Jung-Ah Kang

Renal cell carcinoma escapes death by p53 depletion through transglutaminase 2-chaperoned autophagy.

In renal cell carcinoma, transglutaminase 2 (TGase 2) crosslinks p53 in autophagosomes, resulting in p53 depletion and the tumor’s evasion of apoptosis. Inhibition of TGase 2 stabilizes p53 and induces tumor cells to enter apoptosis. This study explored the mechanism of TGase 2-dependent p53 degradation. We found that TGase 2 competes with human double minute 2 homolog (HDM2) for binding to p53; promotes autophagy-dependent p53 degradation in renal cell carcinoma (RCC) cell lines under starvation; and binds to p53 and p62 simultaneously without ubiquitin-dependent recognition of p62. The bound complex does not have crosslinking activity. A binding assay using a series of deletion mutants of p62, p53 and TGase 2 revealed that the PB1 (Phox and Bem1p-1) domain of p62 (residues 85–110) directly interacts with the β-barrel domains of TGase 2 (residues 592–687), whereas the HDM2-binding domain (transactivation domain, residues 15–26) of p53 interacts with the N terminus of TGase 2 (residues 1–139). In addition to the increase in p53 stability due to TGase 2 inhibition, the administration of a DNA-damaging anti-cancer drug such as doxorubicin-induced apoptosis in RCC cell lines and synergistically reduced tumor volume in a xenograft model. Combination therapy with a TGase 2 inhibitor and a DNA-damaging agent may represent an effective therapeutic approach for treating RCC.

The kinase PDK1 is essential for B-cell receptor mediated survival signaling.

Phosphoinositide-dependent kinase 1 (PDK1) plays an important role in integrating the T cell antigen receptor (TCR) and CD28 signals to achieve efficient NF-κB activation. PDK1 is also an important regulator of T cell development, mediating pre-TCR induced proliferation signals. However, the role of PDK1 in B cell antigen receptor (BCR) signaling and B cell development remains largely unknown. In this study we provide genetic evidence supporting the role of PDK1 in B cell survival. We found PDK1 is required for BCR mediated survival in resting B cells, likely through regulation of Foxo activation. PDK1-dependent signaling to NF-κB is not crucial to resting B cell viability. However, PDK1 is necessary for triggering NF-κB during B cell activation and is required for activated B cell survival. Together these studies demonstrate that PDK1 is essential for BCR-induced signal transduction to Foxo and NF-κB and is indispensable for both resting and activated B cell survival.

Cereblon negatively regulates TLR4 signaling through the attenuation of ubiquitination of TRAF6

Cereblon (CRBN) is a substrate receptor protein for the CRL4A E3 ubiquitin ligase complex. In this study, we report on a new regulatory role of CRBN in TLR4 signaling. CRBN overexpression leads to suppression of NF-κB activation and production of pro-inflammatory cytokines including IL-6 and IL-1β in response to TLR4 stimulation. Biochemical studies revealed interactions between CRBN and TAK1, and TRAF6 proteins. The interaction between CRBN and TAK1 did not affect the association of the TAB1 and TAB2 proteins, which have pivotal roles in the activation of TAK1, whereas the CRBN-TRAF6 interaction critically affected ubiquitination of TRAF6 and TAB2. Binding mapping results revealed that CRBN interacts with the Zinc finger domain of TRAF6, which contains the ubiquitination site of TRAF6, leading to attenuation of ubiquitination of TRAF6 and TAB2. Functional studies revealed that CRBN-knockdown THP-1 cells show enhanced NF-κB activation and p65- or p50-DNA binding activities, leading to up-regulation of NF-κB-dependent gene expression and increased pro-inflammatory cytokine levels in response to TLR4 stimulation. Furthermore, Crbn−/− mice exhibit decreased survival in response to LPS challenge, accompanied with marked enhancement of pro-inflammatory cytokines, such as TNF-α and IL-6. Taken together, our data demonstrate that CRBN negatively regulates TLR4 signaling via attenuation of TRAF6 and TAB2 ubiquitination.

