FBXO3 promotes ubiquitylation and transcriptional activity of AIRE. The study found that the E3 ubiquitin ligase FBXO3 interacts with phosphorylated residues on AIRE and promotes its ubiquitylation. This post-translational modification increases the interaction between AIRE and P-TEFb, potentiating their transcriptional activity on tissue-specific antigen genes in the thymus. Knockdown of FBXO3 decreased ubiquitylation and transcriptional activity of AIRE.
This document reports on a study examining the effects of somatostatin (SST) on human B lymphoblasts. The key findings are:
1) SST stimulates phospholipase C activity and increases cytosolic calcium levels in B lymphoblasts, likely through coupling of SSTR2A to the G protein Gα16.
2) SST activates extracellular signal-regulated kinases and induces increased DNA synthesis, proliferation, and immunoglobulin formation in B lymphoblasts.
3) These stimulatory effects of SST on early signal transduction pathways are accompanied by increased cell proliferation and immunoglobulin production in human B lymphoblasts, indicating SST exerts growth factor-like
1) PSF, a pre-mRNA splicing factor, was identified as a constituent of PER complexes purified from mouse tissues.
2) PSF recruits the SIN3A-HDAC complex to the Per1 promoter in a circadian manner through interactions with PER complexes.
3) Recruitment of the SIN3A-HDAC complex by the PER complex leads to histone deacetylation at the Per1 promoter, providing a molecular mechanism for circadian clock negative feedback through rhythmic repression of Per1 transcription.
Early growth response gene 1 mediated apoptosis is essential for transforming...Tiensae Teshome
This study examined the role of Early Growth Response gene 1 (Egr-1) in Transforming Growth Factor beta 1 (TGF-β1)-induced apoptosis and pulmonary fibrosis using a novel triple transgenic mouse model. The results demonstrated that TGF-β1 caused epithelial apoptosis, followed by inflammation and pulmonary fibrosis. Null mutation of Egr-1 or caspase inhibition blocked TGF-β1-induced apoptosis and significantly reduced fibrosis. This establishes that Egr-1-mediated apoptosis is required for TGF-β1 to induce pulmonary fibrosis and remodeling in this model.
This study investigated the effects of administering a synthetic peptide of CD36 (a receptor for thrombospondin-1) along with bleomycin to induce lung injury in rats. The results showed that administering the CD36 peptide inhibited the activation of latent TGF-β1 by alveolar macrophages, reduced inflammation and connective tissue synthesis in the lung compared to bleomycin alone. This suggests that the thrombospondin-1 and CD36 interaction plays a key role in the in vivo activation of latent TGF-β1 during lung injury, and that activated TGF-β1 from alveolar macrophages drives pulmonary inflammation and fibrosis.
Summer research Homogenity in Th9 cultures 2Daniel Gomes
This document summarizes research from the Kaplan Lab on T helper 9 (Th9) cell differentiation and function. The lab found that culturing naive CD4+ T cells under Th9-polarizing conditions (IL-4 and TGFβ) resulted in a homogenous population of IL-9-secreting cells. However, using an IL-9 reporter mouse, they found that the Th9 genetic program was largely intact in IL-9-negative cells, suggesting it is not restricted only to IL-9-secreting cells. The lab aims to better understand Th9 cell differentiation and the roles they play in disease.
The symphony of the ninth, Th9 cells, by Dr.Pavulraj.S, veterinary pathologistPavulraj Selvaraj
Th9 cells are a subset of CD4+ T helper cells that secrete interleukin-9 (IL-9) as their signature cytokine. They develop in response to transforming growth factor beta (TGF-β) and interleukin-4 (IL-4) signals, which activate transcription factors that regulate IL-9 production. These include PU.1 induced by TGF-β and STAT6, IRF4, and GATA3 induced by IL-4. Th9 cells play roles in autoimmune diseases, allergy, and parasite expulsion by promoting inflammation through IL-9 stimulation of mast cell growth. However, their pathogenic mechanisms are still being defined, and blocking IL-9 is being explored as a potential
This document summarizes research on interleukin-9 (IL-9), a multifunctional cytokine that plays important roles in conditions like airway inflammation and asthma. The study found that transforming growth factor-beta (TGF-β) can "reprogram" the differentiation of T helper 2 cells and promote a IL-9-producing T cell subset. The researchers investigated IL-9 signaling pathways and used mouse models to examine the effects of IL-9 on intestinal nematode infection and autoimmune encephalomyelitis. They analyzed gene expression and cytokine production from T cells cultured under various conditions to identify factors that induce IL-9 production.
Identification of Potent Phosphodiesterase Inhibitors that Demonstrate Cyclic...Trang Luc
This document describes the identification of potent phosphodiesterase (PDE) inhibitors that demonstrate cyclic nucleotide-dependent functions in apicomplexan parasites. The most potent inhibitor identified was 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO), which potently inhibited recombinant P. falciparum Pf PDEα and induced protein kinase G (PKG)-dependent egress in T. gondii and P. falciparum by promoting microneme secretion. BIPPO also promoted cAMP-dependent phosphorylation of a P. falciparum ligand critical for host cell invasion,
This document reports on a study examining the effects of somatostatin (SST) on human B lymphoblasts. The key findings are:
1) SST stimulates phospholipase C activity and increases cytosolic calcium levels in B lymphoblasts, likely through coupling of SSTR2A to the G protein Gα16.
2) SST activates extracellular signal-regulated kinases and induces increased DNA synthesis, proliferation, and immunoglobulin formation in B lymphoblasts.
3) These stimulatory effects of SST on early signal transduction pathways are accompanied by increased cell proliferation and immunoglobulin production in human B lymphoblasts, indicating SST exerts growth factor-like
1) PSF, a pre-mRNA splicing factor, was identified as a constituent of PER complexes purified from mouse tissues.
2) PSF recruits the SIN3A-HDAC complex to the Per1 promoter in a circadian manner through interactions with PER complexes.
3) Recruitment of the SIN3A-HDAC complex by the PER complex leads to histone deacetylation at the Per1 promoter, providing a molecular mechanism for circadian clock negative feedback through rhythmic repression of Per1 transcription.
Early growth response gene 1 mediated apoptosis is essential for transforming...Tiensae Teshome
This study examined the role of Early Growth Response gene 1 (Egr-1) in Transforming Growth Factor beta 1 (TGF-β1)-induced apoptosis and pulmonary fibrosis using a novel triple transgenic mouse model. The results demonstrated that TGF-β1 caused epithelial apoptosis, followed by inflammation and pulmonary fibrosis. Null mutation of Egr-1 or caspase inhibition blocked TGF-β1-induced apoptosis and significantly reduced fibrosis. This establishes that Egr-1-mediated apoptosis is required for TGF-β1 to induce pulmonary fibrosis and remodeling in this model.
This study investigated the effects of administering a synthetic peptide of CD36 (a receptor for thrombospondin-1) along with bleomycin to induce lung injury in rats. The results showed that administering the CD36 peptide inhibited the activation of latent TGF-β1 by alveolar macrophages, reduced inflammation and connective tissue synthesis in the lung compared to bleomycin alone. This suggests that the thrombospondin-1 and CD36 interaction plays a key role in the in vivo activation of latent TGF-β1 during lung injury, and that activated TGF-β1 from alveolar macrophages drives pulmonary inflammation and fibrosis.
Summer research Homogenity in Th9 cultures 2Daniel Gomes
This document summarizes research from the Kaplan Lab on T helper 9 (Th9) cell differentiation and function. The lab found that culturing naive CD4+ T cells under Th9-polarizing conditions (IL-4 and TGFβ) resulted in a homogenous population of IL-9-secreting cells. However, using an IL-9 reporter mouse, they found that the Th9 genetic program was largely intact in IL-9-negative cells, suggesting it is not restricted only to IL-9-secreting cells. The lab aims to better understand Th9 cell differentiation and the roles they play in disease.
The symphony of the ninth, Th9 cells, by Dr.Pavulraj.S, veterinary pathologistPavulraj Selvaraj
Th9 cells are a subset of CD4+ T helper cells that secrete interleukin-9 (IL-9) as their signature cytokine. They develop in response to transforming growth factor beta (TGF-β) and interleukin-4 (IL-4) signals, which activate transcription factors that regulate IL-9 production. These include PU.1 induced by TGF-β and STAT6, IRF4, and GATA3 induced by IL-4. Th9 cells play roles in autoimmune diseases, allergy, and parasite expulsion by promoting inflammation through IL-9 stimulation of mast cell growth. However, their pathogenic mechanisms are still being defined, and blocking IL-9 is being explored as a potential
This document summarizes research on interleukin-9 (IL-9), a multifunctional cytokine that plays important roles in conditions like airway inflammation and asthma. The study found that transforming growth factor-beta (TGF-β) can "reprogram" the differentiation of T helper 2 cells and promote a IL-9-producing T cell subset. The researchers investigated IL-9 signaling pathways and used mouse models to examine the effects of IL-9 on intestinal nematode infection and autoimmune encephalomyelitis. They analyzed gene expression and cytokine production from T cells cultured under various conditions to identify factors that induce IL-9 production.
Identification of Potent Phosphodiesterase Inhibitors that Demonstrate Cyclic...Trang Luc
This document describes the identification of potent phosphodiesterase (PDE) inhibitors that demonstrate cyclic nucleotide-dependent functions in apicomplexan parasites. The most potent inhibitor identified was 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO), which potently inhibited recombinant P. falciparum Pf PDEα and induced protein kinase G (PKG)-dependent egress in T. gondii and P. falciparum by promoting microneme secretion. BIPPO also promoted cAMP-dependent phosphorylation of a P. falciparum ligand critical for host cell invasion,
This document summarizes a study examining the role of the long non-coding RNA Saf and splicing factor 45 in regulating alternative splicing of the Fas pre-mRNA and production of soluble Fas protein. The main findings are:
1) Saf is predominantly localized to the nucleus of cells. Overexpression of Saf in cell lines resulted in minimal effects on global gene expression.
2) Saf interacts with Fas pre-mRNA and splicing factor 45. This interaction promotes the skipping of exon 6 in the Fas pre-mRNA, leading to increased production of soluble Fas protein.
3) Soluble Fas protein protects cells from Fas ligand-induced apoptosis by binding to the ligand and blocking
Proinflammatory Consequences of FasL Expression in the HeartElizabeth Berry
This document summarizes research on the effects of transgenic expression of Fas ligand (FasL) specifically in heart muscle cells. The researchers generated several lines of transgenic mice with different copy numbers of a FasL transgene driven by a heart-specific promoter. They found that FasL expression in the heart caused mild leukocyte infiltration and interstitial fibrosis but, surprisingly, no cardiomyocyte apoptosis or necrosis. Instead, the hearts developed cardiac hypertrophy accompanied by upregulation of proinflammatory cytokines. The degree of proinflammatory changes correlated with transgene copy number, indicating a dose-dependent response to FasL expression level. This suggests that tissue-specific factors can modulate the proinflammatory effects of FasL.
