SAM68 directs STING signaling to apoptosis in macrophages.

DNA is a danger signal sensed by cGAS to engage signaling through STING to activate innate immune functions. The best-studied downstream responses to STING activation include expression of type I interferon and inflammatory genes, but STING also activates other pathways, including apoptosis. Here, we report that STING-dependent induction of apoptosis in macrophages occurs through the intrinsic mitochondrial pathway and is mediated via IRF3 but acts independently of gene transcription. By intersecting four mass spectrometry datasets, we identify SAM68 as crucial for the induction of apoptosis downstream of STING activation. SAM68 is essential for the full activation of apoptosis. Still, it is not required for STING-mediated activation of IFN expression or activation of NF-κB. Mechanistic studies reveal that protein trafficking is required and involves SAM68 recruitment to STING upon activation, with the two proteins associating at the Golgi or a post-Golgi compartment. Collectively, our work identifies SAM68 as a STING-interacting protein enabling induction of apoptosis through this DNA-activated innate immune pathway.

Bioorthogonal protein labelling enables the study of antigen processing of citrullinated and carbamylated auto-antigens.

Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation.

A STING antagonist modulating the interaction with STIM1 blocks ER-to-Golgi trafficking and inhibits lupus pathology.

BACKGROUND: Nucleic acids are potent stimulators of type I interferon (IFN-I) and antiviral defense, but may also promote pathological inflammation. A range of diseases are characterized by elevated IFN-I, including systemic lupus erythematosus (lupus). The DNA-activated cGAS-STING pathway is a major IFN-I-inducing pathway, and activation of signaling is dependent on trafficking of STING from the ER to the Golgi. METHODS: Here we used cell culture systems, a mouse lupus model, and material from lupus patients, to explore the mode of action of a STING antagonistic peptide, and its ability to modulate disease processes. FINDINGS: We report that the peptide ISD017 selectively inhibits all known down-stream activities of STING, including IFN-I, inflammatory cytokines, autophagy, and apoptosis. ISD017 blocks the essential trafficking of STING from the ER to Golgi through a mechanism dependent on the STING ER retention factor STIM1. Importantly, ISD017 blocks STING activity in vivo and ameliorates disease development in a mouse model for lupus. Finally, ISD017 treatment blocks pathological cytokine responses in cells from lupus patients with elevated IFN-I levels. INTERPRETATION: These data hold promise for beneficial use of STING-targeting therapy in lupus. FUNDING: The Novo Nordisk Foundation, The European Research Council, The Lundbeck Foundation, European Union under the Horizon 2020 Research, Deutsche Forschungsgemeinschaft, Chulalongkorn University.

Synthesis of Asparagine Derivatives Harboring a Lewis X Type DC-SIGN Ligand and Evaluation of their Impact on Immunomodulation in Multiple Sclerosis.

The protein myelin oligodendrocyte glycoprotein (MOG) is a key component of myelin and an autoantigen in the disease multiple sclerosis (MS). Post-translational N-glycosylation of Asn31 of MOG seems to play a key role in modulating the immune response towards myelin. This is mediated by the interaction of Lewis-type glycan structures in the N-glycan of MOG with the DC-SIGN receptor on dendritic cells (DCs). Here, we report the synthesis of an unnatural Lewis X (LeX )-containing Fmoc-SPPS-compatible asparagine building block (SPPS=solid-phase peptide synthesis), as well as asparagine building blocks containing two LeX -derived oligosaccharides: LacNAc and Fucα1-3GlcNAc. These building blocks were used for the glycosylation of the immunodominant portion of MOG (MOG31-55 ) and analyzed with respect to their ability to bind to DC-SIGN in different biological setups, as well as their ability to inhibit the citrullination-induced aggregation of MOG31-55 . Finally, a cytokine secretion assay was carried out on human monocyte-derived DCs, which showed the ability of the neoglycopeptide decorated with a single LeX to alter the balance of pro- and anti-inflammatory cytokines, inducing a tolerogenic response.

Conditionally Controlling Human TLR2 Activity via Trans-Cyclooctene Caged Ligands.

