Aire in Autoimmunity.

The role of the autoimmune regulator (Aire) in central immune tolerance and thymic self-representation was first described more than 20 years ago, but fascinating new insights into its biology continue to emerge, particularly in the era of advanced single-cell genomics. We briefly describe the role of human genetics in the discovery of Aire, as well as insights into its function gained from genotype-phenotype correlations and the spectrum of Aire-associated autoimmunity-including insights from patients with Aire mutations with broad and diverse implications for human health. We then highlight emerging trends in Aire biology, focusing on three topic areas. First, we discuss medullary thymic epithelial diversity and the role of Aire in thymic epithelial development. Second, we highlight recent developments regarding the molecular mechanisms of Aire and its binding partners. Finally, we describe the rapidly evolving biology of the identity and function of extrathymic Aire-expressing cells (eTACs), and a novel eTAC subset called Janus cells, as well as their potential roles in immune homeostasis.

The transcriptional regulator Aire coopts the repressive ATF7ip-MBD1 complex for the induction of immunotolerance.

The maintenance of immunological tolerance requires the deletion of self-reactive T cells in the thymus. The expression of genes encoding tissue-specific antigens (TSAs) by thymic epithelial cells is critical for this process and depends on activity of the transcriptional regulator Aire; however, the molecular mechanisms Aire uses to target loci encoding TSAs are unknown. Here we identified two Aire-interacting proteins known to be involved in gene repression, ATF7ip and MBD1, that were required for Aire's targeting of loci encoding TSAs. Moreover, Mbd1(-/-) mice developed pathological autoimmunity and had a defect in Aire-dependent thymic expression of genes encoding TSAs, which underscores the importance of Aire's interaction with the ATF7ip-MBD1 protein complex in maintaining central tolerance.

ATF7ip Targets Transposable Elements for H3K9me3 Deposition to Modify CD8+ T Cell Effector and Memory Responses.

CD8+ T cells are critical for the immune response to pathogens and tumors, and CD8+ T cell memory protects against repeat infections. In this study, we identify the activating transcription factor 7 interacting protein (ATF7ip) as a critical regulator of CD8+ T cell immune responses. Mice with a T cell-specific deletion of ATF7ip have a CD8+ T cell-intrinsic enhancement of Il7r expression and Il2 expression leading to enhanced effector and memory responses. Chromatin immunoprecipitation sequencing studies identified ATF7ip as a repressor of Il7r and Il2 gene expression through the deposition of the repressive histone mark H3K9me3 at the Il7r gene and Il2-Il21 intergenic region. Interestingly, ATF7ip targeted transposable elements for H3K9me3 deposition at both the IL7r locus and the Il2-Il21 intergenic region, indicating that ATF7ip silencing of transposable elements is important for regulating CD8+ T cell function. These results demonstrate a new epigenetic pathway by which IL-7R and IL-2 production are constrained in CD8+ T cells, and this may open up new avenues for modulating their production.

Functional proteomic analysis of long-term growth factor stimulation and receptor tyrosine kinase coactivation in Swiss 3T3 fibroblasts.

In Swiss 3T3 fibroblasts, long-term stimulation with PDGF, but not insulin-like growth factor 1 (IGF-1) or EGF, results in the establishment of an elongated migratory phenotype, characterized by the formation of retractile dendritic protrusions and absence of actin stress fibers and focal adhesion complexes. To identify receptor tyrosine kinase-specific reorganization of the Swiss 3T3 proteome during phenotypic differentiation, we compared changes in the pattern of protein synthesis and phosphorylation during long-term exposure to PDGF, IGF-1, EGF, and their combinations using 2DE-based proteomics after (35)S- and (33)P-metabolic labeling. One hundred and five differentially regulated proteins were identified by mass spectrometry and some of these extensively validated. PDGF stimulation produced the highest overall rate of protein synthesis at any given time and induced the most sustained phospho-signaling. Simultaneous activation with two or three of the growth factors revealed both synergistic and antagonistic effects on protein synthesis and expression levels with PDGF showing dominance over both IGF-1 and EGF in generating distinct proteome compositions. Using signaling pathway inhibitors, PI3K was identified as an early site for signal diversification, with sustained activity of the PI3K/AKT pathway critical for regulating late protein synthesis and phosphorylation of target proteins and required for maintaining the PDGF-dependent motile phenotype. Several proteins were identified with novel PI3K/Akt-dependent synthesis and phosphorylations including eEF2, PRS7, RACK-1, acidic calponin, NAP1L1, Hsp73, and fascin. The data also reveal induction/suppression of key F-actin and actomyosin regulators and chaperonins that enable PDGFR to direct the assembly of a motile cytoskeleton, despite simultaneous antagonistic signaling activities. Together, the study demonstrates that long-term exposure to different growth factors results in receptor tyrosine kinase-specific regulation of relatively small subproteomes, and implies that the strength and longevity of receptor tyrosine kinase-specific signals are critical in defining the composition and functional activity of the resulting proteome.

