid) is widely used for the acute treatment of inflammation and the management of cardiovascular disease. More recently, it has also been shown to reduce the risk of a variety of cancers. The anti-inflammatory properties of aspirin in pain-relief, cardio-protection, and chemoprevention are well-known to result from the covalent inhibition of cyclooxygenase enzymes through nonenzymatic acetylation of key serine residues. However, any additional molecular mechanisms that may contribute to the beneficial effects of aspirin remain poorly defined. Interestingly, studies over the past 50 years using radiolabeled aspirin demonstrated that other proteins are acetylated by aspirin and enrichment with antiacetyl-lysine antibodies identified 33 potential targets of aspirin-dependent acetylation. Herein we describe the development of an alkyne-modified aspirin analogue (AspAlk) as a chemical reporters of aspirin-dependent acetylation in living cells. When combined with the Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition, this chemical reporter allowed for the robust in-gel fluorescent detection of acetylation and the subsequent enrichment and identification of 120 proteins, 112 of which have not been previously reported to be acetylated by aspirin in cellular or in vivo contexts. Finally, AspAlk was shown to modify the core histone proteins, implicating aspirin as a potential chemical-regulator of transcription.