CARMA1 mediates T cell receptor (TCR)-induced NF-kappaB activation. However, how TCR links to CARMA1 in the signaling pathway is not clear. Here, we show that CARMA1 is inducibly phosphorylated after TCR-CD28 costimulation. This phosphorylation is likely induced by PKCtheta, since PKCtheta induces phosphorylation of CARMA1 in vitro and in vivo. Our results indicate that the PKCtheta-induced phosphorylation of CARMA1 likely occurs on Ser552 on the Linker region of CARMA1. Importantly, expression of CARMA1 mutant, in which Ser552 is mutated, fails to mediate TCR-induced NF-kappaB activation in CARMA1-deficient T cells. The functional defect of this CARMA1 mutant is likely due to the fact that this mutant cannot be phosphorylated at the critical residue, thereby failing to recruit the downstream signaling components into the immunological synapse. Together, our studies provide the first genetic evidence that the phosphorylation of CARMA1 plays a critical role in the TCR signaling pathway.

SummaryReprogrammed cellular metabolism is a common characteristic observed in various cancers1,2. However, whether metabolic changes directly regulate cancer development and progression remains poorly understood. Here we show that BCAT1, a cytosolic aminotransferase for the branched-chain amino acids (BCAAs), is aberrantly activated and functionally required for chronic myeloid leukemia (CML). BCAT1 is up-regulated during CML progression and promotes BCAA production in leukemia cells by aminating the branched-chain keto acids. Blocking BCAT1 expression or enzymatic activity induces cellular differentiation and impairs the propagation of blast crisis CML (BC-CML) both in vitro and in vivo. Stable isotope tracer experiments combined with NMR-based metabolic analysis demonstrate the intracellular production of BCAAs by BCAT1. Direct supplementation with BCAAs ameliorates the defects caused by BCAT1 knockdown, indicating that BCAT1 exerts its oncogenic function via BCAA production in BC-CML cells. Importantly, BCAT1 expression not only is activated in human BC-CML and de novo acute myeloid leukemia but also predicts disease outcome in patients. As an upstream regulator of BCAT1 expression, we identified Musashi2 (MSI2), an oncogenic RNA binding protein that is required for BC-CML. MSI2 is physically associated with the BCAT1 transcript and positively regulates its protein expression in leukemia. Taken together, this work reveals that altered BCAA metabolism activated through the MSI2-BCAT1 axis drives cancer progression in myeloid leukemia.

Stimulation of cells with tumor necrosis factor (TNF␣) triggers a recruitment of various signaling molecules, such as RIP, to the TNF␣ receptor 1 complex, leading to activation of NF-B. Previous studies indicate that RIP plays an essential role for TNF␣-induced NF-B activation, but the molecular mechanism by which RIP mediates TNF␣ signals to activate NF-B is not fully defined. Earlier studies suggest that RIP undergoes a ligand-dependent ubiquitination. However, it remains to be determined whether the ubiquitination of RIP is required for TNF␣-induced NF-B activation. In this study, we have identified Lys 377 of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-B activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNF␣ receptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNF␣-induced NF-B activation.NF-B is a homo-or heterodimeric transcription factor that controls the expression of various genes involved in inflammatory, apoptotic, and immune responses. In resting cells, the activity of NF-B is controlled through its cytoplasmic sequestration by a family of inhibitors, IB (Inhibitor of NF-B) (1). In response to extracellular stimuli, IB proteins are phosphorylated by the IB kinase (IKK) 2 complex, then ubiquitinated, and rapidly degraded, which leads to the nuclear localization and activation of NF-B (2). One of the most potent NF-B activators is the proinflammatory cytokine, tumor necrosis factor (TNF)␣. TNF␣ functions through the TNF receptors, mainly TNFR1, on the cell surface. The binding of TNF␣ to TNFR1 causes trimerization of the receptor and recruitment of the adaptor protein TRADD (TNF receptor-associated death domain) (3). TRADD further recruits TARF2 (TNF receptor-associated factor 2) (4), FADD (Fas-associated death domain) (4), and RIP (receptor-interacting protein) (5, 6).RIP is a serine/threonine kinase that plays an essential role in TNF␣-induced NF-B activation (6, 7). It contains an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain. Earlier studies indicate that the RIP kinase domain is dispensable for the activation of NF-B (6, 8), whereas its death domain is required for the association with the upstream signaling component TRADD (5). The intermediate domain is required for RIP-mediated NF-B activation (6) and plays an important role for interacting with its downstream signaling components such as NEMO, the regulatory subunit of the IKK complex, and other molecules (9). Therefore, RIP likely functions as an adaptor molecule to mediate the TNF␣ signaling cascade. Although genetic studies have demonstrated that TAK1 (tumor growth factor ␤-activated kinase 1) and MEKK3 are involved in mediating TNF␣-induced NF-B activation downstream of RIP (10 -15), the mechanism by which RIP activates these downstream kinases is not fully def...