This study investigated how oxidative stress activates the PI3K pathway in neurons affected by neurodegenerative diseases. The researchers found that ingestion of the oxidative stress inducer Paraquat in Drosophila larvae caused axonal transport defects and neuronal cell death. Expressing active PI3K suppressed Paraquat-mediated cell death but not axonal blocks, indicating PI3K acts downstream of transport defects. Expression of active PI3K also suppressed cell death from polyQ protein expression but did not affect associated transport defects. Dominant negative PI3K disrupted normal transport of huntingtin protein, linking PI3K directly to transport. Together, the findings suggest axonal transport defects activate the PI3K pathway to decrease oxidative stress-induced
THRAP3 interacts with HELZ2 and plays a novel role in adipocyte differentiation. Researchers identified THRAP3, a component of the Mediator complex, as a protein that stably associates with HELZ2. THRAP3 interacts directly with HELZ2 and enhances PPARγ-mediated transcriptional activation in combination with HELZ2. Knockdown of THRAP3 in 3T3-L1 preadipocytes attenuated adipocyte differentiation and lipid droplet accumulation, suggesting THRAP3 plays an indispensable role in terminal adipocyte differentiation by enhancing PPARγ-mediated gene activation with HELZ2.
A new effector pathway links ATM kinase with the DNA damage responseCostas Demonacos
The related kinases ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) phosphorylate a limited number of downstream protein targets in response to DNA damage. Here we report a new pathway in which ATM kinase signals the DNA damage response by targeting the transcriptional cofactor Strap. ATM phosphorylates Strap at a serine residue, stabilizing nuclear Strap and facilitating formation of a stress-responsive co-activator complex. Strap activity enhances p53 acetylation, and augments the response to DNA damage. Strap remains localized in the cytoplasm in cells derived from ataxia telangiectasia individuals with defective ATM, as well as in cells expressing a Strap mutant that cannot be phosphorylated by ATM. Targeting Strap to the nucleus reinstates protein stabilization and activates the DNA damage response. These results indicate that the nuclear accumulation of Strap is a critical regulator in the damage response, and argue that this function can be assigned to ATM through the DNA damage-dependent phosphorylation of Strap.
This document summarizes research finding that elevated activity of the Akt protein protects prostate cancer LNCaP cells from apoptosis induced by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). The researchers found that LNCaP cells have high constitutive Akt activity due to lack of the PTEN lipid phosphatase. Inhibiting PI3-kinase, which activates Akt, sensitized LNCaP cells to TRAIL-induced apoptosis. TRAIL alone activated caspases 8 and XIAP in LNCaP cells but failed to induce full apoptosis. Combining TRAIL with Akt inhibitors allowed cleavage of BID and subsequent mitochondrial apoptosis steps. Akt inhibition of BID cleavage appears to mediate the protective effect
Normal human prostate epithelial cells (PrEC) are sensitive to TRAIL-induced apoptosis, contrary to previous beliefs. TRAIL treatment of PrEC leads to DNA fragmentation and cell death. While PrEC are sensitive, other primary cell types like fibroblasts and endothelial cells are resistant. PrEC contain fewer decoy receptors that inhibit TRAIL's apoptotic signal compared to resistant cells. Inhibition of protein synthesis enhances TRAIL's toxicity in PrEC, suggesting short-lived proteins inhibit aspects of TRAIL-induced apoptosis in these cells. This has implications for using TRAIL as a potential prostate cancer treatment.
Cells respond to nutrient deprivation a variety of ways. In addition to global down regulation of cap-dependent protein
synthesis mediated by the GCN2 and mTO RC1 signaling pathways, a catabolic process autophagy is upregulated to
provide internal building blocks and energy needed to sustain viability. It has recently been shown that during nutrient
deprivation tRNAs accumulate in the nucleus, but the functional role of this accumulation remains unknown. This study
investigates whether subcellular localization of tRNAs plays a role in signaling nutritional stress and autophagy. We report
that human fibroblasts that accumulate tRNA in the nucleus due to downregulation of their transportin, Xpo-t, show
reduced mTO RC1 activity and upregulated autophagy. This suggests that sub-cellular localization of tRNAs may regulate
an unicellular response to starvation independently of the cellular nutritional status.
This study investigated the role of the C-terminal region of the human telomerase catalytic subunit (hTERT) in telomerase activity and function. The authors generated 41 mutant versions of hTERT where every six amino acids in the C-terminal region were substituted. These mutant hTERT proteins were tested for their ability to immortalize human cells and for telomerase catalytic activity in vitro. Several mutations were identified that inactivated both biological and catalytic functions, while others had little effect or generated catalytically active but biologically inert proteins. This suggests distinct regions in the C-terminus are important for the biochemical and biological roles of telomerase.
Role of stat3 protein & thelper 17 cell in psoriasis development by yousryM.YOUSRY Abdel-Mawla
This document discusses the role of STAT3 and Th17 cells in the development of psoriasis. It describes how STAT3 signaling regulates the differentiation of naive T cells into Th17 cells through the production of cytokines like IL-6, IL-21, and IL-23. Th17 cells secrete pro-inflammatory cytokines like IL-17 and IL-22 that contribute to psoriasis by stimulating keratinocyte growth and activation. The overexpression of Th17 cells and cytokines in the skin is thought to play a key role in the pathogenesis of psoriasis.
1) The study investigated the role of the LAT-PLCγ1 interaction in γδ T cell development and homeostasis by crossing LATY136F mice, which lack this interaction, with TCRβ−/− mice.
2) Results showed the LATY136F mutation partially blocked γδ T cell development in the thymus. However, epithelial γδ T cells were still present in the skin and intestine.
3) Interestingly, a population of CD4+ γδ T cells in the spleen and lymph nodes of LATY136F/TCRβ−/− mice underwent rapid proliferation, producing elevated IL-4 and causing autoimmunity. This suggested LAT signaling functions differently in distinct
This study characterized the aggregation and toxicity of the Machado Joseph Disease (MJD)-associated ataxin-3 protein expressed in different tissues of C. elegans. The researchers found that expressing a mutant fragment of ataxin-3 tagged with YFP led to polyglutamine length-dependent aggregation in body wall muscle cells and neurons over time. Toxicity, as measured by motility assays, also increased with polyglutamine length in muscles but had little effect in neurons. Surprisingly, ataxin-3 expression did not impair the organism's heat shock response despite clear neuronal aggregation. The study provides insights into tissue-specific effects of ataxin-3 aggregation and toxicity.
This study investigated the functional redundancy of the four aspartic proteinases (plasmepsins) found in the digestive vacuole of the malaria parasite Plasmodium falciparum. The researchers disrupted each of the plasmepsin genes (PfPM1, PfPM2, PfPM4, and PfHAP) through genetic engineering. They found that while disruption of PfPM1 and PfPM4 resulted in reduced parasite growth, none of the plasmepsins were essential for survival. This suggests the plasmepsins can compensate for each other, likely due to their structural and catalytic similarities. The study implies that effective antimalarial drugs will need to inhibit multiple plasmepsin family members.
The document summarizes a study that found:
1) Interleukin-12 (IL-12) in dendritic cells traffics through late endocytic vesicles marked by the SNARE protein VAMP7.
2) Dendritic cells from VAMP7 knockout mice show partially impaired secretion of bioactive IL-12/p70 dimer.
3) At the immune synapse between dendritic cells and T cells, IL-12 containing vesicles rapidly redistribute and their release is entirely dependent on VAMP7, leading to reduced T cell acquisition of effector functions when stimulated with VAMP7 knockout dendritic cells.
This document summarizes a study investigating the role of a Plasmodium falciparum S33 proline aminopeptidase (PfPAP) in changes to host red blood cell deformability. The key findings are:
1) PfPAP contains a predicted protein export element suggesting it is exported into infected red blood cells. In silico modeling and recombinant protein studies confirmed PfPAP is a proline aminopeptidase.
2) Genetic deletion of PfPAP in P. falciparum led to an increase in the deformability of infected red blood cells and reduced adherence of infected cells to the endothelial cell receptor CD36 under flow conditions.
3) These results suggest PfP
The document reviews the current literature on prion protein (PrP) function and the pathogenesis of prion diseases, focusing on two areas: the unfolded protein response (UPR) and PrP interactions with metal ions. It describes PrP genetics and structure, the UPR pathways involving IRE1, PERK and ATF6, links between the UPR and prions, and the roles of copper and zinc binding in PrP function and prion diseases. The aim is to establish connections between the UPR and PrP metal ion interactions to further our understanding of PrP pathogenesis and native function
This document summarizes a study that created an inducible mouse model expressing pathological human tau (PH-Tau) to investigate the effects of tau phosphorylation levels on cognition and neurodegeneration. The study found that even low levels of PH-Tau expression (4% of total tau) led to cognitive deficits through synaptic dysfunction, while higher levels (14% of total tau) induced neuronal death and further cognitive impairment. This suggests that abnormal tau can cause cognitive impairment through two different mechanisms depending on its expression level - synaptic dysfunction at low levels and neuronal loss at high levels.
STAT 3 and Other Target Proteins: New Concepts in Psoriasis Pathogenesis & Therapy
This document discusses STAT3 signaling and its role in psoriasis pathogenesis. It summarizes that:
1) STAT3 is activated by cytokines and growth factors and forms dimers that enter the nucleus and activate gene transcription.
2) STAT3 signaling is involved in processes like proliferation and differentiation of keratinocytes. Its dysregulation contributes to psoriasis pathogenesis.
3) Psoriasis is a chronic inflammatory skin disease involving excessive keratinocyte proliferation, abnormal differentiation, and immune system involvement. Understanding STAT3 signaling may provide novel therapeutic targets for psoriasis treatment.
The researchers engineered a genetic toolset called pB-Tet-GOI for flexible control of transgene expression in stem and progenitor-derived cell lineages. The system incorporates the latest tetracycline transactivator and reverse transactivator variants to provide regulated transgene expression upon addition or removal of doxycycline. It allows for doxycycline-induced, doxycycline-suppressed, doxycycline-resistant (constitutive), and doxycycline-induced/constitutive regulation of transgenes. Initial tests showed the system provides inducible transgene expression with minimal leakiness and can be used to bidirectionally express reporters and genes of interest to direct cell differentiation.
