Termination of STING responses is mediated via ESCRT-dependent degradation

Published online 4 May 2023cGAS-STING signalling is induced by detection of foreign or mislocalised host double-stranded (ds)DNA within the cytosol. STING acts as the major signalling hub, where it controls production of type I interferons and inflammatory cytokines. Basally, STING resides on the ER membrane. Following activation STING traffics to the Golgi to initiate downstream signalling and subsequently to endolysosomal compartments for degradation and termination of signalling. While STING is known to be degraded within lysosomes, the mechanisms controlling its delivery remain poorly defined. Here we utilised a proteomics-based approach to assess phosphorylation changes in primary murine macrophages following STING activation. This identified numerous phosphorylation events in proteins involved in intracellular and vesicular transport. We utilised high-temporal microscopy to track STING vesicular transport in live macrophages. We subsequently identified that the endosomal complexes required for transport (ESCRT) pathway detects ubiquitinated STING on vesicles, which facilitates the degradation of STING in murine macrophages. Disruption of ESCRT functionality greatly enhanced STING signalling and cytokine production, thus characterising a mechanism controlling effective termination of STING signalling.Katherine R Balka, Rajan Venkatraman, Tahnee L Saunders, Angus Shoppee, Ee Shan Pang, Zoe Magill, Jihane Homman-Ludiye, Cheng Huang, Rachael M Lane, Harrison M York, Peck Tan, Ralf B Schittenhelm, Senthil Arumugam, Benjamin T Kile, Meredith O, Keeffe, Dominic De Nard

Characterization of Mammalian Regulatory Complexes at Single-Locus Resolution Using TINC

Book series - Print ISSN: 1064-3745 ; Electronic ISSN: 1940-6029In mammalian cells, multiprotein complexes form at specific genomic regulatory elements (REs) to control gene expression, which in turn is ultimately responsible for cellular identity. Consequently, insight into the molecular composition of these regulatory complexes is of major importance for our understanding of any physiological or pathological cellular state or transition. However, it remains extremely difficult to identify the protein complex(es) assembled at a specific RE in the mammalian genome using conventional approaches. We therefore developed a novel single locus isolation technique based on Transcription Activator-Like Effector (TALE) proteins termed TALE-mediated isolation of nuclear chromatin (TINC). When coupled with high-resolution mass spectrometry, TINC enables the identification and characterization of protein complexes formed at any RE of interest. Using the Nanog promoter in mouse embryonic stem cells as proof of concept, this chapter describes in detail the novel TINC methodology as well as subsequent mass spectrometric considerations.Anja S. Knaupp, Ralf B. Schittenhelm, and Jose M. Pol

Mitochondrial dysfunction caused by outer membrane vesicles from Gram-negative bacteria activates intrinsic apoptosis and inflammation

Published17 August 2020Sensing of microbes activates the innate immune system, depending on functional mitochondria. However, pathogenic bacteria inhibit mitochondrial activity by delivering toxins via outer membrane vesicles (OMVs). How macrophages respond to pathogenic microbes that target mitochondria remains unclear. Here, we show that macrophages exposed to OMVs from Neisseria gonorrhoeae, uropathogenic Escherichia coli and Pseudomonas aeruginosa induce mitochondrial apoptosis and NLRP3 inflammasome activation. OMVs and toxins that cause mitochondrial dysfunction trigger inhibition of host protein synthesis, which depletes the unstable BCL-2 family member MCL-1 and induces BAK-dependent mitochondrial apoptosis. In parallel with caspase-11-mediated pyroptosis, mitochondrial apoptosis and potassium ion efflux activate the NLRP3 inflammasome after OMV exposure in vitro. Importantly, in the in vivo setting, the activation and release of interleukin-1β in response to N. gonorrhoeae OMVs is regulated by mitochondrial apoptosis. Our data highlight how innate immune cells sense infections by monitoring mitochondrial health.Pankaj Deo, Seong H. Chow, Mei-Ling Han, Mary Speir, Cheng Huang, Ralf B. Schittenhelm, Subhash Dhital, Jack Emery, Jian Li, Benjamin T. Kile, James E. Vince, Kate E. Lawlor and Thomas Nadere

SMOC1 is a glucose-responsive hepatokine and therapeutic target for glycemic control

