HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)–dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses

The Hedgehog Signaling Pathway Emerges as a Pathogenic Target

The Hedgehog (Hh) signaling pathway plays an essential role in the growth, development, and homeostatis of many tissues in vertebrates and invertebrates. Much of what is known about Hh signaling is in the context of embryonic development and tumor formation. However, a growing body of evidence is emerging indicating that Hh signaling is also involved in postnatal processes such as tissue repair and adult immune responses. To that extent, Hh signaling has also been shown to be a target for some pathogens that presumably utilize the pathway to control the local infected environment. In this review, we discuss what is currently known regarding pathogenic interactions with Hh signaling and speculate on the reasons for this pathway being a target. We also hope to shed light on the possibility of using small molecule modulators of Hh signaling as effective therapies for a wider range of human diseases beyond their current use in a limited number of cancers

Processing of the Drosophila Hedgehog Signaling Effector Ci-155 to the Repressor Ci-75 Is Mediated by Direct Binding to the SCF Component Slimb

SummarySignaling by extracellular Hedgehog (Hh) molecules is crucial for the correct allocation of cell fates and patterns of cell proliferation in humans and other organisms [1, 2]. Responses to Hh are universally mediated by regulating the activity and the proteolysis of the Gli family of transcriptional activators such that they induce target genes only in the presence of Hh [1, 3]. In the absence of Hh, the sole Drosophila Gli homolog, Cubitus interruptus (Ci), undergoes partial proteolysis to Ci-75, which represses key Hh target genes [4]. This processing requires phosphorylation of full-length Ci (Ci-155) by protein kinase A (PKA), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3), as well as the activity of Slimb [5–7]. Slimb is homologous to vertebrate β-TRCP1, which binds as part of an SCF (Skp1/Cullin1/F-box) complex to a defined phosphopeptide motif to target proteins for ubiquitination and subsequent proteolysis [8–10]. Here, we show that phosphorylation of Ci at the specific PKA, GSK-3, and CK1 sites required in vivo for partial proteolysis stimulates binding to Slimb in vitro. Furthermore, a consensus Slimb/β-TRCP1 binding site from another protein can substitute for phosphorylated residues of Ci-155 to direct conversion to Ci-75 in vivo. From this, we conclude that Slimb binds directly to phosphorylated Ci-155 to initiate processing to Ci-75. We also explore the phosphorylated motifs in Ci that are recognized by Slimb and provide some evidence that silencing of Ci-155 by phosphorylation may involve more than binding to Slimb

Phosphoproteome Profiling of the Macrophage Response to Different Toll-Like Receptor Ligands Identifies Differences in Global Phosphorylation Dynamics

Toll-like receptors (TLRs) are among the first sensors that detect infection and drive immune response. Macrophages encountering a pathogen are usually stimulated not by one TLR, but by a combination of TLRs engaged by distinct microbe ligands. To understand the integrated signaling under complex conditions, we investigated the differences in the phosphoprotein signaling cascades triggered by TLR2, TLR4, and TLR7 ligands using a single responding cell population. We performed a global, quantitative, early poststimulation kinetic analysis of the mouse macrophage phosphoproteome using stable isotope labeling with amino acids coupled to phosphopeptide enrichment and high-resolution mass spectrometry. For each TLR ligand, we found marked elevation of phosphorylation of cytoskeleton components, GTPases of the Rho family, and phospholipase C signaling pathway proteins. Phosphorylation of proteins involved in phagocytosis was only seen in response to TLR2 and TLR4 but not to TLR7 activation. Changes in the phosphorylation of proteins involved in endocytosis were delayed in response to TLR2 as compared to TLR4 ligands. These findings reveal that the phosphoproteomic response to stimulation of distinct TLRs varies both in the major modification targets and the phosphorylation dynamics. These results advance the understanding of how macrophages sense and respond to a diverse set of TLR stimuli

Phosphoproteome Profiling of the Macrophage Response to Different Toll-Like Receptor Ligands Identifies Differences in Global Phosphorylation Dynamics

Toll-like receptors (TLRs) are among the first sensors that detect infection and drive immune response. Macrophages encountering a pathogen are usually stimulated not by one TLR, but by a combination of TLRs engaged by distinct microbe ligands. To understand the integrated signaling under complex conditions, we investigated the differences in the phosphoprotein signaling cascades triggered by TLR2, TLR4, and TLR7 ligands using a single responding cell population. We performed a global, quantitative, early poststimulation kinetic analysis of the mouse macrophage phosphoproteome using stable isotope labeling with amino acids coupled to phosphopeptide enrichment and high-resolution mass spectrometry. For each TLR ligand, we found marked elevation of phosphorylation of cytoskeleton components, GTPases of the Rho family, and phospholipase C signaling pathway proteins. Phosphorylation of proteins involved in phagocytosis was only seen in response to TLR2 and TLR4 but not to TLR7 activation. Changes in the phosphorylation of proteins involved in endocytosis were delayed in response to TLR2 as compared to TLR4 ligands. These findings reveal that the phosphoproteomic response to stimulation of distinct TLRs varies both in the major modification targets and the phosphorylation dynamics. These results advance the understanding of how macrophages sense and respond to a diverse set of TLR stimuli

