TRIM65 E3 ligase targets VCAM-1 degradation to limit LPS-induced lung inflammation

Although the adhesion molecules-mediated leukocyte adherence and infiltration into tissues is an important step of inflammation,the post-translational regulation of these proteins on the endothelial cells is poorly understood.Here,we report that TRIM65,an ubiquitin E3 iigase of tripartite protein family,selectively targets vascular cell adhesion molecule 1(VCAM-1)and promotes its ubiq-uitination and degradation,by which it critically controls the duration and magnitude of sepsis-induced pulmonary inflammation.TRIM65 is constitutively expressed in human vascular endothelial cells.During TNFa-induced endothelial activation,the protein levels ofTRIM65 and VCAM-1 are inversely correlated.Expression of wild-type TRIM65,but not expression of aTRIM65 mutant that lacks E3 ubiquitin ligase function in endothelial cells,promotes VCAM-1 ubiquitination and degradation,whereas small interference RNA-mediated knockdown of TRIM65 attenuates VCAM-1 protein degradation.Further experiments show that TRIM65 directly interacts with VCAM-1 protein and directs its polyubiquitination,by which TRIM65 controls monocyte adherence and infiltration into tissues during inflammation.Importantly,TRIM65-deficient mice are more sensitive to lipopolysaccharide-induced death,due to sustained and severe pulmonary inflammation.Taken together,our studies suggest that TRIM65-mediated degradation of VCAM-1 represents a potential mechanism that controls the duration and magnitude of infiammation.

Non-proteolytic ubiquitination of OTULIN regulates NF-κB signaling pathway

NF-κB signaling regulates diverse processes such as cell death,inflammation,immunity,and cancer.The activity of NF-κB is controlled by methionine 1-linked linear polyubiquitin,which is assembled by the linear ubiquitin chain assembly complex(LUBAC)and the ubiquitin-conjugating enzyme UBE2L3.Recent studies found that the deubiquitinase OTULIN breaks the linear ubiquitin chain,thus inhibiting NF-κB signaling.Despite the essential role of OTULIN in NF-κB signaling has been established,the regulatory mechanism for OTULIN is not well elucidated.To discover the potential regulators of OTULIN,we analyzed the OTULIN protein complex by proteomics and revealed several OTULIN-binding proteins,including LUBAC and tripartite motif-containing protein 32(TRIM32).TRIM32 is known to activate NF-κB signaling,but the mechanism is not dear.Genetic complement experiments found that TRIM32 is upstream of OTULIN and TRIM32-mediated NF-κB activation is dependent on OTULIN.Mutagenesis of the E3 ligase domain showed that the E3 ligase activity is essential for TRIM32-mediated NF-κB activation.Further experiments found that TRIM32 conjugates polyubiquitin onto OTULIN and the polyubiquitin blocks the interaction between HOIP and OTULIN,thereby activating NF-κB signaling.Taken together,we report a novel regulatory mechanism by which TRIM32-mediated non-proteolytic ubiquitination of OTULIN impedes the access of OTULIN to the LUBAC and promotes NF-κB activation.

Transformation of plant young proembryos by electroporation

It is first reported that plant young proembryos expressed exogenous reporter genes by electroporation. Young proembryos with 8-32 cells and globular proembryos with 250-400 cells could be isolated by enzymatic maceration combined with microdissection. After electroporation with GUS or GFP genes, the proembryos were cultured for 1 -2 d in KM8p medium. At the field strength of electroporation 500-1 500 V/cm, blue reaction of GUS or green fluorescence of GFP could be observed in the proembryos. The highest transient expression frequency of young proembryos (2.2%) was obtained at the field strength of 750 V/cm, whereas the highest frequency of globular proembryos (5.9%) was obtained at the field strength of 1 250 V/cm. Taking the proportion of transformed cells in the whole cells of proembryos as efficient transformation frequency, the efficient transformation frequency of the young proembryos was 7 times that of the globular proembryos.

Enhancement of the angular rotation measurement sensitivity based on SU(2)and SU(1,1) interferometers

We investigate the sensitivity of the angular rotation measurement with the method of homodyne detection in SU(2) and SU(1,1) interferometers by employing orbital angular momentum(OAM). By combining a coherent beam with a vacuum beam in an SU(2) interferometer, we get the sensitivity of the angular rotation measurement as 1/(2N^(1/2)l). We can surpass the limit of the angular rotation measurement in an SU(1,1) interferometer by combining a coherent beam with a vacuum beam or a squeezed vacuum beam when the probe beam has OAM. Without injection, the sensitivity can reach 1/(2N^(1/2)l). In addition, by employing another construction of an SU(1,1) interferometer where the pump beam has OAM, with the same injection of an SU(1,1) interferometer, the sensitivity of the angular rotation measurement can be improved by a factor of 2, reaching 1/(4Nl). The results confirm the potential of this technology for precision measurements in angular rotation measurements.