Transition from Heterotypic to Homotypic PDK1 Homodimerization Is Essential for TCR-Mediated NF-κB Activation

Strong NF-κB activation requires ligation of both the CD28 coreceptor and TCR. Phosphoinositide-dependent kinase 1 (PDK1) acts as a scaffold by binding both protein kinase Cθ (PKCθ) and CARMA1, and is therefore essential for signaling to NF-κB. In this article, we demonstrate the importance of PDK1 Thr513 phosphorylation in regulating the intermolecular organization of PDK1 homodimers. Thr513 is directly involved in heterotypic PDK1 homodimer formation, in which binding is mediated through the pleckstrin homology (PH) and kinase domains. Upon activation, phosphorylated Thr513 instead mediates homotypic intermolecular binding through the PH domains. Consequently, cell-permeable peptides with a Thr513 to Ile derivative (protein transduction domain [PTD]-PDK1-Thr513-Ile) bound the kinase domain, whereas a Thr513-to-Asp peptide (PTD-PDK1-Thr513-Asp) bound the PH domain. PTD-PDK1-Thr513-Ile blocked binding between PDK1 and PKCθ, phosphorylation of PKCθ Thr538, and activation of both NF-κB and AKT. In contrast, PTD-PDK1- Thr513-Asp selectively inhibited binding between PDK1 and CARMA1, and blocked TCR/CD28-induced NF-κB activation. Therefore, Thr513 phosphorylation regulates a critical intermolecular switch governing PDK1 homodimer structure and the capacity to interact with downstream signaling pathway components. Given the pleiotropic functions of PDK1, these data may open the door to the development of immunosuppressive therapies that selectively target the PDK1 to NF-κB pathway in T cell activation.

Epigenetic regulation of Kcna3-encoding Kv1.3 potassium channel by cereblon contributes to regulation of CD4+ T-cell activation.

Significance In resting T cells, CRBN normally represses expression of the Kv1.3 potassium channel by regulating histone modifications to prevent hyperactivation of T cells. It does this by controlling recruitment of EZH1 to the potassium channel region of the Kcna3 gene (or locus). However, lack of CRBN causes up-regulation of Kv1.3 expression, which in turn increases potassium flux, thereby triggering increased calcium flux during T-cell activation. The role of cereblon (CRBN) in T cells is not well understood. We generated mice with a deletion in Crbn and found cereblon to be an important antagonist of T-cell activation. In mice lacking CRBN, CD4+ T cells show increased activation and IL-2 production on T-cell receptor stimulation, ultimately resulting in increased potassium flux and calcium-mediated signaling. CRBN restricts T-cell activation via epigenetic modification of Kcna3, which encodes the Kv1.3 potassium channel required for robust calcium influx in T cells. CRBN binds directly to conserved DNA elements adjacent to Kcna3 via a previously uncharacterized DNA-binding motif. Consequently, in the absence of CRBN, the expression of Kv1.3 is derepressed, resulting in increased Kv1.3 expression, potassium flux, and CD4+ T-cell hyperactivation. In addition, experimental autoimmune encephalomyelitis in T-cell–specific Crbn-deficient mice was exacerbated by increased T-cell activation via Kv1.3. Thus, CRBN limits CD4+ T-cell activation via epigenetic regulation of Kv1.3 expression.

Notch1 is an important mediator for enhancing of B-cell activation and antibody secretion by Notch ligand

The roles of Notch1 and Notch2 in T‐cell function have been well studied, but the functional roles of Notch in B cells have not been extensively investigated, except for Notch2 involvement in peripheral marginal zone B‐cell differentiation. This study examined the roles of Notch1 in murine primary B cells. During B‐cell activation by B‐cell receptor ligation, Notch1 was up‐regulated while Notch2 was not. In addition, Notch1 up‐regulation itself did not contribute to the further activation of B cells, but the Notch ligand was important for Notch1‐mediated further B‐cell activation. Moreover, Notch1 deficiency significantly decreased B‐cell activation and antibody secretion under the presence of Notch ligand. These data suggest that Notch1 is an important mediator for enhancing B‐cell activation and antibody secretion by Notch ligand.

