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Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving direct functional perturbation and/or ligand-induced degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells, underscoring the potential of electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

Summary Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

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.

Aging impairs the activation of stress signaling pathways (SSPs), preventing the induction of longevity mechanisms late in life. Here, we show that the antibiotic minocycline increases lifespan and reduces protein aggregation even in old, SSP-deficient Caenorhabditis elegans by targeting cytoplasmic ribosomes, preferentially attenuating translation of highly translated mRNAs. In contrast to most other longevity paradigms, minocycline inhibits rather than activates all major SSPs and extends lifespan in mutants deficient in the activation of SSPs, lysosomal or autophagic pathways. We propose that minocycline lowers the concentration of newly synthesized aggregation-prone proteins, resulting in a relative increase in protein-folding capacity without the necessity to induce protein-folding pathways. Our study suggests that in old individuals with incapacitated SSPs or autophagic pathways, pharmacological attenuation of cytoplasmic translation is a promising strategy to reduce protein aggregation. Altogether, it provides a geroprotecive mechanism for the many beneficial effects of tetracyclines in models of neurodegenerative disease.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

SummaryAging impairs the activation of Stress Signaling Pathways (SSPs), preventing the induction of longevity mechanisms late in life. Here we show that the antibiotic minocycline increases lifespan and reduces protein aggregation even in old, SSP-deficient C. elegans by targeting cytoplasmic ribosomes, preferentially attenuating translation of highly translated mRNAs. In contrast to most other longevity paradigms, minocycline inhibits rather than activates all major SSPs and extends lifespan in mutants deficient in the activation of SSPs, lysosomal or autophagic pathways. We propose that minocycline lowers the concentration of newly synthesized aggregation-prone proteins, resulting in a relative increase in protein-folding capacity without the necessity to induce protein-folding pathways. Our study suggests that in old individuals with incapacitated SSPs or autophagic pathways, pharmacological attenuation of cytoplasmic translation is a promising strategy to reduce protein aggregation. Altogether, it provides a geroprotecive mechanism for the many beneficial effects of tetracyclines in models of neurodegenerative disease.

Cells produce electrophilic products with the potential to modify and affect the function of proteins. Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify the sites in the proteome that are most sensitive to electrophilic modification requires more quantitative methods. Here we describe a competitive activity-based profiling method for quantifying the reactivity of electrophilic compounds against >1,000 cysteines in parallel in the human proteome. Using this approach, we identified a select set of proteins that constitute 'hot spots' for modification by various lipid-derived electrophiles, including the oxidative stress product 4-hydroxy-2-nonenal (HNE). We show that one of these proteins, ZAK kinase, is labeled by HNE on a conserved, active site-proximal cysteine and that the resulting enzyme inhibition creates a negative feedback mechanism that can suppress the activation of JNK pathways normally induced by oxidative stress.

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 pro-drug that is converted to monomethyl fumarate (MMF). Here, 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 C θ (PKCθ). Furthermore, 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.

The experiments described here clarify the mechanism and origin of the enantioselectivity of the oxidation of racemic secondary alcohols catalyzed by chiral Mn(III)-salen complexes using HOBr, Br(2)/H(2)O/KOAc or PhI(OAc)(2)/H(2)O/KBr as a stoichiometric oxidant. Key points of the proposed pathway include (1) the formation of a Mn(V)-salen dibromide, (2) its subsequent reaction with the alcohol to give an alkoxy-Mn(V) species, and (3) carbonyl-forming elimination to produce the ketone via a highly organized transition state with intramolecular transfer of hydrogen from carbon to an oxygen of the salen ligand.

It is proposed that facial selectivity in the Jacobsen epoxidation is determined by electrostatic and steric factors with a two-step pathway involving a carbocationic intermediate.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system (CNS) that is thought to be caused by a combination of genetic and environmental factors. To date, considerable evidence has associated Epstein-Barr virus (EBV) infection with disease development. However, it remains controversial whether EBV infects multiple sclerosis brain and contributes directly to CNS immunopathology. To assess whether EBV infection is a characteristic feature of multiple sclerosis brain, a large cohort of multiple sclerosis specimens containing white matter lesions (nine adult and three paediatric cases) with a heterogeneous B cell infiltrate and a second cohort of multiple sclerosis specimens (12 cases) that included B cell infiltration within the meninges and parenchymal B cell aggregates, were examined for EBV infection using multiple methodologies including in situ hybridization, immunohistochemistry and two independent real-time polymerase chain reaction (PCR) methodologies that detect genomic EBV or the abundant EBV encoded RNA (EBER) 1, respectively. We report that EBV could not be detected in any of the multiple sclerosis specimens containing white matter lesions by any of the methods employed, yet EBV was readily detectable in multiple Epstein-Barr virus-positive control tissues including several CNS lymphomas. Furthermore, EBV was not detected in our second cohort of multiple sclerosis specimens by in situ hybridization. However, our real-time PCR methodologies, which were capable of detecting very few EBV infected cells, detected EBV at low levels in only 2 of the 12 multiple sclerosis meningeal specimens examined. Our finding that CNS EBV infection was rare in multiple sclerosis brain indicates that EBV infection is unlikely to contribute directly to multiple sclerosis brain pathology in the vast majority of cases.

A detailed stereomechanistic analysis has led to the design of a new tetradentate ligand for the enantioselective Ti(IV)-catalyzed oxidation of unsymmetrical sulfides to sulfoxides with high selectivity. The pathway of this oxidation and the closely related and long-known Kagan–Modena oxidation have been clarified to identify the likely origin of the enantioselectivity.

Lipids comprise over 70% of the myelin sheath but have been largely underinvestigated as autoantigens in multiple sclerosis (MS). This paper cites evidence for the involvement of lipid autoreactivity in MS and details how self lipid cross-reactivity may also contribute to the development of type 1 diabetes and autoimmune thyroid disorders (both of which have been associated with MS). A further analysis of myelin chemistry suggests several mechanisms by which infection may contribute to etiology and trigger lipid autoreactivity via molecular mimicry. This analysis may aid the development of new therapies for autoimmune diseases.

According to the molecular mimicry theory, multiple sclerosis (MS) develops when the immune system mistakenly attacks a component of the myelin sheath that is structurally similar to a foreign epitope. The glycolipid galactocerebroside (GalC) is a major component of myelin. As lipids comprise between 70% and 85% of myelin, glycolipids should be investigated as candidate autoantigens in MS. GalC displays broad structural similarities to the Borrelia burgdorferi glycolipid antigen BbGL-2 and to the Sphingomonas antigen GalAGSL. In principle, therefore, these bacteria may induce an autoimmune attack on the myelin sheath. GalC is also structurally similar to natural killer T (NKT) cell ligand alpha-galactosylceramide (alpha-GalCer). Further studies must be performed to clarify the role of GalC in the activation of NKT cells and the development of MS.