Stat3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in multiple cancers. Inhibition of Stat3 signaling pathways suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting direct inhibition of Stat3 function is a viable therapeutic approach. Herein, we identify a small molecule, C48, as a selective Stat3-family member inhibitor. To determine its mechanism of action, we used site-directed mutagenesis and multiple biochemical techniques to show that C48 alkylates Cys468 in Stat3, a residue at the DNA-binding interface. We further demonstrate that C48 blocks accumulation of activated Stat3 in the nucleus in tumor cell lines that over-express active Stat3 leading to impressive inhibition of tumor growth in mouse models. Collectively, these findings suggest Cys468 in Stat3 represents a novel site for therapeutic intervention and demonstrates the promise of alkylation as a potentially effective chemical approach for Stat3-dependent cancers. KeywordsStat3; small molecule inhibitor; alkylation; cysteine; cancer; drug development Signal Transducers and Activators of Transcription (STATs) comprise a family of transcription factors that transmit signal responses triggered by cytokines, growth factors and hormones (1-3). Upon ligand engagement, STATs are recruited to cell surface receptors and become phosphorylated at a single carboxy-terminal tyrosine by receptor-intrinsic, receptor-associated or non-receptor tyrosine kinases. Tyrosine phosphorylation of STATs then initiates their nuclear translocation, resulting in binding of activated STAT dimers to specific promoter regions in DNA and regulation of target gene expression (4-7). Although STATs exist as non-phosphorylated and phosphorylated dimers, it has been speculated that the conformational change that occurs after tyrosine phosphorylation stabilizes the dimer and improves its ability to bind to specific DNA regions (8-13).The STAT family includes seven members, namely Stat5a, Stat5b, and Stat6 (1). In normal cells, STAT signaling is usually transient and tightly regulated by cytokines

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Rationale Oxidative stress is an important contributing factor in a number of human pathologies ranging from atherosclerosis to cancer progression; however, the mechanisms underlying tissue protection from oxidation products are poorly understood. Oxidation of membrane phospholipids, containing the polyunsaturated fatty acid DHA, results in the accumulation of an end product, 2-(ω-carboxyethyl)pyrrole (CEP), which was shown to have pro-angiogenic and pro-inflammatory functions. While CEP is continuously accumulated during chronic processes such as tumor progression and atherosclerosis, its levels during wound healing return to normal when the wound is healed, suggesting the existence of a specific clearance mechanism. Objective To identify the cellular and molecular mechanism for CEP clearance. Methods and Results Here we show that macrophages are able to bind, scavenge, and metabolize carboxyethylpyrrole derivatives of proteins but not structurally similar ethylpyrrole derivatives, demonstrating the high specificity of the process. F4/80hi and M2-skewed macrophages are much more efficient at CEP binding and scavenging compared to F4/80lo and M1-skewed macrophages. Depletion of macrophages leads to increased CEP accumulation in vivo. CEP binding and clearance are dependent on two receptors expressed by macrophages, CD36 and TLR2. While knockout of each individual receptor results in diminished CEP clearance, the lack of both receptors almost completely abrogates macrophages’ ability to scavenge CEP derivatives of proteins. Conclusions Our study demonstrates the mechanisms of recognition, scavenging, and clearance of pathophysiologically active products of lipid oxidation in vivo, thereby contributing to tissue protection against products of oxidative stress.

Angiotensin-converting enzyme (ACE) converts angiotensin I into the potent vasoconstrictor angiotensin II, which regulates blood pressure. However, ACE activity is also essential for other physiological functions, presumably through processing of peptides unrelated to angiotensin. The goal of this study was to identify novel natural substrates and products of ACE through a series of mass-spectrometric experiments. This included comparing the ACE-treated and untreated plasma peptidomes of ACE-knockout (KO) mice, validation with select synthetic peptides, and a quantitative in vivo study of ACE substrates in mice with distinct genetic ACE backgrounds. In total, 244 natural peptides were identified ex vivo as possible substrates or products of ACE, demonstrating high promiscuity of the enzyme. ACE prefers to cleave substrates with Phe or Leu at the C-terminal P2′ position and Gly in the P6 position. Pro in P1′ and Iso in P1 are typical residues in peptides that ACE does not cleave. Several of the novel ACE substrates are known to have biological activities, including a fragment of complement C3, the spasmogenic C3f, which was processed by ACE ex vivo and in vitro. Analyses with N-domain-inactive (NKO) ACE allowed clarification of domain selectivity toward substrates. The in vivo ACE-substrate concentrations in WT, transgenic ACE-KO, NKO, and CKO mice correspond well with the in vitro observations in that higher levels of the ACE substrates were observed when the processing domain was knocked out. This study highlights the vast extent of ACE promiscuity and provides a valuable platform for further investigations of ACE functionality.

Colonization of the gut by certain probiotic Lactobacillus reuteri strains has been associated with reduced risk of inflammatory diseases and colorectal cancer. Previous studies pointed to a functional link between immunomodulation, histamine production, and folate metabolism, the central 1‐carbon pathway for the transfer of methyl groups. Using mass spectrometry and NMR spectroscopy, we analyzed folate metabolites of L. reuteri strain 6475 and discovered that the bacterium produces a 2‐carbon‐transporting folate in the form of 5, 10‐ethenyl‐tetrahydrofolyl polyglutamate. Isotopic labeling permitted us to trace the source of the 2‐carbon unit back to acetate of the culture medium. We show that the 2C folate cycle of L. reuteri is capable of transferring 2 carbon atoms to homocysteine to generate the unconventional amino acid ethionine, a known immunomodulator. When we treated monocytic THP‐1 cells with ethionine, their transcription of TNF‐α was inhibited and cell proliferation reduced. Mass spectrometry of THP‐1 histones revealed incorporation of ethionine instead of methionine into proteins, a reduction of histone‐methylation, and ethylation of histone lysine residues. Our findings suggest that the microbiome can expose the host to ethionine through a novel 2‐carbon transporting variant of the folate cycle and modify human chromatin via ethylation.—Roth, D., Chiang, A. J., Hu, W., Gugiu, G. B., Morra, C. N., Versalovic, J., Kalkum, M. The two‐carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation. FASEB J. 33,3536‐3548 (2019). http://www.fasebj.org