SummaryWe investigated the significance of the platelet indices, mean platelet volume (MPV), platelet size deviation width (PDW), and platelet‐large cell ratio (P‐LCR), in the diagnosis of thrombocytopenia by comparing these levels in 40 patients with hypo‐productive thrombocytopenia (aplastic anaemia; AA) and 39 patients with hyper‐destructive thrombocytopenia (immune thrombo‐cytopenia; ITP). The sensitivity and specificity of platelet indices to make a diagnosis of ITP were also compared. All platelet indices were significantly higher in ITP than in AA, and platelet indices showed sufficient sensitivity and specificity. The area under the curve (AUC) of the receiver operating characteristics curve of platelet indices was large enough to enable the diagnosis of ITP. P‐LCR and PDW had the largest AUCs, which indicated that these values were very reliable for immune thrombocytopenia. Our results suggest that these indices provide clinical information about the underlying conditions of thrombocytopenia. More attention should be paid to these indices in the diagnosis of thrombocytopenia.

Human immunodeficiency virus, type 1 (HIV-1) Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion. Vif functions to counteract an anti-HIV-1 cellular factor in non-permissive cells, CEM15/Apobec-3G, which shares a cytidine deaminase motif. CEM15/Apobec-3G deaminates dC to dU in the minus strand DNA of HIV-1, resulting in G to A hypermutation in the plus strand DNA. In this study, we have done the mutagenesis analysis on two cytidine deaminase motifs in CEM15/Apobec-3G and examined their antiviral functions as well as the DNA editing activity. Point mutations in the C-terminal active site such as E259Q and C291A almost completely abrogated the antiviral function, while those in the N-terminal active site such as E67Q and C100A retained this activity to a lesser extent as compared with that of the wild type. The DNA editing activities of E67Q and E259Q mutants were both retained but impaired to the same extent. This indicates that the enzymatic activity of this protein is essential but not a sole determinant of the antiviral activity. Furthermore, all the deletion mutants tested in this study lost the antiviral activity because of the loss of the activity for dimerization, suggesting that the entire protein structure is necessary for the antiviral function.

Langerhans cell histiocytosis (LCH) is a rare systemic disorder characterized by the accumulation of CD1a+/Langerin+ LCH cells and wide‐ranging organ involvement. Langerhans cell histiocytosis was formerly referred to as histiocytosis X, until it was renamed in 1987. Langerhans cell histiocytosis β was named for its morphological similarity to skin Langerhans cells. Studies have shown that LCH cells originate from myeloid dendritic cells rather than skin Langerhans cells. There has been significant debate regarding whether LCH should be defined as an immune disorder or a neoplasm. A breakthrough in understanding the pathogenesis of LCH occurred in 2010 when a gain‐of‐function mutation in BRAF (V600E) was identified in more than half of LCH patient samples. Studies have since reported that 100% of LCH cases show ERK phosphorylation, indicating that LCH is likely to be a clonally expanding myeloid neoplasm. Langerhans cell histiocytosis is now defined as an inflammatory myeloid neoplasm in the revised 2016 Histiocyte Society classification. Randomized trials and novel approaches have led to improved outcomes for pediatric patients, but no well‐defined treatments for adult patients have been developed to date. Although LCH is not fatal in all cases, delayed diagnosis or treatment can result in serious impairment of organ function and decreased quality of life. This study summarizes recent advances in the pathophysiology and treatment of adult LCH, to raise awareness of this “orphan disease”.