Ikaros is a principal regulator of Aire+ mTEC homeostasis, thymic mimetic cell diversity, and central tolerance.

Mutations in the gene encoding the zinc-finger transcription factor Ikaros (IKZF1) are found in patients with immunodeficiency, leukemia, and autoimmunity. Although Ikaros has a well-established function in modulating gene expression programs important for hematopoietic development, its role in other cell types is less well defined. Here, we uncover functions for Ikaros in thymic epithelial lineage development in mice and show that Ikzf1 expression in medullary thymic epithelial cells (mTECs) is required for both autoimmune regulator-positive (Aire+) mTEC development and tissue-specific antigen (TSA) gene expression. Accordingly, TEC-specific deletion of Ikzf1 in mice results in a profound decrease in Aire+ mTECs, a global loss of TSA gene expression, and the development of autoimmunity. Moreover, Ikaros shapes thymic mimetic cell diversity, and its deletion results in a marked expansion of thymic tuft cells and muscle-like mTECs and a loss of other Aire-dependent mimetic populations. Single-cell analysis reveals that Ikaros modulates core transcriptional programs in TECs that correlate with the observed cellular changes. Our findings highlight a previously undescribed role for Ikaros in regulating epithelial lineage development and function and suggest that failed thymic central tolerance could contribute to the autoimmunity seen in humans with IKZF1 mutations.

Republication: Targeting PI3KC2ß Impairs Proliferation and Survival in Acute Leukemia, Brain Tumours and Neuroendocrine Tumours.

BACKGROUND: Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. MATERIALS AND METHODS: The expression pattern and functions of the class II PI3KC2ß isoform were investigated in a panel of tumour samples and cell lines. RESULTS: Overexpression of PI3KC2ß was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2ß or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2ß also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. CONCLUSION: Together, these data show that PI3KC2ß contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.

Major role of epidermal growth factor receptor and Src kinases in promoting oxidative stress-dependent loss of adhesion and apoptosis in epithelial cells.

A growing body of evidence suggests that reactive oxygen species are critical components of cell signaling pathways, in particular regulating protein phosphorylation events. Here, we show that oxidative stress in response to hydrogen peroxide treatment of human epithelial cells induces robust tyrosine phosphorylation on multiple proteins. Using an anti-phosphotyrosine purification and liquid chromatography-tandem mass spectrometry approach, we have identified many of these H(2)O(2)-induced tyrosine-phosphorylated proteins. Importantly, we show that epidermal growth factor receptor (EGFR) and Src are the primary upstream kinases mediating these events through their redox activation. The finding that many of the identified proteins have functions in cell adhesion, cell-cell junctions, and the actin cytoskeleton prompted us to examine stress-induced changes in adhesion. Immunofluorescence analysis showed that H(2)O(2) alters cell adhesion structures and the actin cytoskeleton causing loss of adhesion and apoptosis. Remarkably, these cellular changes could be attenuated by inhibition of EGFR and Src, identifying these kinases as targets to block oxidative damage. In summary, our data demonstrate that EGFR and Src together play a central role in oxidative stress-induced phosphorylation, which in turn results in loss of adhesion, morphological changes, and cell damage in epithelial cells. These data also provide a general model for redox signaling in other cell systems.

mTORbeta splicing isoform promotes cell proliferation and tumorigenesis.

The mTOR (mammalian target of rapamycin) promotes growth in response to nutrients and growth factors and is deregulated in numerous pathologies, including cancer. The mechanisms by which mTOR senses and regulates energy metabolism and cell growth are relatively well understood, whereas the molecular events underlining how it mediates survival and proliferation remain to be elucidated. Here, we describe the existence of the mTOR splicing isoform, TOR beta, which, in contrast to the full-length protein (mTOR alpha), has the potential to regulate the G(1) phase of the cell cycle and to stimulate cell proliferation. mTOR beta is an active protein kinase that mediates downstream signaling through complexing with Rictor and Raptor proteins. Remarkably, overexpression of mTOR beta transforms immortal cells and is tumorigenic in nude mice and therefore could be a proto-oncogene.

Phosphoinositide 3-Kinase C2beta regulates cytoskeletal organization and cell migration via Rac-dependent mechanisms.