This study aimed to identify domains in the N-terminal region of the human telomerase catalytic subunit (hTERT) that are required for telomerase activity and function in vivo. The researchers introduced mutations substituting six amino acid stretches throughout the N-terminal region of hTERT and assessed the ability of the mutant enzymes to maintain telomeres and support cellular proliferation. They identified four domains essential for in vitro and in vivo enzyme activity, two of which were required for binding to the hTERT RNA subunit. Additionally, they discovered a novel domain, termed DAT, where mutations left the enzyme catalytically active but unable to function in vivo, suggesting it is involved in other aspects of telomere elongation beyond catalytic
This document describes a study that used bioinformatics tools to model the 3D structure of the Hc-Stp1 protein from the parasite Haemonchus contortus. The researchers first identified a homologous protein, Tv-Stp1 from Trichostrongylus vitrinus, to use as a template. They then generated a multiple sequence alignment of Hc-Stp1 and the template protein 3e7a. Secondary structure was predicted using PSIPRED and a homology model of the Hc-Stp1 structure was created using MODELLER. The model was analyzed using Ramachandran plots which showed 3 outliers requiring further investigation before the model could be used as a drug target
This document discusses cloning the mouse Hist2H4 gene, which encodes the histone H4 protein. Histones are proteins that package DNA into nucleosomes. The goals are to clone two fragments of the Hist2H4 gene - a short 984bp fragment containing the stem-loop sequence and a long 2686bp fragment containing both the stem-loop and polyadenylation signals. Primers were designed and PCR was used to amplify the genomic regions from mouse DNA. The clones will be used to further study histone mRNA regulation in proliferating and non-proliferating cells.
This document summarizes a study examining the role of the long non-coding RNA Saf and splicing factor 45 in regulating alternative splicing of the Fas pre-mRNA and production of soluble Fas protein. The main findings are:
1) Saf is predominantly localized to the nucleus of cells. Overexpression of Saf in cell lines resulted in minimal effects on global gene expression.
2) Saf interacts with Fas pre-mRNA and splicing factor 45. This interaction promotes the skipping of exon 6 in the Fas pre-mRNA, leading to increased production of soluble Fas protein.
3) Soluble Fas protein protects cells from Fas ligand-induced apoptosis by binding to the ligand and blocking
Proinflammatory Consequences of FasL Expression in the HeartElizabeth Berry
This document summarizes research on the effects of transgenic expression of Fas ligand (FasL) specifically in heart muscle cells. The researchers generated several lines of transgenic mice with different copy numbers of a FasL transgene driven by a heart-specific promoter. They found that FasL expression in the heart caused mild leukocyte infiltration and interstitial fibrosis but, surprisingly, no cardiomyocyte apoptosis or necrosis. Instead, the hearts developed cardiac hypertrophy accompanied by upregulation of proinflammatory cytokines. The degree of proinflammatory changes correlated with transgene copy number, indicating a dose-dependent response to FasL expression level. This suggests that tissue-specific factors can modulate the proinflammatory effects of FasL.
This study investigated how oxidative stress activates the PI3K pathway in neurons affected by neurodegenerative diseases. The researchers found that ingestion of the oxidative stress inducer Paraquat in Drosophila larvae caused axonal transport defects and neuronal cell death. Expressing active PI3K suppressed Paraquat-mediated cell death but not axonal blocks, indicating PI3K acts downstream of transport defects. Expression of active PI3K also suppressed cell death from polyQ protein expression but did not affect associated transport defects. Dominant negative PI3K disrupted normal transport of huntingtin protein, linking PI3K directly to transport. Together, the findings suggest axonal transport defects activate the PI3K pathway to decrease oxidative stress-induced
THRAP3 interacts with HELZ2 and plays a novel role in adipocyte differentiation. Researchers identified THRAP3, a component of the Mediator complex, as a protein that stably associates with HELZ2. THRAP3 interacts directly with HELZ2 and enhances PPARγ-mediated transcriptional activation in combination with HELZ2. Knockdown of THRAP3 in 3T3-L1 preadipocytes attenuated adipocyte differentiation and lipid droplet accumulation, suggesting THRAP3 plays an indispensable role in terminal adipocyte differentiation by enhancing PPARγ-mediated gene activation with HELZ2.
A new effector pathway links ATM kinase with the DNA damage responseCostas Demonacos
The related kinases ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) phosphorylate a limited number of downstream protein targets in response to DNA damage. Here we report a new pathway in which ATM kinase signals the DNA damage response by targeting the transcriptional cofactor Strap. ATM phosphorylates Strap at a serine residue, stabilizing nuclear Strap and facilitating formation of a stress-responsive co-activator complex. Strap activity enhances p53 acetylation, and augments the response to DNA damage. Strap remains localized in the cytoplasm in cells derived from ataxia telangiectasia individuals with defective ATM, as well as in cells expressing a Strap mutant that cannot be phosphorylated by ATM. Targeting Strap to the nucleus reinstates protein stabilization and activates the DNA damage response. These results indicate that the nuclear accumulation of Strap is a critical regulator in the damage response, and argue that this function can be assigned to ATM through the DNA damage-dependent phosphorylation of Strap.
This document summarizes research finding that elevated activity of the Akt protein protects prostate cancer LNCaP cells from apoptosis induced by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). The researchers found that LNCaP cells have high constitutive Akt activity due to lack of the PTEN lipid phosphatase. Inhibiting PI3-kinase, which activates Akt, sensitized LNCaP cells to TRAIL-induced apoptosis. TRAIL alone activated caspases 8 and XIAP in LNCaP cells but failed to induce full apoptosis. Combining TRAIL with Akt inhibitors allowed cleavage of BID and subsequent mitochondrial apoptosis steps. Akt inhibition of BID cleavage appears to mediate the protective effect
Normal human prostate epithelial cells (PrEC) are sensitive to TRAIL-induced apoptosis, contrary to previous beliefs. TRAIL treatment of PrEC leads to DNA fragmentation and cell death. While PrEC are sensitive, other primary cell types like fibroblasts and endothelial cells are resistant. PrEC contain fewer decoy receptors that inhibit TRAIL's apoptotic signal compared to resistant cells. Inhibition of protein synthesis enhances TRAIL's toxicity in PrEC, suggesting short-lived proteins inhibit aspects of TRAIL-induced apoptosis in these cells. This has implications for using TRAIL as a potential prostate cancer treatment.
Cells respond to nutrient deprivation a variety of ways. In addition to global down regulation of cap-dependent protein
synthesis mediated by the GCN2 and mTO RC1 signaling pathways, a catabolic process autophagy is upregulated to
provide internal building blocks and energy needed to sustain viability. It has recently been shown that during nutrient
deprivation tRNAs accumulate in the nucleus, but the functional role of this accumulation remains unknown. This study
investigates whether subcellular localization of tRNAs plays a role in signaling nutritional stress and autophagy. We report
that human fibroblasts that accumulate tRNA in the nucleus due to downregulation of their transportin, Xpo-t, show
reduced mTO RC1 activity and upregulated autophagy. This suggests that sub-cellular localization of tRNAs may regulate
an unicellular response to starvation independently of the cellular nutritional status.
This study investigated the role of the C-terminal region of the human telomerase catalytic subunit (hTERT) in telomerase activity and function. The authors generated 41 mutant versions of hTERT where every six amino acids in the C-terminal region were substituted. These mutant hTERT proteins were tested for their ability to immortalize human cells and for telomerase catalytic activity in vitro. Several mutations were identified that inactivated both biological and catalytic functions, while others had little effect or generated catalytically active but biologically inert proteins. This suggests distinct regions in the C-terminus are important for the biochemical and biological roles of telomerase.
Role of stat3 protein & thelper 17 cell in psoriasis development by yousryM.YOUSRY Abdel-Mawla
This document discusses the role of STAT3 and Th17 cells in the development of psoriasis. It describes how STAT3 signaling regulates the differentiation of naive T cells into Th17 cells through the production of cytokines like IL-6, IL-21, and IL-23. Th17 cells secrete pro-inflammatory cytokines like IL-17 and IL-22 that contribute to psoriasis by stimulating keratinocyte growth and activation. The overexpression of Th17 cells and cytokines in the skin is thought to play a key role in the pathogenesis of psoriasis.
1) The study investigated the role of the LAT-PLCγ1 interaction in γδ T cell development and homeostasis by crossing LATY136F mice, which lack this interaction, with TCRβ−/− mice.
2) Results showed the LATY136F mutation partially blocked γδ T cell development in the thymus. However, epithelial γδ T cells were still present in the skin and intestine.
3) Interestingly, a population of CD4+ γδ T cells in the spleen and lymph nodes of LATY136F/TCRβ−/− mice underwent rapid proliferation, producing elevated IL-4 and causing autoimmunity. This suggested LAT signaling functions differently in distinct
This study characterized the aggregation and toxicity of the Machado Joseph Disease (MJD)-associated ataxin-3 protein expressed in different tissues of C. elegans. The researchers found that expressing a mutant fragment of ataxin-3 tagged with YFP led to polyglutamine length-dependent aggregation in body wall muscle cells and neurons over time. Toxicity, as measured by motility assays, also increased with polyglutamine length in muscles but had little effect in neurons. Surprisingly, ataxin-3 expression did not impair the organism's heat shock response despite clear neuronal aggregation. The study provides insights into tissue-specific effects of ataxin-3 aggregation and toxicity.
This study investigated the functional redundancy of the four aspartic proteinases (plasmepsins) found in the digestive vacuole of the malaria parasite Plasmodium falciparum. The researchers disrupted each of the plasmepsin genes (PfPM1, PfPM2, PfPM4, and PfHAP) through genetic engineering. They found that while disruption of PfPM1 and PfPM4 resulted in reduced parasite growth, none of the plasmepsins were essential for survival. This suggests the plasmepsins can compensate for each other, likely due to their structural and catalytic similarities. The study implies that effective antimalarial drugs will need to inhibit multiple plasmepsin family members.
The document summarizes a study that found:
1) Interleukin-12 (IL-12) in dendritic cells traffics through late endocytic vesicles marked by the SNARE protein VAMP7.
2) Dendritic cells from VAMP7 knockout mice show partially impaired secretion of bioactive IL-12/p70 dimer.
3) At the immune synapse between dendritic cells and T cells, IL-12 containing vesicles rapidly redistribute and their release is entirely dependent on VAMP7, leading to reduced T cell acquisition of effector functions when stimulated with VAMP7 knockout dendritic cells.