Intertissue communication is a fundamental feature of metabolic regulation, and the liver is central to this process. We have identified sparc-related modular calcium-binding protein 1 (SMOC1) as a glucose-responsive hepatokine and regulator of glucose homeostasis. Acute intraperitoneal administration of SMOC1 improved glycemic control and insulin sensitivity in mice without changes in insulin secretion. SMOC1 exerted its favorable glycemic effects by inhibiting adenosine 3',5'-cyclic monophosphate (cAMP)-cAMP-dependent protein kinase (PKA)-cAMP response element-binding protein (CREB) signaling in the liver, leading to decreased gluconeogenic gene expression and suppression of hepatic glucose output. Overexpression of SMOC1 in the liver or once-weekly intraperitoneal injections of a stabilized SMOC1-FC fusion protein induced durable improvements in glucose tolerance and insulin sensitivity in db/db mice, without adverse effects on adiposity, liver histopathology, or inflammation. Furthermore, circulating SMOC1 correlated with hepatic and systemic insulin sensitivity and was decreased in obese, insulin-resistant humans. Together, these findings identify SMOC1 as a potential pharmacological target for the management of glycemic control in type 2 diabetes.Magdalene K. Montgomery, Jacqueline Bayliss, Camille Devereux, Ayenachew Bezawork-Geleta ... Scott L. Townley, Luke A. Selth ... et al

Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.Xiaodong Liu, Christian M Nefzger, Fernando J Rossello, Joseph Chen, Anja S Knaupp, Jaber Firas ... et al

Suppressing fatty acid uptake has therapeutic effects in preclinical models of prostate cancer

Metabolism alterations are hallmarks of cancer, but the involvement of lipid metabolism in disease progression is unclear. We investigated the role of lipid metabolism in prostate cancer using tissue from patients with prostate cancer and patient-derived xenograft mouse models. We showed that fatty acid uptake was increased in human prostate cancer and that these fatty acids were directed toward biomass production. These changes were mediated, at least partly, by the fatty acid transporter CD36, which was associated with aggressive disease. Deleting Cd36 in the prostate of cancer-susceptible Pten⁻⁄⁻ mice reduced fatty acid uptake and the abundance of oncogenic signaling lipids and slowed cancer progression. Moreover, CD36 antibody therapy reduced cancer severity in patient-derived xenografts. We further demonstrated cross-talk between fatty acid uptake and de novo lipogenesis and found that dual targeting of these pathways more potently inhibited proliferation of human cancer-derived organoids compared to the single treatments. These findings identify a critical role for CD36-mediated fatty acid uptake in prostate cancer and suggest that targeting fatty acid uptake might be an effective strategy for treating prostate cancer.Matthew J. Watt, Ashlee K. Clark, Luke A. Selth, Vanessa R. Haynes, Natalie Lister, Richard Rebello, Laura H. Porter, Birunthi Niranjan, Sarah T. Whitby, Jennifer Lo, Cheng Huang, Ralf B. Schittenhelm, Kimberley E. Anderson, Luc Furic, Poornima R. Wijayaratne, Maria Matzaris, Magdalene K. Montgomery, Melissa Papargiris, Sam Norden, Maria Febbraio, Gail P. Risbridger, Mark Frydenberg, Daniel K. Nomura, Renea A. Taylo

A Natural Peptide Antigen within the Plasmodium Ribosomal Protein RPL6 Confers Liver TRM Cell-Mediated Immunity against Malaria in Mice

Liver-resident memory CD8+ T (TRM) cells remain in and constantly patrol the liver to elicit rapid immunity upon antigen encounter and can mediate efficient protection against liver-stage Plasmodium infection. This finding has prompted the development of immunization strategies where T cells are activated in the spleen and then trapped in the liver to form TRM cells. Here, we identify PbRPL6120-127, a H2-Kb-restricted epitope from the putative 60S ribosomal protein L6 (RPL6) of Plasmodium berghei ANKA, as an optimal antigen for endogenous liver TRM cell generation and protection against malaria. A single dose vaccination targeting RPL6 provided effective and prolonged sterilizing immunity against high dose sporozoite challenges. Expressed throughout the parasite life cycle, across Plasmodium species, and highly conserved, RPL6 exhibits strong translation potential as a vaccine candidate. This is further advocated by the identification of a broadly conserved, immunogenic HLA-A∗02:01-restricted epitope in P. falciparum RPL6