Toll-like receptors (TLRs) are key pathogen sensors of the immune system. Their activation results in the production of cytokines, chemokines, and costimulatory molecules that are crucial for innate and adaptive immune responses. In recent years, specific (sub)-cellular location and timing of TLR activation have emerged as parameters for defining the signaling outcome and magnitude. To study the subtlety of this signaling, we here report a new molecular tool to control the activation of TLR2 via "click-to-release"-chemistry. We conjugated a bioorthogonal trans-cyclooctene (TCO) protecting group via solid support to a critical position within a synthetic TLR2/6 ligand to render the compound unable to initiate signaling. The TCO-group could then be conditionally removed upon addition of a tetrazine, resulting in restored agonist activity and TLR2 activation. This approach was validated on RAW264.7 macrophages and various murine primary immune cells as well as human cell line systems, demonstrating that TCO-caging constitutes a versatile approach for generating chemically controllable TLR2 agonists.

On the electron withdrawing nature of ethers in glycosylation chemistry.

The present paper is a commentary on the electronic effects that protecting groups exert on glycosylation chemistry. Specifically, its purpose is to rectify the misguided use of the term electron donating benzyl groups, which hardly makes sense in the context of protecting groups on alcohols in saturated systems such as carbohydrates. It is argued that benzyl ethers (OBn) should rightfully be referred to as being inductively electron withdrawing, even if they are less so than benzoyl esters (OBz).

Going Native: Synthesis of Glycoproteins and Glycopeptides via Native Linkages To Study Glycan-Specific Roles in the Immune System.

Glycosylation plays a myriad of roles in the immune system: Certain glycans can interact with specific immune receptors to kickstart a pro-inflammatory response, whereas other glycans can do precisely the opposite and ameliorate the immune response. Specific glycans and glycoforms can themselves become the targets of the adaptive immune system, leading to potent antiglycan responses that can lead to the killing of altered self- or pathogenic species. This hydra-like set of roles glycans play is of particular importance in cancer immunity, where it influences the anticancer immune response, likely playing pivotal roles in tumor survival or clearance. The complexity of carbohydrate biology requires synthetic access to glycoproteins and glycopeptides that harbor homogeneous glycans allowing the probing of these systems with high precision. One particular complicating factor in this is that these synthetic structures are required to be as close to the native structures as possible, as non-native linkages can themselves elicit immune responses. In this Review, we discuss examples and current strategies for the synthesis of natively linked single glycoforms of peptides and proteins that have enabled researchers to gain new insights into glycoimmunology, with a particular focus on the application of these reagents in cancer immunology.

Amyloid-like Behavior of Site-Specifically Citrullinated Myelin Oligodendrocyte Protein (MOG) Peptide Fragments inside EBV-Infected B-Cells Influences Their Cytotoxicity and Autoimmunogenicity.

Multiple sclerosis (MS) is an autoimmune disorder manifested via chronic inflammation, demyelination, and neurodegeneration inside the central nervous system. The progressive phase of MS is characterized by neurodegeneration, but unlike classical neurodegenerative diseases, amyloid-like aggregation of self-proteins has not been documented. There is evidence that citrullination protects an immunodominant peptide of human myelin oligodendrocyte glycoprotein (MOG34-56) against destructive processing in Epstein-Barr virus-infected B-lymphocytes (EBV-BLCs) in marmosets and causes exacerbation of ongoing MS-like encephalopathies in mice. Here we collected evidence that citrullination of MOG can also lead to amyloid-like behavior shifting the disease pathogenesis toward neurodegeneration. We observed that an immunodominant MOG peptide, MOG35-55, displays amyloid-like behavior upon site-specific citrullination at positions 41, 46, and/or 52. These amyloid aggregates are shown to be toxic to the EBV-BLCs and to dendritic cells at concentrations favored for antigen presentation, suggesting a role of amyloid-like aggregation in the pathogenesis of progressive MS.

Why Is Direct Glycosylation with N-Acetylglucosamine Donors Such a Poor Reaction and What Can Be Done about It?