This document summarizes a study investigating the role of a Plasmodium falciparum S33 proline aminopeptidase (PfPAP) in changes to host red blood cell deformability. The key findings are:
1) PfPAP contains a predicted protein export element suggesting it is exported into infected red blood cells. In silico modeling and recombinant protein studies confirmed PfPAP is a proline aminopeptidase.
2) Genetic deletion of PfPAP in P. falciparum led to an increase in the deformability of infected red blood cells and reduced adherence of infected cells to the endothelial cell receptor CD36 under flow conditions.
3) These results suggest PfP
The document reviews the current literature on prion protein (PrP) function and the pathogenesis of prion diseases, focusing on two areas: the unfolded protein response (UPR) and PrP interactions with metal ions. It describes PrP genetics and structure, the UPR pathways involving IRE1, PERK and ATF6, links between the UPR and prions, and the roles of copper and zinc binding in PrP function and prion diseases. The aim is to establish connections between the UPR and PrP metal ion interactions to further our understanding of PrP pathogenesis and native function
This document summarizes a study that created an inducible mouse model expressing pathological human tau (PH-Tau) to investigate the effects of tau phosphorylation levels on cognition and neurodegeneration. The study found that even low levels of PH-Tau expression (4% of total tau) led to cognitive deficits through synaptic dysfunction, while higher levels (14% of total tau) induced neuronal death and further cognitive impairment. This suggests that abnormal tau can cause cognitive impairment through two different mechanisms depending on its expression level - synaptic dysfunction at low levels and neuronal loss at high levels.
STAT 3 and Other Target Proteins: New Concepts in Psoriasis Pathogenesis & Therapy
This document discusses STAT3 signaling and its role in psoriasis pathogenesis. It summarizes that:
1) STAT3 is activated by cytokines and growth factors and forms dimers that enter the nucleus and activate gene transcription.
2) STAT3 signaling is involved in processes like proliferation and differentiation of keratinocytes. Its dysregulation contributes to psoriasis pathogenesis.
3) Psoriasis is a chronic inflammatory skin disease involving excessive keratinocyte proliferation, abnormal differentiation, and immune system involvement. Understanding STAT3 signaling may provide novel therapeutic targets for psoriasis treatment.
The researchers engineered a genetic toolset called pB-Tet-GOI for flexible control of transgene expression in stem and progenitor-derived cell lineages. The system incorporates the latest tetracycline transactivator and reverse transactivator variants to provide regulated transgene expression upon addition or removal of doxycycline. It allows for doxycycline-induced, doxycycline-suppressed, doxycycline-resistant (constitutive), and doxycycline-induced/constitutive regulation of transgenes. Initial tests showed the system provides inducible transgene expression with minimal leakiness and can be used to bidirectionally express reporters and genes of interest to direct cell differentiation.
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J. biol. chem. 2016-shao-jbc.m116.724401
1. 1
FBXO3 Promotes Ubiquitylation and Transcriptional Activity of AIRE
Wei Shao1
, Kristina Zumer2
, Koh Fujinaga1
, B. Matija Peterlin1*
1
Departments of Medicine, Microbiology and Immunology
University of California at San Francisco, San Francisco, CA 94143-0703
2
Max-Planck-Institute for Biophysical Chemistry, Department of Molecular Biology, Am Fassberg 11,
37077 Göttingen, Germany
Running Title: FBXO3 activates AIRE
Keywords: AIRE, FBXO3, ubiquitylation, transcription, P-TEFb
*
To whom correspondence should be addressed: Rm U432, Box 0703, 533 Parnassus Ave, San Francisco,
CA 9413-0703. TEL: 415-502-1905; FAX: 415-502-1901; Email: matija.peterlin@ucsf.edu
ABSTRACT
The autoimmune regulator (AIRE) is a
transcription factor, which is expressed in
medullary thymic epithelial cells. It directs the
expression of otherwise tissue specific antigens
(TSAs), which leads to the elimination of
autoreactive T cells during development. AIRE
is modified post-translationally by
phosphorylation and ubiquitylation. In this
report, we connected these modifications. AIRE,
which is phosphorylated on two specific
residues near its N-terminus then binds to the
F-box protein 3 (FBXO3) E3 ubiquitin ligase.
In turn, this SCFFBXO3
(SKP1-CUL1-F box)
complex ubiquitylates AIRE, increases its
binding to the positive transcription elongation
factor b (P-TEFb) and potentiates its
transcriptional activity. Since P-TEFb is
required for the transition from initiation to
elongation of transcription, this interaction
ensures proper expression of AIRE-responsive
TSAs in the thymus.
INTRODUCTION
The autoimmune regulator (AIRE) is a
transcription factor (TF) that regulates central
tolerance in the thymus (1-4). In its absence,
affected individuals suffer from a congenital
autoimmunity, the autoimmune poly-
endocrinopathy-candidiasis-ectodermal dystrophy
(APECED) also known as the autoimmune
polyglandular syndrome type 1 (APS1) (5,6).
Expressed in medullary thymic epithelial cells
(mTECs), AIRE directs the expression of
otherwise tissue-specific antigens (TSAs). Their
presentation to developing T cells allows self-
reacting cells to be eliminated. In these cells,
AIRE is recruited to the stalled RNA polymerase
II (RNAPII) on TSA promoters by the unmodified
histone H3K4 and DNA-dependent protein kinase
(DNA-PK) (7-10). DNA-PK also phosphorylates
AIRE for increased transcriptional activity (11).
AIRE binds to the positive transcription elongation
factor b (P-TEFb), which modifies RNAPII for
elongation and co-transcriptional processing of
TSA genes (7,8,12). Ectopic expression of AIRE
also activates TSA genes in HEK 293T (293T)
(13) and mouse 1C6 mTEC cells (8).
Transcriptional activation is a complex process
between TFs and RNAPII. Recent evidence
suggests that on most promoters, be they
transcribed or not, RNAPII is already present;
however, it elongates only on active genes (14).
Other transcription units can be activated
following the recruitment of P-TEFb, which
phosphorylates negative elongation factors and the
C-terminal domain (CTD) of RNAPII (15). Many
TFs, which contain acidic or amphipathic
transcription activation domains (TADs) are also
activated by ubiquitylation, especially via
phosphorylated degrons that increase their potency
and limit their duration of action (16,17).
Previously, we demonstrated that the
ubiquitylation of VP16 increased its binding to P-
TEFb. Indeed, the TAD and monoubiquitin on
VP16 bound to cyclin boxes and the extreme C-
terminus of cyclin T1 (CycT1), respectively (18).
These interactions were not constrained spatially
and besides increased binding they also relieved
the autoinhibition of P-TEFb by the C-terminus of
CycT1 (18).
Of interest, AIRE contains a similar TAD near its
C-terminus (9). AIRE is also ubiquitylated (19-21).
Indeed, an early report suggested that the plant
homeodomain 1 (PHD1) in AIRE is a RING
domain and that AIRE is itself an E3 ubiquitin
http://www.jbc.org/cgi/doi/10.1074/jbc.M116.724401The latest version is at
JBC Papers in Press. Published on June 30, 2016 as Manuscript M116.724401
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
byguestonJuly14,2016http://www.jbc.org/Downloadedfrom
2. 2
ligase (21). Subsequent reports disputed this
finding and the structure of PHD1 proved to be
distinct from a RING finger (22). Additionally, the
PHD1 lacked E3 ligase activity (19). Nevertheless,
the N-terminus of AIRE is ubiquitylated (20). In
addition, AIRE is phosphorylated on two residues,
threonine at position 68 (T68) and serine at
position 156 (S156), which when mutated to
alanine abrogated AIRE’s transcriptional activity
(11). In this report, we discovered the E3 ligase
responsible for the ubiquitylation of AIRE.
Revealed by our proteomic studies, FBXO3 was
found to interact with these phosphorylated T68
and S156 residues, promote the ubiquitylation of
AIRE and lead to its rapid degradation. In addition,
this post-translational modification increased
interactions between AIRE and P-TEFb and
potentiated their transcriptional activity on TSA
genes.
EXPERIMENTAL PROCEDURES
Cell culture and transfection
HEK 293T (293T) cells and 1C6 mTECs were
cultured in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% fetal calf serum (FCS) at
37 °C and 5% CO2. Transfection of plasmid DNA
was performed using Lipofectamine 2000 (Life
Technologies) according to the manufacturer’s
instructions. Small interfering RNA (siRNA) was
transfected for 72 h using Lipofectamine
RNAiMAX transfection reagent (Life
Technologies) according to the manufacturer’s
instructions. Validated siRNA targeting human
FBXO3 was purchased from Qiagen
(GeneSolution GS26273) (23), siRNA targeting
mouse FBXO3 was purchased from Invitrogen
(FBXO3MSS226550) and control siRNA was
purchased from Santa Cruz Biotechnology (sc-
37007). Mouse thymus samples were collected
from C57BL/6J mice from the Jackson Laboratory.
Mice were maintained in specific pathogen-free
facilities and were handled in accordance with
protocols by the Institutional Animal Care and Use
Committee of the University of California, San
Francisco.
Plasmids and antibodies
FLAG-AIRE and INSLuc plasmids were
described previously (9). Plasmids pSI-AIRE-WT,
pSI-AIRE-T68A and pSI-S156A were kind gifts
of Pärt Peterson (University of Tartu, Tartu,
Estonia). mHis:AIRET68A, mHis:AIRES156A
and mHis:AIRET68AS156A were cloned from
pSI-AIRE-T68A and pSI-AIRE-S156A into
pcDNA3.1B (-) after the mHis epitope tag. The
WT mHis:AIRE and mutant mHis:AIRET68E
S156E plasmids were generated by PCR and
cloned into pcDNA3.1B (-) as above. v:FBXO3
was a kind gift from Rama Mallampalli
(University of Pittsburgh, Pittsburgh, USA).
f:FBXO3, h:FBXO3, m:CUL1 and His:Ub were
all created by cloning them into pcDNA3.1 vector
with CMV promoter and the appropriate epitope
tags. Deletion mutants of AIRE were created by
inserting stop codons into f:AIRE using Stratagene
XL Site-Directed Mutagenesis Kit. h:Ub was
created by cloning ubiquitin into pMT2 vector
with CMV promoter and 3’ HA tag.