Receptor-linked class I phosphoinositide 3-kinases (PI3Ks) induce assembly of signal transduction complexes through protein-protein and protein-lipid interactions that mediate cell proliferation, survival, and migration. Although class II PI3Ks have the potential to make the same phosphoinositides as class I PI3Ks, their precise cellular role is currently unclear. In this report, we demonstrate that class II phosphoinositide 3-kinase C2beta (PI3KC2beta) associates with the Eps8/Abi1/Sos1 complex and is recruited to the EGF receptor as part of a multiprotein signaling complex also involving Shc and Grb2. Increased expression of PI3KC2beta stimulated Rac activity in A-431 epidermoid carcinoma cells, resulting in enhanced membrane ruffling and migration speed of the cells. Conversely, expression of dominant negative PI3KC2beta reduced Rac activity, membrane ruffling, and cell migration. Moreover, PI3KC2beta-overexpressing cells were protected from anoikis and displayed enhanced proliferation, independently of Rac function. Taken together, these findings suggest that PI3KC2beta regulates the migration and survival of human tumor cells by distinct molecular mechanisms.

Gene and protein expression profiling of human ovarian cancer cells treated with the heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin.

The promising antitumor activity of 17-allylamino-17-demethoxygeldanamycin (17AAG) results from inhibition of the molecular chaperone heat shock protein 90 (HSP90) and subsequent degradation of multiple oncogenic client proteins. Gene expression microarray and proteomic analysis were used to profile molecular changes in the A2780 human ovarian cancer cell line treated with 17AAG. Comparison of results with an inactive analogue and an alternative HSP90 inhibitor radicicol indicated that increased expression of HSP72, HSC70, HSP27, HSP47, and HSP90beta at the mRNA level were on-target effects of 17AAG. HSP27 protein levels were increased in tumor biopsies following treatment of patients with 17AAG. A group of MYC-regulated mRNAs was decreased by 17AAG. Of particular interest and novelty were changes in expression of chromatin-associated proteins. Expression of the heterochromatin protein 1 was increased, and expression of the histone acetyltransferase 1 and the histone arginine methyltransferase PRMT5 was decreased by 17AAG. PRMT5 was shown to be a novel HSP90-binding partner and potential client protein. Cellular protein acetylation was reduced by 17AAG, which was shown to have an antagonistic interaction on cell proliferation with the histone deacetylase inhibitor trichostatin A. This mRNA and protein expression analysis has provided new insights into the complex molecular pharmacology of 17AAG and suggested new genes and proteins that may be involved in response to the drug or be potential biomarkers of drug action.

Gene Expression Meta-Analysis Reveals Concordance in Gene Activation, Pathway, and Cell-Type Enrichment in Dermatomyositis Target Tissues.

OBJECTIVE: We conducted a comprehensive gene expression meta-analysis in dermatomyositis (DM) muscle and skin tissues to identify shared disease-relevant genes and pathways across tissues. METHODS: Six publicly available data sets from DM muscle and two from skin were identified. Meta-analysis was performed by first processing data sets individually then cross-study normalization and merging creating tissue-specific gene expression matrices for subsequent analysis. Complementary single-gene and network analyses using Significance Analysis of Microarrays (SAM) and Weighted Gene Co-expression Network Analysis (WGCNA) were conducted to identify genes significantly associated with DM. Cell-type enrichment was performed using xCell. RESULTS: There were 544 differentially expressed genes (FC ≥ 1.3, q < 0.05) in muscle and 300 in skin. There were 94 shared upregulated genes across tissues enriched in type I and II interferon (IFN) signaling and major histocompatibility complex (MHC) class I antigen-processing pathways. In a network analysis, we identified eight significant gene modules in muscle and seven in skin. The most highly correlated modules were enriched in pathways consistent with the single-gene analysis. Additional pathways uncovered by WGCNA included T-cell activation and T-cell receptor signaling. In the cell-type enrichment analysis, both tissues were highly enriched in activated dendritic cells and M1 macrophages. CONCLUSION: There is striking similarity in gene expression across DM target tissues with enrichment of type I and II IFN pathways, MHC class I antigen-processing, T-cell activation, and antigen-presenting cells. These results suggest IFN-γ may contribute to the global IFN signature in DM, and altered auto-antigen presentation through the class I MHC pathway may be important in disease pathogenesis.

The epigenetic regulator ATF7ip inhibits Il2 expression, regulating Th17 responses.