The reaction center (RC) of photosynthetic bacteria is a membrane protein complex that promotes a light-induced charge separation during the primary process of photosynthesis. In the photosynthetic electron transfer chain, the soluble electron carrier proteins transport electrons to the RC and reduce the photo-oxidized special-pair of bacteriochlorophyll. The high-potential iron-sulfur protein (HiPIP) is known to serve as an electron donor to the RC in some species, where the c-type cytochrome subunit, the peripheral subunit of the RC, directly accepts electrons from the HiPIP. Here we report the crystal structures of the RC and the HiPIP from Thermochromatium (Tch.) tepidum, at 2.2-Å and 1.5-Å resolution, respectively. Tch. tepidum can grow at the highest temperature of all known purple bacteria, and the Tch. tepidum RC shows some degree of stability to high temperature. Comparison with the RCs of mesophiles, such as Blastochloris viridis, has shown that the Tch. tepidum RC possesses more Arg residues at the membrane surface, which might contribute to the stability of this membrane protein. The RC and the HiPIP both possess hydrophobic patches on their respective surfaces, and the HiPIP is expected to interact with the cytochrome subunit by hydrophobic interactions near the heme-1, the most distal heme to the special-pair. In photosynthetic purple bacteria, the electron transfer reactions of photosynthesis are performed by the following three components: the photosynthetic reaction center (RC), the cytochrome (Cyt) bc 1 complex, and the soluble electron carrier protein. First, the RC promotes the light-induced charge separation across the plasma membrane, which results in the oxidation of the special-pair and the reduction of the quinone to the quinol. The quinol then leaves the RC and moves to the Cyt bc 1 complex through the quinone pool of the plasma membrane. Second, the Cyt bc 1 complex reoxidizes the quinol to the quinone, and the released electrons are transferred to the soluble electron carriers. Third, the soluble electron carriers transport the electrons to the RC through the periplasmic space. Finally, the photo-oxidized special-pair is reduced by the soluble electron carriers, and the RC comes back to the initial state. In the course of the oxidation and the reduction of the quinones, the transmembrane electrochemical gradient of the protons is formed, and its energy is used to produce ATP by ATP synthase.Thermochromatium (Tch.; formerly Chromatium) tepidum is a purple sulfur bacterium originally isolated from the hot springs in Yellowstone National Park (1, 2) and belongs to the ␥-subclass. Tch. tepidum is a thermophilic bacterium and can grow at the highest temperature of all known purple bacteria. The optimum growth temperature is 48-50°C, the maximum temperature 58°C. The RC from Tch. tepidum is stable up to 70°C in chromatophore and to 48°C in detergent-micelle (3). The RC is the first membrane protein whose three-dimensional structure has been determined at an atomic resolution (4, 5), and the...

Thermophilic purple sulfur bacterium, Thermochromatium tepidum, can grow at temperatures up to 58 degrees C and exhibits an unusual Qy absorption at 915 nm for the core light-harvesting complex (LH1), an approximately 35-nm red shift from those of its mesophilic counterparts. We demonstrate in this study, using a highly purified LH1-reaction center complex, that the LH1 Qy transition is strongly dependent on metal cations and Ca2+ is involved in the unusual red shift. Removal of the Ca2+ resulted in formation of a species with the LH1 Qy absorption at 880 nm, and addition of the Ca2+ to the 880-nm species recovered the native 915-nm form. Interchange between the two forms is fully reversible. Based on spectroscopic and isothermal titration calorimetry analyses, the Ca2+ binding to the LH1 complex was estimated to occur in a stoichiometric ratio of Ca2+/alphabeta-subunit = 1:1 and the binding constant was in 10(5) m(-1) order of magnitude, which is comparable with those for EF-hand Ca2+-binding proteins. Despite the high affinity, conformational changes in the LH1 complex upon Ca2+ binding were small and occurred slowly, with a typical time constant of approximately 6 min. Replacement of the Ca2+ with other metal cations caused blue shifts of the Qy bands depending on the property of the cations, indicating that the binding site is highly selective. Based on the amino acid sequences of the LH1 complex, possible Ca2+-binding sites are proposed that consist of several acidic amino acid residues near the membrane interfaces of the C-terminal region of the alpha-polypeptide and the N-terminal region of the beta-polypeptide.