Antibodies used for western blotting, co-
immunoprecipitation or immunofluorescence
were: AIRE (H300, sc-33188 and D17, sc-17986,
Santa Cruz), AIRE (phospho S156) antibody
(ab78211, Abcam), Cullin 1 (D-5, sc-17775, Santa
Cruz), c-Myc (9E10, HRP, ab62928, Abcam),
CycT1 (H-245, sc-10750, Santa Cruz), FBXO3
(H300, sc-134722, Santa Cruz), FLAG M2-
Peroxidase (A8592, HRP, Sigma), FLAG M2-
Agarose from mouse (A2220, Sigma), GAPDH
(6C5, sc-32233, Santa Cruz), His (H3, sc-8036,
Santa Cruz), HA (Y-11, sc-805, Santa Cruz),
RBX1 (34-2500, Invitrogen), UBC4 (Y20, sc-
100617, Santa Cruz), ubiquitin (P4D1, sc-8017,
Santa Cruz), V5-HRP antibodies (R961-25, Life
Technologies) as well as rabbit and mouse control
IgG (Santa Cruz).
Mass spectrometry
Immunopurification and mass spectrometry (MS)
were carried out as previously described (24).
293T cells transfected with f:AIRE were lysed and
the supernatants were incubated with FLAG M2
affinity gel (Sigma). After extensive washes,
bound proteins were eluted and digested using
trypsin (Promega). The peptide mixture was
analyzed by liquid chromatography-tandem mass
spectrometry (LC-MS/MS) on a Thermo Scientific
Velos Pro ion trap mass spectrometry system
equipped with a Proxeon Easy nLC high-pressure
liquid chromatography and autosampler system.
The MS data was searched using a database
containing Swiss-Prot human protein sequence for
protein identification.
Immunofluorescence assays (IFAs)
For IFAs, 293T cells were cultured on the
Collagen I 22mm Round Coverslip (354089;
Corning) in 6 well plates. Cells were transfected
with mHis:AIRE and h:FBXO3. Two days after
transfection, cells were washed by PBS and fixed
byguestonJuly14,2016http://www.jbc.org/Downloadedfrom
3. 3
in 4% formaldehyde for 30 min at room
temperature. They were then incubated with 0.2%
Triton X-100 in PBS for 5 min. Reactions were
then blocked for 1 h in PBS containing 5% BSA.
Antibodies were diluted in 1% BSA in PBS. The
primary anti-His and anti-HA antibodies were
added and incubated at 4 °C overnight. Goat anti-
mouse secondary antibodies, which were
conjugated with green Alexa Fluor 488 dye
(56881A; Invitrogen) and donkey anti-rabbit
secondary antibodies, which were conjugated with
red Alexa fluor 594 (404239; Invitrogen) were
then performed for 2 h at room temperature. DAPI
(P36962; Life Technologies) was used for nuclear
staining. Images were captured on an Olympus
microscope and analyzed using Image-Pro Plus
(version 6.2) software.
Immunoprecipitations
Immunoprecipitations were performed as
described before (24). 293T cells were lysed on
ice using lysis buffer (50 mM Tris-HCl, pH 7.5,
150 mM NaCl, 0.5% NP-40, 1.0% Triton X-100)
or RIPA buffer (50 mM Tris-HCl, pH 8.0, 150
mM NaCl, 5 mM EDTA, 0.1% SDS, 1.0% NP-40,
0.5% sodium deoxycholate) supplement with the
protease inhibitor cocktail (78430; Thermo), then
sonicated with Sonic Dismembrator 100 (Fisher
Scientific) by three cycles of 5 s on and 30 s off at
an intensity of 3 and incubated on ice for 30 min.
Extracts were then precleared with protein A- or
G- Sepharose beads (Invitrogen). After
centrifugation, whole cell lysates were incubated
with appropriate primary antibodies or antibodies-
conjugated beads overnight at 4 °C. After
incubations with primary antibodies, lysates were
centrifuged and supernatants were incubated with
Protein A- or G-Sepharose beads for 2 h at 4 °C.
Beads were washed 3 times using lysis buffer, 5
min every time at 4 °C. Calf intestinal phosphatase
(CIP, M0290S, NEB) treatment was performed by
adding 10 units of CIP to whole cell lysates at
37 °C for 30 min. For native IP using mouse
thymus, 10 thymi were lysed using pestles (1415-
5390; USA Scientific) in 1.5 ml tubes. The lysate
was sonicated as above described, and then the
lysates were centrifuged and precleared by protein
A Sepharose beads (Invitrogen). Lysates were then
incubated with anti-FBXO3 antibodies overnight.
Beads were washed 3 times using the lysis buffer.
Immunoprecipitated complexes were boiled in
SDS sample buffer (Laemmli sample buffer with
β-mercaptoethanol) and analyzed by western
blotting.
Chromatin immunoprecipitations
Chromatin immunoprecipitations (ChIPs) were
performed as described previously (9,24). 293T
cells were transfected using Lipofectamine 2000
or Lipofectamine RNAiMAX transfection reagent
according to the manufacturer’s instructions. 293T
cells were harvested and cross-linked with 1%
formaldehyde at room temperature for 10 min and
stopped by adding glycine to a final concentration
of 125 mM. Cells were lysed in RIPA buffer and
sonicated at power 4 five times for 10 s each by
using a Sonic Dismembrator model 100 (Fisher
Scientific). Lysates were precleared by protein G
Sepharose beads (Invitrogen). Chromatin
immunoprecipitations were performed using anti-
FLAG beads or anti-His beads.
Immunoprecipitated DNA was detected by qPCR
using SensiFAST SYBR Lo-ROX Kit (Bioline) on
an Mx3005p machine (Stratagene). Precipitated
DNA was quantified relative to input and is
presented as fold over IgG control. The following
primers were used: for KRT14 promoter, forward
(5’-GGAAAGTGCCAGACCCGCCC) and
reverse (5’-GGAGGGAGGTGAGCGAGCGA);
S100A8 promoter, forward (5’-
CTGGGCTGCTGGCATCCACT ) and reverse
(5’-GGCCTGACCACCAATGCAGGG).
In vivo ubiquitylation assay
For in vivo ubiquitylation assays, cells were
treated with 1 µM MG132 (M7449; Sigma) for 4 h
before harvesting, and then cells were lysed in 1%
SDS buffer (50 mM Tris-HCl pH 7.5, 150 mM
NaCl, 1% SDS, 10 mM DTT) and boiled for 30
min. The lysates were centrifuged and diluted 1:10
with lysis buffer (50 mM Tri-HCl pH 7.5, 150 mM
NaCl, 1 mM EDTA, 1% Triton X-100). The
diluted lysates were immunoprecipitated with anti-
FLAG beads overnight at 4 °C. Myc, His epitope-
tagged AIRE protein was isolated as described
above. After SDS-PAGE, the membrane was
incubated with indicated antibodies.
Luciferase assay
293T cells were transfected with Lipofectamine
2000 to co-express AIRE proteins and INSLuc in
6 well plates. Cells were harvested after 48 h and
luciferase assays were performed using dual
luciferase kit (Promega) according to the
manufacturer’s instructions. Firefly luciferase
activity readings of each sample were normalized
to total protein in the whole extract to determine
relative luciferase activities. Total protein was
measured using NanoDrop Lite spectrophotometer
(Thermo).
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RNA isolation and RT-PCR
293T cells or 1C6 mTECs were washed by cold
PBS and harvested in Trizol reagent (Life
Technologies). RNA was isolated according to the
manufacturer’s instructions. 2 µg RNA was
reverse transcribed with SuperScript III Reverse
Transcriptase (Life Technologies) and random
primers. Levels of expression of AIRE-dependent
and -independent genes were determined by real-
time PCR and normalized to glyceraldehyde-3-
phosphate dehydrogenase (GAPDH). Real-time
PCR was performed using SensiFAST SYBR Lo-
ROX Kit (Bioline) on an Mx3005p machine
(Stratagene). Relative activations represent the
ratio between FBXO3 siRNA knockdown and
siRNA control, which were calculated using the
threshold cycles (ΔΔCT) method (10). The Primers
used in this study have been published (13,25-28).
Protein stability assay
293T cells were transfected with mHis:AIRE,
control siRNA or siRNA targeting FBXO3 for 48
h, and then cells were treated with translation
inhibitor cycloheximide (CHX, 100 µg/ml, Sigma)
(29). Cells were washed by cold PBS and
collected at time points 0 h, 1 h, 4 h, 8 h, then
lysed and subjected to western blotting, using
GAPDH as the control. Protein signals were
quantified by Image J (30).
Bioinformatics
AIRE protein sequences from Homo sapiens, Pan
troglodytes, Macaca mulatta, Rattus norvegicus,
Mus musculus, Canis lupus were obtained from
NCBI and aligned using MegAlign by Clustal W
method (Dnastar, Madison, WI).
RESULTS
AIRE interacts with FBXO3
To find additional AIRE-interacting proteins, we
performed proteomic studies with the FLAG
epitope-tagged AIRE protein, which was
expressed transiently in 293T cells. Besides many
proteins already identified by others (13), we
found one E3 ligase, FBXO3 (Table 1). FBXO3
contains 471 residues and migrates with an
apparent molecular mass of 55 kDa. It forms a
SCF E3 ubiquitin ligase complex with SKP1,
Cullin1 (CUL1) and ring-box 1 (RBX1). By
ubiquitylating F-box and leucine-rich repeat
protein 2 (FBXL2), FBXO3 plays a role in
cytokine-mediated inflammation (31). FBXL2 also
ubiquitylates, degrades and inhibits tumor necrosis
factor receptor-associated factors (TRAFs). By
removing it, FBXO3 increases TRAF signaling
(31,32). FBXO3 also ubiquitylates and degrades
homeodomain interacting protein kinase 2
(HIPK2) and P300 (33). By ubiquitylating and
degrading SMAD-specific E3 ubiquitin protein
ligase 1 (SMURF1), FBXO3 also increases BMP
signaling (34). In all these areas, FBXO3 appears
to play an active role in gene expression. However,
FBXO3 had not previously been implicated in
effects of AIRE.
To confirm this association of FBXO3, we co-
expressed transiently the Myc, His epitope-tagged
AIRE protein (mHis:AIRE) with the FLAG
epitope-tagged FBXO3 protein (f:FBXO3) in
293T cells. In addition, since FBXO3 is found in
association with CUL1 and RBX1, we co-
expressed the Myc epitope-tagged CUL1
(m:CUL1) protein in these cells (33). Anti-His
immunoprecipitations were then probed with
appropriate antibodies by western blotting.
Presented in Fig. 1A, lane 3, AIRE co-
immunoprecipitated with FBXO3, CUL1 and
RBX1, but not with UBC4. In the absence of
AIRE, none of these proteins were detected (Fig.
1A, vector, lane 4). To confirm this association
further, we also co-expressed the FLAG epitope-
tagged AIRE protein (f:AIRE) with the HA
epitope-tagged FBXO3 protein (h:FBXO3) and
found the same association (Fig. 1B, lane 3).
