Viral RNA recognition by LGP2 and MDA5, and activation of signaling through step-by-step conformational changes

細胞内のウイルスを認識する蛋白質の仕組みを解明 --ウイルスから我々の体を守る影のヒーロー--. 京都大学プレスリリース. 2020-12-04.Cytoplasmic RIG-I-like receptor (RLR) proteins in mammalian cells recognize viral RNA and initiate an antiviral response that results in IFN-β induction. Melanoma differentiation-associated protein 5 (MDA5) forms fibers along viral dsRNA and propagates an antiviral response via a signaling domain, the tandem CARD. The most enigmatic RLR, laboratory of genetics and physiology (LGP2), lacks the signaling domain but functions in viral sensing through cooperation with MDA5. However, it remains unclear how LGP2 coordinates fiber formation and subsequent MDA5 activation. We utilized biochemical and biophysical approaches to observe fiber formation and the conformation of MDA5. LGP2 facilitated MDA5 fiber assembly. LGP2 was incorporated into the fibers with an average inter-molecular distance of 32 nm, suggesting the formation of hetero-oligomers with MDA5. Furthermore, limited protease digestion revealed that LGP2 induces significant conformational changes on MDA5, promoting exposure of its CARDs. Although the fibers were efficiently dissociated by ATP hydrolysis, MDA5 maintained its active conformation to participate in downstream signaling. Our study demonstrated the coordinated actions of LGP2 and MDA5, where LGP2 acts as an MDA5 nucleator and requisite partner in the conversion of MDA5 to an active conformation. We revealed a mechanistic basis for LGP2-mediated regulation of MDA5 antiviral innate immune responses

Characterization of single actomyosin rigor bonds: load dependence of lifetime and mechanical properties.

Load dependence of the lifetime of the rigor bonds formed between a single myosin molecule (either heavy meromyosin, HMM, or myosin subfragment-1, S1) and actin filament was examined in the absence of nucleotide by pulling the barbed end of the actin filament with optical tweezers. For S1, the relationship between the lifetime (tau) and the externally imposed load (F) at absolute temperature T could be expressed as tau(F) = tau(0).exp(-F.d/k(B)T) with tau(0) of 67 s and an apparent interaction distance d of 2.4 nm (k(B) is the Boltzmann constant). The relationship for HMM was expressed by the sum of two exponentials, with two sets of tau(0) and d being, respectively, 62 s and 2.7 nm, and 950 s and 1.4 nm. The fast component of HMM coincides with tau(F) for S1, suggesting that the fast component corresponds to single-headed binding and the slow component to double-headed binding. These large interaction distances, which may be a common characteristic of motor proteins, are attributed to the geometry for applying an external load. The pulling experiment has also allowed direct estimation of the number of myosin molecules interacting with an actin filament. Actin filaments tethered to a single HMM molecule underwent extensive rotational Brownian motion, indicating a low torsional stiffness for HMM. From these results, we discuss the characteristics of interaction between actin and myosin, with the focus on the manner of binding of myosin

Mercury transformation behavior on a bench scale coal combustion furnace

The mercury release behavior in bituminous coals, and the partitioning rate of mercury in solids and gaseous in flue gases have measured to develop technologies for evaluating the partitioning of mercury in coal combustion process and develop in-situ adsorption and removal technologies using three kinds of experiment equipments - a thermo-balance, a drop-tube furnace (DTF), a bench-scale pulverized coal combustion fumace. The results showed that about 20 to 60% of the mercury in coal was released between 573 K and 673 K, which was the range of temperature in which the release of the volatile matter of coal began. And more than 90% of the mercury was released at 773 K, the temperature at which the release of the volatile matter was completed. The rate of mercury partitioned into bottom ash in a bench-scale pulverized coal combustion furnace was the smallest irrespective of the type of coal. The rate of mercury partitioned into cyclone ash was also low for all types of coal with values generally below 10%. The rest of the mercury was partitioned into mercury in gaseous form, but the rate partitioned into dust, oxidized mercury and elemental mercury varied slightly depending on the flue gas temperature and the type of coal.

Different role of Apaf-1 in positive selection, negative selection and death by neglect in foetal thymic organ culture

Apoptotic protease-activating factor 1 (Apaf-1) is a component of the apoptosome which is required for the activation of procaspase-9. As Apaf-1 knockout (KO) (Apaf-1(-/-)) mice die before birth, the role of Apaf-1 during thymic selection was investigated using 5 day foetal thymic organ culture (FTOC) of thymi obtained at gestational day 15. There was a lower ratio of CD4 single-positive (SP) to CD8 SP cells and decreased apoptosis of CD4(+) CD8(+) (DP) thymocytes from Apaf-1 (-/-) mice compared with wild-type. To determine if these defects resulted in increased production of neglected thymocytes, the Apaf-1 (-/-)mice were crossed with the T-cell receptor (TCR)- alpha-chain KO mice. There was no difference in thymocyte development in the thymi of TCR-alpha (-/-) Apaf-1 (-/-) and TCR-alpha (-/-) Apaf-1 (+/+) mice 5 days after FTOC. To determine if Apaf-1 is involved in apoptosis during death by negative or positive selection, FTOC of the thymus of Apaf-1 D- -/-(b)/HY TCR-alphabeta transgenic (Tg) mice was carried out. There was decreased apoptosis of the HY clonal-specific M33(+) thymocytes and an increased percentage of the autoreactive CD8(+) M33(+) thymocytes in male, but not female Apaf-1 (-/-) D-b/HY TCR Tg mice. Our data suggest that Apaf-1 is not involved in positive selection or death by neglect, but may have a partial role in negative selection during early thymic T-cell development