IFIT1 Exerts Opposing Regulatory Effects on the Inflammatory and Interferon Gene Programs in LPS-Activated Human Macrophages

Summary: Activation of the TLR4 signaling pathway by lipopolysaccharide (LPS) leads to induction of both inflammatory and interferon-stimulated genes, but the mechanisms through which these coordinately activated transcriptional programs are balanced to promote an optimal innate immune response remain poorly understood. In a genome-wide small interfering RNA (siRNA) screen of the LPS-induced tumor necrosis factor α (TNF-α) response in macrophages, we identify the interferon-stimulated protein IFIT1 as a negative regulator of the inflammatory gene program. Transcriptional profiling further identifies a positive regulatory role for IFIT1 in type I interferon expression, implicating IFIT1 as a reciprocal modulator of LPS-induced gene classes. We demonstrate that these effects of IFIT1 are mediated through modulation of a Sin3A-HDAC2 transcriptional regulatory complex at LPS-induced gene loci. Beyond the well-studied role of cytosolic IFIT1 in restricting viral replication, our data demonstrate a function for nuclear IFIT1 in differential transcriptional regulation of separate branches of the LPS-induced gene program. : John et al. describe a function for IFIT1 in the innate immune response. Previously considered an antiviral protein, IFIT1 is identified as a reciprocal modulator of bacterially induced pro-inflammatory and interferon genes and shown to associate with chromatin regulators to modulate transcription and the host response to bacterial infection. Keywords: interferon, IFIT1, TLR4, interferon gene program, pro-inflammatory gene program, RNAi screen, TNF-

The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major.

For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo

HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)–dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.This article is published as Cook, Sarah A., William A. Comrie, M. Cecilia Poli, Morgan Similuk, Andrew J. Oler, Aiman J. Faruqi, Douglas B. Kuhns et al. "HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease." Science 369, no. 6500 (2020): 202-207. doi: 10.1126/science.aay5663.</p

Ubiquitin-Mediated Response to Microsporidia and Virus Infection in <i>C. elegans</i>

<div><p>Microsporidia comprise a phylum of over 1400 species of obligate intracellular pathogens that can infect almost all animals, but little is known about the host response to these parasites. Here we use the whole-animal host <i>C. elegans</i> to show an <i>in vivo</i> role for ubiquitin-mediated response to the microsporidian species <i>Nematocida parisii</i>, as well to the Orsay virus, another natural intracellular pathogen of <i>C. elegans</i>. We analyze gene expression of <i>C. elegans</i> in response to <i>N. parisii</i>, and find that it is similar to response to viral infection. Notably, we find an upregulation of SCF ubiquitin ligase components, such as the cullin ortholog <i>cul-6</i>, which we show is important for ubiquitin targeting of <i>N. parisii</i> cells in the intestine. We show that ubiquitylation components, the proteasome, and the autophagy pathway are all important for defense against <i>N. parisii</i> infection. We also find that SCF ligase components like <i>cul-6</i> promote defense against viral infection, where they have a more robust role than against <i>N. parisii</i> infection. This difference may be due to suppression of the host ubiquitylation system by <i>N. parisii</i>: when <i>N. parisii</i> is crippled by anti-microsporidia drugs, the host can more effectively target pathogen cells for ubiquitylation. Intriguingly, inhibition of the ubiquitin-proteasome system (UPS) increases expression of infection-upregulated SCF ligase components, indicating that a trigger for transcriptional response to intracellular infection by <i>N. parisii</i> and virus may be perturbation of the UPS. Altogether, our results demonstrate an <i>in vivo</i> role for ubiquitin-mediated defense against microsporidian and viral infections in <i>C. elegans</i>.</p></div

Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam domain (PF) enrichment analysis of <i>C. elegans</i> genes upreguated by <i>N. parisii</i> infection.

<p>GO term, KEGG pathway, and Pfam domain enrichment was analyzed using online DAVID Bioinformatics Resources 6.7. To eliminate redundancy, each term had at least 30% of associated genes not associated with any other term with a more significant P-value. *For each gene group the number of genes included in the analysis out of the total differentially expressed genes is indicated.</p