FBXO3 could also immunoprecipitate AIRE (Fig.
1C). For this panel, we co-expressed f:FBXO3 and
mHis:AIRE and analyzed anti-FLAG
immunoprecipitations (Fig. 1C, lane 3). Finally,
we isolated 10 thymi from young mice and
immunoprecipitated the endogenous FBXO3
protein. These western blots were then probed
with anti-AIRE antibodies. In Fig. 1D, lane 2, the
endogenous FBXO3 protein interacted with the
native AIRE protein. We conclude that AIRE and
FBXO3 interact with each other in transformed
cells as well as in the relevant tissue of the host.
Finally, immunofluorescence assays (IFAs)
revealed that AIRE and FBXO3 co-localize in the
same nuclear organelles in 293T cells (Fig. 1E).
These subcellular organelles are enriched for
splicing factors and represent sites of active
transcription (35). Importantly, this intracellular
localization is in agreement with all previous
reports (33,35,36). Thus, not only do AIRE and
FBXO3 associate with each other and the rest of
the E3 ligase complex, but they also co-localize in
areas of active transcription. These findings
encouraged us to look more deeply into the role of
FBXO3 in regulating the expression of TSA genes.
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It is important to note that cells expressing AIRE
stop growing and many undergo apoptosis. This
cell cycle arrest prevents the continuous
cultivation of AIRE-expressing cells in culture. In
the thymus, these dying cells are cleared by
professional antigen presenting cells, which
present engulfed and processed TSA peptides on
major histocompatibility antigens to developing T
cells for the elimination of autoreactive cells. This
process directs central tolerance in the thymus
(3,4,37). Nevertheless, the transient expression of
AIRE in other epithelial or endothelial cells leads
to the activation of TSA genes.
N-terminus of AIRE binds to FBXO3
Next, we wanted to map surfaces on AIRE that
interact with FBXO3. To this end, we generated
several deletion mutant AIRE proteins (Fig. 2A).
Below the well-described domains of AIRE are
depicted fragments that contain N-terminal,
middle and C-terminal sequences of AIRE. The
FLAG epitope tag was placed at the N-terminus of
these truncated AIRE proteins. They were co-
expressed with the V5 epitope-tagged FBXO3
(v:FBXO3) protein in 293T cells. Of interest, N-
terminal fragments of AIRE were unstable (Fig.
2B, f:AIRE panels, lanes 2, 3, 7 and 8).
Nevertheless, only they interacted with v:FBXO3
(Fig. 2B, v:FBXO3 panel, lanes 7 and 8). We
conclude that N-Terminal 207 residues in AIRE
interact with FBXO3.
Doubly phosphorylated AIRE binds better to
FBXO3
As mentioned in the Introduction, the N-terminus
of AIRE contains two residues that are
phosphorylated by DNA-PK for full activity (11).
They are T68 and S156 and are contained in
sequences that bind to FBXO3. To determine if
the phosphorylation of AIRE is required for
binding to FBXO3, we first treated cell lysates
with calf intestinal phosphatase (CIP) prior to the
immunoprecipitation. As presented in Fig. 3A,
lane 3, the dephosphorylated AIRE protein bound
only weakly to FBXO3. The removal of the
phosphate group was confirmed with specific
antibodies against the phosphorylated S156
residue in AIRE (Fig. 3A, pAIRES156 panel, lane
3). Next, we changed T68 and S156 to alanine,
which cannot be phosphorylated, and the
phosphomimetic glutamic acid, creating the
mutant mHis:AIRET68A, mHis:AIRES156A,
mHis:AIRET68AS156A and mHis:AIRET68E
S156E proteins. Whereas the mutant
mHis:AIRET68ES156E protein bound FBXO3 as
well as its wild type (WT) counterpart (Fig. 3B,
lanes 2 and 6), these interactions were diminished
greatly with the mutant mHis:AIRET68AS156A
protein (Fig. 3B, lanes 2 and 5). Single mutant
AIRET68A or AIRES156A proteins bound
FBXO3 5-fold less well than the WT AIRE
protein (Fig. 3B, lanes 2, 3 and 4). Thus, AIRE
binds to FBXO3 via phosphorylated T68 and S156
residues and both are required for optimal
interactions between these two proteins.
FBXO3 promotes the ubiquitylation of AIRE
FBXO3 is an E3 ubiquitin ligase (23,31,33,34). To
determine if it is responsible for the ubiquitylation
of AIRE, we co-expressed f:AIRE and the His
epitope-tagged ubiquitin (His:Ub) in 293T cells.
These cells were also exposed to DMSO (control)
or MG132, which is a proteasome inhibitor. As
expected, in its absence, the ubiquitylated AIRE
protein was degraded and little to no
ubiquitylation was observed on AIRE (Fig. 4A,
lane 1). In contrast, in the presence of MG132,
AIRE’s ubiquitylation became apparent with the
endogenous FBXO3 protein (Fig. 4A, lane 2). In
Fig. 4B, we observed that the depletion of FBXO3
with siFBXO3 RNA reduced AIRE ubiquitylation
(Fig. 4B, lane 2). Finally, we examined if T68 and
S156 residues contribute to this ubiquitylation of
AIRE. As presented in Fig. 4C (upper h:Ub panel,
lane 2), when these residues were changed to the
phosphomimetic glutamic acid, the ubiquitylation
of AIRE was increased. In contrast, when they
were mutated to alanine, the mutant
mHis:AIRET68AS156A protein had decreased
ubiquitylation (Fig. 4C, h:Ub panel, lane 3). Thus,
the phosphorylation of T68 and S156 not only
allows for the binding to FBXO3 but also leads to
the ubiquitylation of AIRE.
FBXO3 destabilizes AIRE
As inferred from Fig. 4, the ubiquitylated AIRE
protein could be destabilized. To determine the
rate of decay of AIRE in the presence and absence
of FBXO3, we performed steady state and decay
experiments. When the endogenous FBXO3 was
depleted with siRNA, levels of AIRE increased
(Fig. 5A, top panel, lane 2). To distinguish
between increased synthesis and decreased rates of
decay, we also treated cells with cycloheximide
and followed the expression of mHis:AIRE by
western blotting. With endogenous FBXO3, the
half-life of AIRE was 5 h. In the absence of
FBXO3, this half-life was increased 2 to 3-fold
(Fig. 5B and C). Thus, FBXO3 not only
ubiquitylates but also destabilizes the AIRE
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protein in cells. Since T68 and S156 are found in
conserved sequences rich in proline, glutamic acid,
serine and threonine residues (PEST) in
vertebrates (Fig. 6), we conclude that they are
contained in degrons.
FBXO3 is required for AIRE’s transcriptional
activity
There is extensive literature on TADs, which exist
in proteins that contain degrons and function better
when ubiquitylated. They include cJun, cFos,
cMyc, p53 and VP16 (16-18). To determine if
AIRE also acts in this fashion, we co-expressed
mHis:AIRE and the human insulin promoter
linked to the luciferase reporter gene (INSLuc)
plasmid target in 293T cells. The insulin gene is a
bona fide target of AIRE and is expressed in
AIRE-expressing mTECs (38). First, we
investigated transcriptional activity of the WT and
two mutant AIRE proteins on INSLuc. As
presented in Fig. 7A, whereas the WT mHis:AIRE
and mutant mHis:AIRET68ES156E proteins
activated INSLuc 25- and 37.5-fold, respectively,
the mutant mHis:AIRET68AS156A only increased
the luciferase activity 5-fold. Of note, levels of
AIRE proteins also paralleled their relative
degradation, such that the mutant
mHis:AIRET68AS156A achieved the highest
levels of expression in these cells (Fig. 7A,
mHis:AIRE panel). Next, we repeated these report
assays in WT or FBXO3-depleted cells (by
siFBXO3 RNA). As presented in Fig. 7B (bar 2),
the depletion of FBXO3 resulted in 4-fold
decreased activation of the human insulin
promoter. Note also that levels of the WT
mHis:AIRE protein increased in these cells (Fig.
7B, mHis:AIRE panel, lane 2). We conclude that
the ubiquitylation of AIRE is important for its
transcriptional activity on a validated target
promoter.
FBXO3 increases AIRE’s transcriptional
activity
To validate our findings with a transiently
expressed plasmid target, we also performed
studies on host cell genes in 293T cells (Fig. 7C)
and continuously growing 1C6 cells (Fig. 7D).
Although isolated as mTECs, continuously
growing 1C6 cells no longer express the
endogenous AIRE protein. However, when AIRE
is introduced into these cells, it activates TSA
genes (8,13). Levels of expression were
determined by RT-qPCR. In 293T cells, the
ectopically expressed mHis:AIRE activated two
AIRE-responsive genes, S100A8 and KRT14 but
not two AIRE-unresponsive genes, CCNH and
S100A10 (Fig. 7C). When FBXO3 was depleted in
these cells by siFBXO3 RNA, the expression of
S100A8 and KRT14 was decreased up to 4-fold
(Fig. 7C, bars 2 and 3), while that of CCNH and
S100A10 remained unchanged (Fig. 7C, bars 4 and
5). Levels of mHis:AIRE also increased upon the
depletion of FBXO3 (Fig. 7C, mHis:AIRE panel,
lane 2). The same situation was observed in 1C6
cells. In these cells, we investigated the expression
of Spt1, Krt14, Ins1, Ins2 and Gad67 genes, of
which the first four are AIRE-responsive. Upon
the depletion of FBXO3, a similar reduction in the
expression of AIRE-responsive genes but not of
Gad67 was observed in these cells (Fig. 7D, bars 2
to 6). Again, levels of mHis:AIRE increased upon
the absence of FBXO3. Thus, transcriptional
activity of AIRE depends upon its post-
translational modification also on genes in
chromatin.
Ubiquitylation of AIRE increases the binding
and recruitment of P-TEFb to target genes
The ubiquitylation of TFs increases the
recruitment of the proteasome and P-TEFb to
transcription units (16-18). Although the
recruitment of the 19S rather than the complete
26S proteasome suffices, this assembly is thought
to help clean up and degrade histones and other
misfolded proteins that are generated during active
transcription (16,17). In addition, we found that
ubiquitylated TFs also bind better to CycT1, which
together with CDK9 forms P-TEFb (18). To
determine if this ubiquitylation of AIRE also
increases its interactions with P-TEFb, we
performed binding studies between f:AIRE and the
endogenous CycT1 protein. Indeed, mHis:AIRE
and the mutant mHis:AIRET68ES156E proteins
bound to CycT1 better than the mutant
mHis:AIRET68AS156A protein (Fig. 8A, left top
CycT1 panel, lanes 1-3). Densitometric analyses
revealed that the WT mHis:AIRE protein bound
CycT1 about 3-fold better than the mutant
mHis:AIRET68AS156A protein and that the
mutant mHis:AIRET68ES156E protein bound
CycT1 about 5-fold better than its WT counterpart
(Fig. 8A). In addition, f:AIRE bound to CycT1 4-
fold better in the presence of FBXO3 than in its
absence (Fig. 8B, top CycT1 panel, lanes 1 and 2).