Peroxisome-localized hepatitis Bx protein increases the invasion property of hepatocellular carcinoma cells

HBx acts as a multifunctional regulator that modulates various cellular responses, which can lead to development and progression of hepatocellular carcinoma (HCC). Here, we show that the HBx protein is also localized to peroxisomes, and this increases cellular reactive oxygen species (ROS) to levels that are higher than when HBx is localized to other organelles. The elevated ROS strongly activated nuclear factor (NF)-κB. In addition, the peroxisome-localized HBx increased the expressions of matrix metalloproteinases and decreased the expression of E-cadherin, which increased the invasive ability of HCC cells. Thus, a specific distribution of HBx to peroxisomes may contribute to HCC progression by increasing the invasive ability of HCC cells through elevation of the cellular ROS level.

Tofacitinib relieves symptoms of stimulator of interferon genes (STING)–associated vasculopathy with onset in infancy caused by 2 de novo variants in TMEM173

4. Kukkonen AK, Pelkonen AS, M€akinen-Kiljunen S, Voutilainen H, M€akel€a MJ. Ara h 2 and Ara 6 are the best predictors of severe peanut allergy: a double-blind placebo-controlled study. Allergy 2015;11:1239-45. 5. Nozawa A, Okamoto Y, Mov erare R, Borres MP, Kurihara K. Monitoring Ara h 1, 2 and 3-sIgE and sIgG4 antibodies in peanut allergic children receiving oral rush immunotherapy. Pediatr Allergy Immunol 2014;25:323-8. 6. Vickery BP, Scurlock AM, Kulis M, Steele PH, Kamilaris J, Berglund JP, et al. Sustained unresponsiveness to peanut in subjects who have completed peanut oral immunotherapy. J Allergy Clin Immunol 2014;133:468-75. 7. Krause S, Reese G, Randow S, Zennaro D, Quaratino D, Palazzo P, et al. Lipid transfer protein (Ara h 9) as a new peanut allergen relevant for a Mediterranean allergic population. J Allergy Clin Immunol 2009;124:771-8.e5. 8. Glaumann S, Nilsson C, Asarnoj A, Mov erare R, Johansson SG, Borres MP, et al. IgG4 antibodies and peanut challenge outcome in children IgE-sensitized to peanut. Pediatr Allergy Immunol 2015;26:386-9. 9. Santos AF, James LK, Bahnson HT, Shamji MH, Couto-Francisco NC, Islam S, et al. IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens. J Allergy Clin Immunol 2015;135:1249-56.

Secretion of IL‐1β from imatinib‐resistant chronic myeloid leukemia cells contributes to BCR–ABL mutation‐independent imatinib resistance

Some cases of chronic myelogenous leukemia are resistant to tyrosine kinase inhibitors (TKIs) independently of mutation in BCR–ABL, but the detailed mechanism underlying this resistance has not yet been elucidated. In this study, we generated a TKI‐resistant CML cell line, K562R, that lacks a mutation in BCR–ABL. Interleukin‐1β (IL‐1β) was more highly expressed in K562R than in the parental cell line K562S, and higher levels of IL‐1β contributed to the imatinib resistance of K562R. In addition, IL‐1β secreted from K562R cells affected stromal cell production of CXCL11, which in turn promoted migration of K562R cells into the stroma. Thus, elevated IL‐1β production from TKI‐resistant K562R cells may contribute to TKI resistance by increasing cell viability and promoting cell migration.

Intestinal Intraepithelial TCRγδ + T Cells are Activated by Normal Commensal Bacteria

TCRγδ+ T cells play a critical role in protecting the intestinal mucosa against pathogenic infection. In the absence of infection, TCRγδ+ T cell activation must be continuously regulated by T regulatory cells (Treg) to prevent the development of colitis. However, the activation of intestinal TCRγδ+ T cells under normal conditions has not been clearly resolved. In order to determine TCRγδ+ T cell activation in vivo, we designed an NF-κB based reporter system. Using the recombinant lentiviral method, we delivered the NF-κB reporter to isolated TCRγδ+ T cells, which were then adoptively transferred into normal mice. Our data indicate that the NF-κB activation level in TCRγδ+ T cells is higher in the intestinal intraepithelial layer than in the lamina propria region. In addition, the surface expression level of lymphocyte activation marker CD69 in TCRγδ+ T cells is also higher in the intestinal intraepithelial layer and this activation was reduced by Sulfatrim treatment which removes of commensal bacteria. Collectively, our data indicate that the TCRγδ+ T cell population attached to the intestinal lumen is constitutively activated even by normal commensal bacteria.