The monosaccharide N-acetyl-d-glucosamine (GlcNAc) is an abundant building block in naturally occurring oligosaccharides, but its incorporation by chemical glycosylation is challenging since direct reactions are low yielding. This issue, generally agreed upon to be caused by an intermediate 1,2-oxazoline, is often bypassed by introducing extra synthetic steps to avoid the presence of the NHAc functional group during glycosylation. The present paper describes new fundamental mechanistic insights into the inherent challenges of performing direct glycosylation with GlcNAc. These results show that controlling the balance of oxazoline formation and glycosylation is key to achieving acceptable chemical yields. By applying this line of reasoning to direct glycosylation with a traditional thioglycoside donor of GlcNAc, which otherwise affords poor glycosylation yields, one may obtain useful glycosylation results.

6-Deoxyhexoses from l-Rhamnose in the Search for Inducers of the Rhamnose Operon: Synergy of Chemistry and Biotechnology.

In the search for alternative non-metabolizable inducers in the l-rhamnose promoter system, the synthesis of fifteen 6-deoxyhexoses from l-rhamnose demonstrates the value of synergy between biotechnology and chemistry. The readily available 2,3-acetonide of rhamnonolactone allows inversion of configuration at C4 and/or C5 of rhamnose to give 6-deoxy-d-allose, 6-deoxy-d-gulose and 6-deoxy-l-talose. Highly crystalline 3,5-benzylidene rhamnonolactone gives easy access to l-quinovose (6-deoxy-l-glucose), l-olivose and rhamnose analogue with C2 azido, amino and acetamido substituents. Electrophilic fluorination of rhamnal gives a mixture of 2-deoxy-2-fluoro-l-rhamnose and 2-deoxy-2-fluoro-l-quinovose. Biotechnology provides access to 6-deoxy-l-altrose and 1-deoxy-l-fructose.

Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the rhaBAD Promoter.

External control of gene expression is crucial in synthetic biology and biotechnology research and applications, and is commonly achieved using inducible promoter systems. The E. coli rhamnose-inducible rhaBAD promoter has properties superior to more commonly used inducible expression systems, but is marred by transient expression caused by degradation of the native inducer, l-rhamnose. To address this problem, 35 analogues of l-rhamnose were screened for induction of the rhaBAD promoter, but no strong inducers were identified. In the native configuration, an inducer must bind and activate two transcriptional activators, RhaR and RhaS. Therefore, the expression system was reconfigured to decouple the rhaBAD promoter from the native rhaSR regulatory cascade so that candidate inducers need only activate the terminal transcription factor RhaS. Rescreening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterized further to determine the strength, kinetics, and concentration-dependence of induction; whether the inducer was used as a carbon source by E. coli; and the modality (distribution) of induction among populations of cells. l-Mannose was found to be the most useful orthogonal inducer, providing an even greater range of induction than the native inducer l-rhamnose, and crucially, allowing sustained induction instead of transient induction. These findings address the key limitation of the rhaBAD expression system and suggest it may now be the most suitable system for many applications.

A protocol for metal triflate catalyzed direct glycosylations with GalNAc 1-OPiv donors.

Herein we report on the development of novel glycosylation methodology for the concise synthesis of naturally occurring glycoconjugate motifs containing N-acetylgalactosamine (GalNAc) from the cheaper and commercially available N-acetylglucosamine (GlcNAc). The stereoselective glycosylations proceed with catalytic amounts of a promoter and without the need for N-protection other than the biologically relevant N-acetyl group. Among the catalysts explored, both Bi(OTf)3 and Fe(OTf)3 were found to be highly active Lewis acids for this reaction. It was also found that other less reactive metal triflates such as those of Cu(II) and Yb(III) can be beneficial in glycosylation reactions on more demanding glycosyl acceptors. We have furthermore demonstrated that it is possible to control the anomeric stereoselectivity in the glycosylation via postglycosylation in situ anomerization to obtain good yields of α-galactosides. The present protocol was used to prepare important naturally occurring carbohydrate motifs, including a trisaccharide fragment of the naturally occurring marine sponge clarhamnoside.