This improved binding should increase the
transcriptional activity of AIRE.
To this end, we also examined the recruitment of
P-TEFb to AIRE-responsive genes and performed
chromatin immunoprecipitations (ChIPs) on
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KRT14 and S100A8 promoters (Fig. 8C and D).
Anti-His immunoprecipitations revealed no
difference in amounts of WT mHis:AIRE and
mutant mHis:AIRET68ES156E or mHis:AIRET68
AS156A proteins at these promoters (Fig. 8C, left
panel, all bars). In contrast, levels of CycT1 were
attenuated with the mutant mHis:AIRET68AS156
A protein (Fig. 8C, right panel, gray bars). The
same situation was observed with the depletion of
FBXO3 using siFBXO3 RNA (Fig. 8D, left and
right panels, black bars). Since AIRE does not
travel with elongating RNAPII, we did not
investigate the body or 3’ ends of these genes (8).
We conclude that the ubiquitylation of AIRE
increases the binding between AIRE and CycT1
and the recruitment of P-TEFb to AIRE-
responsive promoters.
DISCUSSION
In this study, we identified and validated FBXO3
as the E3 ubiquitin ligase that modifies AIRE post-
translationally and increases its activity on TSA
genes. Following mass spectrometry, further
binding studies indicated that N-terminal
sequences in AIRE bind to FBXO3. FBXO3 was
also found to promote the ubiquitylation of AIRE.
Moreover, two phosphorylated residues were
required for this binding. Dephosphorylating
AIRE and mutating T68 and S156 residues to
alanine abrogated the binding between AIRE and
FBXO3. It also stabilized the AIRE protein and
diminished its transcriptional activity. Mutating
the same residues to glutamic acid, which is
phosphomimetic, not only destabilized AIRE but
also increased its transcriptional activity. Of note,
depleting FBXO3 in 293T and 1C6 cells
attenuated the ability of AIRE to activate TSA
genes. Finally, we found that AIRE’s
phosphorylation is important for its binding to
FBXO3 and its ubiquitylation. AIRE’s
ubiquitylation increased its interactions with
CycT1 and recruited more P-TEFb to AIRE
responsive genes. P-TEFb is required to modify
stalled RNAPII and transcription complexes for
efficient elongation and co-transcriptional
processing of target genes. We conclude that
FBXO3 is the E3 ligase that modifies AIRE for its
transcriptional activity and for central tolerance in
the thymus.
AIRE is expressed in the small subpopulation of
mTECs in the thymus. Thus, it is impossible to
collect enough mTECs for mechanistic studies. In
addition, AIRE-expressing cells stop growing and
most undergo apoptosis. For continuous growth in
culture, mTECs, such as 1C6 cells, lose the
expression of the endogenous AIRE protein. In our
study, we expressed AIRE transiently in 293T and
1C6 cells, which is an approach that has been
validated by others (13,19,21,39). Nevertheless,
since FBXO3 is expressed in all tissues and all
cells, we could study the endogenous protein and
perform its knockdowns. Indeed, the endogenous
FBXO3 protein ubiquitylated AIRE efficiently
(Fig. 4). Because of its deleterious effects in cells,
levels of transiently expressed AIRE protein
approximate those of the endogenous AIRE
protein in the thymus, thus further validating these
cell culture models for our studies (9,10). In this
context, it is also likely that the ubiquitylation of
AIRE ensures the optimal expression of TSA
genes before cells stop growing, thus promoting
their ability to orchestrate the process of central
tolerance in the thymus.
Our studies extend and confirm work on the role
of T68 and S156, which are phosphorylated by
DNA-PK for optimal transcriptional activity of
AIRE (11). These residues are located in a
sequence rich in proline, glutamic acid, serine and
threonine residues (PEST), which could contribute
to the rapid degradation of AIRE (Fig. 6) (40).
Previously, others and we found that DNA-PK and
unmodified histone H3K4 were required to recruit
AIRE to TSA genes (10,26,41). DNA-PK is found
at transcription start sites because of DNA breaks
created during initiation of transcription and
topoisomerase relaxation of chromatin. We also
found other members of FBXO3-E3 ligase
complex in our immunoprecipitations, which
include CUL1 and RBX1 (Fig. 1A). Other
components of the SCFFBXO3
include SKP1 and an
E2 neddylation enzyme that modifies CUL1 for
activity (42). An interesting aside to our studies is
that we created a mutant AIRE protein with
increased activity. This mutant AIRET68ES156E
protein was constitutively more active than the
WT AIRE protein. It warrants expression in
transgenic mice, which should become more
tolerant to self. It is also possible that this mutant
AIRET68ES156E protein could abrogate
autoimmunity in yet other mice, such as the NOD
mouse (43).
A recent report suggested that bromodomain
containing 4 (BRD4) bridges interactions between
AIRE and P-TEFb (44). BRD4 bound to
acetylated lysines in the HSR/CARD at the very
N-terminus of AIRE. However, this mapping
differs from studies of an APECED patient’s
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mutation, which identified the TAD at the extreme
C-terminus of the protein (9). In VP16, a similar
TAD and ubiquitin bind to cyclin boxes and the
extreme C-terminus of CycT1, respectively (18).
A critical finding of this new report was decreased
expression of TSA genes after the repeated
administration of I-BET151, which is a BET
bromodomain inhibitor, to young mice (44).
However, these compounds also increase levels of
hexamethylene bis-acetamide inducible 1
(HEXIM1) that suppress the kinase activity of P-
TEFb, which would decrease the expression of
TSA genes (45). Of interest, another study found
that rather than acetylation, it is the deacetylation
by Sirt1 that is essential for AIRE’s transcriptional
activity (46). Nevertheless, others and we found
that AIRE is destabilized upon the
phosphorylation of T68 and S156 (11). Upon this
post-translational modification, we found that
FBXO3 binds and ubiquitylates AIRE. Since TAD
and ubiquitin bind cooperatively to different
surfaces on CycT1, they increase the recruitment
of P-TEFb to target genes (18). It is also possible
that BRD4, ubiquitin and TAD all cooperate or act
individually in different contexts to recruit more
P-TEFb to RNAPII to optimize effects of AIRE on
TSA genes.
In addition to its effects on AIRE, this SCFFBXO3
complex has been reported to increase
proinflammatory responses by eliminating FBXL2,
which inhibits TRAF proteins (31,32). A more
recent study found an association between FBXO3
and SMURF1, which is a Nedd4 family member
(34). By degrading SMURF1, it stabilized
intermediates in BMP signaling cascades, which
include SMAD1/2 and JunB, thus increasing the
responsiveness of target genes. FBXO3 can also
degrade HIPK2 and p300 as well as p62 during
IFN-mediated suppression of the Rift Valley fever
virus (RVFV) (23). These other effects of FBXO3
could complicate knockout studies in transgenic
mice, which would have to be controlled for yet
additional published phenotypes. Nevertheless, we
were able to observe direct transcriptional effect of
AIRE on TSA genes in 293T and 1C6 cells, and
they were specific to transcription units that had
been validated previously by whole genome and
targeted studies from several laboratories (25,47).
It is also possible that these global effects might
render mutations in or inactivation of FBXO3
more deleterious to the organism than those found
in AIRE in APECED/APS1 patients. Thus, it
would be of interest to investigate severe cases of
APECED/APS1 or of other congenital
autoimmunities for possible mutations in or
around the FBXO3 gene.
ACKNOWLEDGEMENT
We thank Dr. Zhiyong Yang for the mouse thymus sample, Pärt Peterson, Rama Mallampalli for reagents
and members of the Peterlin laboratory for helpful discussions and comments on the manuscript. This
work was supported by a grant from the Nora Eccles Treadwell Foundation.
Conflict of interest
The authors declare that they have no conflict of interest with the contents of this article.
Author contributions: WS, KZ performed all experiments. WS, KZ, KF and BMP analyzed the data and
wrote the manuscript.
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Abbreviations: AIRE, autoimmune regulator; FBXO3, F-box protein 3; P-TEFb, positive transcription
elongation factor b; TF, transcription factor; APECED, autoimmune polyendocrinopathy-candidiasis-
ectodermal dystrophy; APS1, autoimmune polyglandular syndrome type 1; TSAs, tissue-specific
antigens; DNA-PK, DNA-dependent protein kinase; CTD, C-terminal domain; RNAPII, RNA
polymerase II; CycT1, cyclin T1; Cdk9, cyclin-dependent kinase 9; TADs, transcription activation
domains; PHD1, plant homeodomain 1; mTECs, medullary thymic epithelial cells; CUL1, Cullin1; RBX1,
ring-box 1; FBXL2, leucine-rich repeat protein 2; HIPK2, homeodomain interacting protein kinase 2;
SMURF1, SMAD-specific E3 ubiquitin protein ligase 1; IFAs, immunofluorescence assays; PEST,
proline, glutamic acid, serine and threonine residues; RVFV, Rift Valley fever virus; CIP, calf intestinal
phosphatase; ChIPs, chromatin immunoprecipitations; HEXIM1, hexamethylene bis-acetamide inducible
1.
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FIGURE LEGENDS
FIGURE 1. AIRE interacts with FBXO3.
(A) AIRE interacts with the SCFFBXO3
complex. Anti-His antibodies immunoprecipitated Myc, His
epitope-tagged AIRE (mHis:AIRE) protein from 293T cell lysates. Western blotting with anti-Myc,
anti-FLAG, anti-RBX1 and anti-Cullin1 antibodies revealed the presence of associated proteins in
immunoprecipitations. Western blotting with anti-UBC4 antibodies represented a negative control.
In the absence of AIRE (vector, lane 4), none of these proteins were detected in our
immunoprecipitations. f:FBXO3, FLAG epitope-tagged FBXO3, m:CUL1, Myc epitope-tagged
Cullin1, α-, anti.
(B) AIRE interacts with FBXO3. FLAG epitope-tagged AIRE protein co-immunoprecipitated the HA
epitope-tagged FBXO3 (h:FBXO3) protein from 293T cell lysates. AIRE and FBXO3 were detected
using anti-FLAG and anti-HA antibodies.