T helper 17 cells (Th17) are critical for fighting infections at mucosal surfaces; however, they have also been found to contribute to the pathogenesis of multiple autoimmune diseases and have been targeted therapeutically. Due to the role of Th17 cells in autoimmune pathogenesis, it is important to understand the factors that control Th17 development. Here we identify the activating transcription factor 7 interacting protein (ATF7ip) as a critical regulator of Th17 differentiation. Mice with T cell-specific deletion of Atf7ip have impaired Th17 differentiation secondary to the aberrant overproduction of IL-2 with T cell receptor (TCR) stimulation and are resistant to colitis in vivo. ChIP-seq studies identified ATF7ip as an inhibitor of Il2 gene expression through the deposition of the repressive histone mark H3K9me3 in the Il2-Il21 intergenic region. These results demonstrate a new epigenetic pathway by which IL-2 production is constrained, and this may open up new avenues for modulating its production.

Structural analysis of PI3-kinase isoforms: identification of residues enabling selective inhibition by small molecule ATP-competitive inhibitors.

A series of small molecule, ATP-competitive phosphoinositide 3-kinase inhibitors have been examined in homology models of the four class I isoforms, p110alpha, p110beta, p110delta and p110gamma. This analysis provided an insight into the mode of binding of these inhibitors to the hinge and to other key regions of the ATP binding site in each of the four subtypes. Significantly, residues were identified that differ between these proteins, and which help explain the isoform-selective inhibition profiles of the compounds.

Exploring the specificity of the PI3K family inhibitor LY294002.

The PI3Ks (phosphatidylinositol 3-kinases) regulate cellular signalling networks that are involved in processes linked to the survival, growth, proliferation, metabolism and specialized differentiated functions of cells. The subversion of this network is common in cancer and has also been linked to disorders of inflammation. The elucidation of the physiological function of PI3K has come from pharmacological studies, which use the enzyme inhibitors Wortmannin and LY294002, and from PI3K genetic knockout models of the effects of loss of PI3K function. Several reports have shown that LY294002 is not exclusively selective for the PI3Ks, and could in fact act on other lipid kinases and additional apparently unrelated proteins. Since this inhibitor still remains a drug of choice in numerous PI3K studies (over 500 in the last year), it is important to establish the precise specificity of this compound. We report here the use of a chemical proteomic strategy in which an analogue of LY294002, PI828, was immobilized onto epoxy-activated Sepharose beads. This affinity material was then used as a bait to fish-out potential protein targets from cellular extracts. Proteins with high affinity for immobilized PI828 were separated by one-dimensional gel electrophoresis and identified by liquid chromatography-tandem MS. The present study reveals that LY294002 not only binds to class I PI3Ks and other PI3K-related kinases, but also to novel targets seemingly unrelated to the PI3K family.

Analysis of pulmonary features and treatment approaches in the COPA syndrome.

The COPA syndrome is a monogenic, autoimmune lung and joint disorder first identified in 2015. This study sought to define the main pulmonary features of the COPA syndrome in an international cohort of patients, analyse patient responses to treatment and highlight when genetic testing should be considered. We established a cohort of subjects (N=14) with COPA syndrome seen at multiple centres including the University of California, San Francisco, CA, USA. All subjects had one of the previously established mutations in the COPA gene, and had clinically apparent lung disease and arthritis. We analysed cohort characteristics using descriptive statistics. All subjects manifested symptoms before the age of 12 years, had a family history of disease, and developed diffuse parenchymal lung disease and arthritis. 50% had diffuse alveolar haemorrhage. The most common pulmonary findings included cysts on chest computed tomography and evidence of follicular bronchiolitis on lung biopsy. All subjects were positive for anti-neutrophil cytoplasmic antibody, anti-nuclear antibody or both and 71% of subjects had rheumatoid factor positivity. All subjects received immunosuppressive therapy. COPA syndrome is an autoimmune disorder defined by diffuse parenchymal lung disease and arthritis. We analysed an international cohort of subjects with genetically confirmed COPA syndrome and found that common pulmonary features included cysts, follicular bronchiolitis and diffuse alveolar haemorrhage. Common extrapulmonary features included early age of onset, family history of disease, autoantibody positivity and arthritis. Longitudinal data demonstrated improvement on chest radiology but an overall decline in pulmonary function despite chronic treatment.

Phosphorylation of NF-kappaB1/p105 by oncoprotein kinase Tpl2: implications for a novel mechanism of Tpl2 regulation.