(C) FBXO3 interacts with AIRE. f:FBXO3 co-immunoprecipitated mHis:AIRE from 293T cell lysates.
(D) Endogenous FBXO3 and AIRE proteins interact in the mouse thymus. Endogenous FBXO3 and
AIRE proteins co-immunoprecipitated from mouse thymus lysates. Input (lower panels, 5% whole
thymus lysate) and IgG control are also presented.
(E) AIRE and FBXO3 co-localize in cells. 293T cells co-expressed Myc, His epitope-tagged AIRE
(green) and HA epitope-tagged FBXO3 (red) proteins. AIRE and FBXO3 were detected with anti-
His and anti-HA antibodies, respectively. The nuclear staining with DAPI is blue. Scale bar, 10 µm.
FIGURE 2. N terminus of AIRE binds to FBXO3.
(A) Schematic representation of wild type (WT) and mutant AIRE proteins. Black lines below the
schematic indicate FLAG epitope-tagged deletion mutant AIRE peptides.
(B) N terminus of AIRE interacts with FBXO3. FLAG epitope-tagged WT and mutant AIRE proteins
(f:AIRE) were co-expressed with the V5 epitope-tagged FBXO3 (v:FBXO3) protein in 293T cells.
Anti-FLAG immunoprecipitations were performed. Lower panels depict total lysates (lanes 1 to 5,
3%) and immunoprecipitated (lanes 6 to 10) WT and mutant AIRE proteins. Upper panels depict the
V5 epitope-tagged FBXO3 protein in lysates (lanes 1 to 5) and co-immunoprecipitations (lanes 6 to
10) with WT and mutant FLAG epitope-tagged AIRE proteins.
FIGURE 3. Doubly phosphorylated AIRE binds better to FBXO3.
(A) Calf intestinal phosphatase (CIP) treatment decreases interactions between AIRE and FBXO3.
f:AIRE proteins were co-expressed with the V5 epitope-tagged FBXO3 (v:FBXO3) protein in 293T
cells. Cell extracts were treated with CIP (lane 3) before anti-FLAG immunoprecipitations. Upper
and lower panels contain specific immunoprecipitations and inputs (3%), respectively. Antibody
detecting the phosphorylated S156 site in AIRE input (pAIRES156, 3%) and IgG control are also
presented.
(B) Progressive alanine substitutions of T68 and S156 in AIRE diminish interactions with FBXO3.
293T cells expressed WT (lane 2) or mutant mHis:AIRET68A (lane 3), mHis:AIRES156A (lane 4),
mHis:AIRET68AS156A (lane 5) and mHis:AIRET68ES156E (lane 6) proteins. Cell lysates were
incubated with anti-His antibodies. Input (lower panels, 3%) and no AIRE (lane 1) controls are also
presented.
FIGURE 4. FBXO3 promotes ubiquitylation of AIRE.
(A) AIRE is ubiquitylated in cells. 293T cells co-expressed the His epitope-tagged ubiquitin and f:AIRE
in the presence of DMSO or MG132. Proteins were detected with anti-FLAG antibodies by western
blotting. Ub depicts the polyubiquitylated AIRE protein. GAPDH served as a loading control.
(B) Depletion of FBXO3 reduces greatly the ubiquitylation of AIRE. 293T cells expressed f:AIRE with
control siRNA (scrambled, lane 1) or siRNA targeting FBXO3 (siFBXO3, lane 2). Lysates were
immunoprecipitated with anti-FLAG antibodies followed by detection with indicated antibodies by
western blotting. Input proteins (f:AIRE and FBXO3) and the loading control (GAPDH) are
presented in the lower three panels.
(C) The mutant mHisAIRET68AS156A protein is only weakly ubiquitylated. 293T cells co-expressed
HA epitope-tagged ubiquitin (h:Ub), f:FBXO3 and WT mHis:AIRE, mutant mHis:AIRET68ES156E
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and mutant mHis:AIRET68AS156A proteins for 48 h. Anti-His antibodies immunoprecipitated
AIRE. Western blotting was performed with anti-Myc, anti-HA and anti-FLAG antibodies. Input
proteins (mHis:AIRE and f:FBXO3) and the loading control (GAPDH) are presented in the lower
three panels.
FIGURE 5. FBXO3 destabilizes AIRE.
(A) Knockdown of FBXO3 increases amounts of steady state AIRE protein. 293T cells co-expressed
mHis:AIRE, control (scrambled) siRNA (siScr) or FBXO3 siRNA (siFBXO3). Whole cell lysates
were probed with anti-Myc or anti-FBXO3 antibodies by western blotting. GAPDH served as a
loading control.
(B) Knockdown of FBXO3 stabilizes AIRE. Cells were transfected with mHis:AIRE, control siRNA
(siScr) or siRNA targeting FBXO3 (siFBXO3) for 48 h. 100 µg/ml cycloheximide (CHX) was added
to cells and the disappearance of AIRE was followed by western blotting with specific antibodies
(anti-Myc, anti-FBXO3 and anti-GAPDH) for indicated times (0, 1, 4 and 8 hours).
(C) Densitometric analysis of mHis:AIRE western blot from panel B. Protein bands were quantified
using ImageJ software and AIRE protein half-life was calculated. Bars represent the standard error
of the mean of three independent experiments (SEM, n=3).
FIGURE 6. T68 and S156 are conserved across species.
Comparisons of conserved T68 and S156 residues in AIRE between species. Sequences from Homo
sapiens, Pan troglodytes, Macaca mulatta, Rattus norvegicus, Mus musculus, Canis lupus, Danio
rerio are presented. The number of proline, glutamic acid, serine and threonine (PEST) residues
surrounding T68 and S156 are indicated on the bottom. White to black bars indicate increased
conservation of amino acids in these FBXO3 proteins.
FIGURE 7. FBXO3 increases AIRE’s transcriptional activity.
(A) Mutations of T68 and S156 influence AIRE’s transcriptional activity. The relative luciferase activity
of INSLuc for WT mHis:AIRE (lane 2) and mutant mHis:AIRET68ES156E (lane 3) or
mHis:AIRET68AS156A proteins (lane 4) is presented as fold-activation above levels of the empty
plasmid vector. Levels of mHis:AIRE and GAPDH (as internal control) proteins are given below the
bar graph.
(B) Knockdown of FBXO3 decreases AIRE’s transcriptional activity. Presented is the relative luciferase
activity of INSLuc, which is induced by AIRE, after FBXO3 knockdown (lane 2) or control (lane 1).
Values are presented as fold-activation above levels of control siRNA. Levels of mHis:AIRE,
FBXO3 and GAPDH (as internal control) proteins are given below the bar graph.
(C) FBXO3 knockdown affects the expression of AIRE-dependent genes in 293T cells. RT-qPCR
analysis was performed on 4 candidate genes after FBXO3 knockdown in 293T cells. 293T cells co-
expressed mHis:AIRE with control (siScr) or FBXO3 targeting siRNAs (siFBXO3) for 72 h before
harvesting. Bottom panels present levels of FBXO3, AIRE and GAPDH (as the internal control)
proteins.
(D) FBXO3 knockdown affects AIRE-dependent genes in the mTECs. RT-qPCR analysis was
performed on 5 candidate genes after FBXO3 knockdown in 1C6 mTECs. 1C6 cells co-expressed
mHis:AIRE with control (siScr) or FBXO3 targeting (siFBXO3) siRNAs for 72 h before harvesting.
Values and western blots are as in panel C.
For panels A to D, bars represent SEM, n=3. A Student’s t-test was preformed to measure the significance
of the data (* P<0.05, ** P<0.01, *** P<0.001).
FIGURE 8. Ubiquitylation of AIRE increases the binding and recruitment of P-TEFb to target genes.
(A) Mutations of T68 and S156 influence AIRE’s interaction with P-TEFb. 293T cells expressed WT
mHis:AIRE or mutant mHis:AIRE proteins for 48 h. Whole cell lysates were immunoprecipitated
with anti-His antibodies, followed by western blotting with indicated antibodies.
(B) Knockdown of FBXO3 decreases interactions between AIRE and P-TEFb. f:AIRE was expressed in
293T cells with control (siScr) or siFBXO3 (siFBXO3) RNAs for 72 h before harvesting. Total cell
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lysates were incubated with anti-FLAG beads. AIRE, the endogenous CycT1 and FBXO3 proteins
were examined with indicated antibodies. Top panels contain immunoprecipitated CycT1 and
f:AIRE proteins. Lower panels contain 3% input CycT1, f:AIRE and FBXO3 proteins.
(C) and (D) Interactions between AIRE and P-TEFb on DNA are decreased by deleterious mutations in
AIRE and the depletion of FBXO3. C. Presented are ChIPs of AIRE and CycT1 at promoters of
TSA genes (KRT14 and S100A8) in cells expressing WT and mutant mHis:AIRE proteins. D.
Presented are ChIPs of AIRE and CycT1 at the promoter of TSA genes (KRT14 and S100A8) in
control or FBXO3-depleted cells. Bars represent the standard error of the mean of three independent
experiments (SEM, n=3). A Student’s t-test was preformed to measure the significance of the data (*
P<0.05, ** P<0.01).
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TABLE 1: AIRE-interacting proteins identified by MS
Gene symbol # unique peptides
1 GCN1L_HUMAN GCN1-like protein 1 44
2 LAP2A_HUMAN Lamina-associated polypeptide 2 isoform alpha 13
3 AIRE_HUMAN Autoimmune regulator 12
4 HNRH1_HUMAN Heterogeneous nuclear ribonucleoprotein H 11
5 FBXO3_HUMAN F-box protein 3 8
6 DDX5_HUMAN Probable ATP-dependent RNA helicase DDX5 5
7 HNRPF_HUMAN Heterogeneous nuclear ribonucleoprotein F 4
8 SMC3_HUMAN Structural maintenance of chromosomes protein 3 3
9 SF3B3_HUMAN Splicing factor 3B subunit 3 3
10 TADBP_HUMAN TAR DNA-binding protein 43 2
11 SUMO2_HUMAN Small ubiquitin-related modifier 2 precursor 2
12 LANC2_HUMAN LanC-like protein 2 2
13 ATR_HUMAN Serine/threonine-protein kinase ATR 2
List of AIRE interacting proteins identified by mass spectrometry (MS). The number of unique peptides
is presented. FBXO3 and its number of peptides are written in bold letters.
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24. Wei Shao, Kristina Zumer, Koh Fujinaga and B. Matija Peterlin
FBXO3 Promotes Ubiquitylation and Transcriptional Activity of AIRE
published online June 30, 2016J. Biol. Chem.
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