The oncoprotein kinase Tpl2 plays an essential role in macrophage activation by the bacterial component lipopolysaccharide (LPS). In response to LPS stimulation, Tpl2 phosphorylates a downstream kinase, MEK1, leading to the activation of ERK signaling pathway. Recent studies demonstrate that the NF-kappaB1 precursor protein p105 functions as an inhibitor of Tpl2 and that the LPS-stimulated Tpl2 activation requires p105 degradation. However, how p105 inhibits the signaling function of Tpl2 is not completely understood. We show here that p105 does not inhibit the intrinsic kinase activity of Tpl2. When complexed with p105, Tpl2 remains catalytically active and uses p105 as a substrate. However, the p105-bound Tpl2 is unable to phosphorylate its physiological target, MEK1. These findings suggest that p105 functions as a competitive inhibitor of Tpl2 that blocks its access by MEK1.

IkappaB kinase is an essential component of the Tpl2 signaling pathway.

IkappaB kinase (IKK), a key regulator of immune and inflammatory responses, is known as an effector kinase mediating activation of the transcription factor NF-kappaB. Whether IKK also participates in other signaling events is not known. Here we show that IKK serves as an essential component of a signaling pathway that involves activation of the Tpl2 kinase and its downstream targets, MEK1 and ERK. Inhibition of IKKbeta in macrophages eliminates Tpl2 activation and ERK phosphorylation induced by lipopolysaccharide and tumor necrosis factor alpha. Using IKK-deficient murine fibroblasts, we further demonstrate that IKKbeta, but not IKKalpha, is required for Tpl2 activation. Moreover, this novel function of IKKbeta appears to involve phosphorylation and degradation of the Tpl2 inhibitor NF-kappaB1/p105. These findings suggest that IKKbeta exerts its immune-regulatory functions by targeting different downstream signaling pathways.

Targeted expression of the class II phosphoinositide 3-kinase in Drosophila melanogaster reveals lipid kinase-dependent effects on patterning and interactions with receptor signaling pathways.

Phosphoinositide 3-kinases (PI3Ks) can be divided into three distinct classes (I, II, and III) on the basis of their domain structures and the lipid signals that they generate. Functions have been assigned to the class I and class III enzymes but have not been established for the class II PI3Ks. We have obtained the first evidence for a biological function for a class II PI3K by expressing this enzyme during Drosophila melanogaster development and by using deficiencies that remove the endogenous gene. Wild-type and catalytically inactive PI3K_68D transgenes have opposite effects on the number of sensory bristles and on wing venation phenotypes induced by modified epidermal growth factor (EGF) receptor signaling. These results indicate that the endogenous PI3K_68D may act antagonistically to the EGF receptor-stimulated Ras-mitogen-activated protein kinase pathway and downstream of, or parallel to, the Notch receptor. A class II polyproline motif in PI3K_68D can bind the Drk adaptor protein in vitro, primarily via the N-terminal SH3 domain of Drk. Drk may thus be important for the localization of PI3K_68D, allowing it to modify signaling pathways downstream of cell surface receptors. The phenotypes obtained are markedly distinct from those generated by expression of the Drosophila class I PI3K, which affects growth but not pattern formation.

Proteomic analysis of UVC irradiation-induced damage of plasma proteins: Serum amyloid P component as a major target of photolysis.

Ultraviolet-C (UVC) irradiation is a pathogen inactivation method used for disinfection of pharmaceutical products derived from human blood. Previous studies have shown that UVC can potentially damage proteins through photolysis or can generate reactive species resulting in protein thiol oxidation. In this study, two fluorescence-based quantitative proteomic approaches were used to assess the effects of a novel UVC-disinfection strategy on human plasma fractions. We show minimal changes in protein content, but gross alterations in protein thiol reactivity, indicative of oxidative damage. We identify a number of the damaged proteins by mass spectrometry, including serum amyloid P component, and further demonstrate UVC-induced photolysis of its disulphide bond.

Identification of novel candidates for replicative senescence by functional proteomics.

To identify the underlying mechanisms that limit the mitotic potential of normal somatic cells, we have undertaken a high resolution differential proteomic analysis aimed at identifying proteins that were differentially expressed upon replicative senescence. Since replicative senescence in heterogeneous primary fibroblast cultures is asynchronous, we analysed a group of conditionally immortalized rat embryo fibroblast cell lines that have previously been shown to undergo synchronous senescence upon inactivation of SV40 tsA58 T antigen. This identified 43 spots that were differentially expressed in these cell lines. Comparison of the identity of these features with those identified in a complimentary independent differential proteomic analysis of replicative senescence, directly in primary rat embryo fibroblasts upon serial passaging, identified nine features that were in common between the two studies even though they had been conducted entirely separately. None of these proteins have previously been recognized to be involved with replicative senescence. Thus, they represent novel starting points for elucidating the underlying mechanism that regulates the finite mitotic life span of somatic cells and how it can be overcome in cancer cells.