Alpha-synuclein dynamics bridge Type-I Interferon response and SARS-CoV-2 replication in peripheral cells

BACKGROUND: Increasing evidence suggests a double-faceted role of alpha-synuclein (α-syn) following infection by a variety of viruses, including SARS-CoV-2. Although α-syn accumulation is known to contribute to cell toxicity and the development and/or exacerbation of neuropathological manifestations, it is also a key to sustaining anti-viral innate immunity. Consistently with α-syn aggregation as a hallmark of Parkinson's disease, most studies investigating the biological function of α-syn focused on neural cells, while reports on the role of α-syn in periphery are limited, especially in SARS-CoV-2 infection. RESULTS: Results herein obtained by real time qPCR, immunofluorescence and western blot indicate that α-syn upregulation in peripheral cells occurs as a Type-I Interferon (IFN)-related response against SARS-CoV-2 infection. Noteworthy, this effect mostly involves α-syn multimers, and the dynamic α-syn multimer:monomer ratio. Administration of excess α-syn monomers promoted SARS-CoV-2 replication along with downregulation of IFN-Stimulated Genes (ISGs) in epithelial lung cells, which was associated with reduced α-syn multimers and α-syn multimer:monomer ratio. These effects were prevented by combined administration of IFN-ß, which hindered virus replication and upregulated ISGs, meanwhile increasing both α-syn multimers and α-syn multimer:monomer ratio in the absence of cell toxicity. Finally, in endothelial cells displaying abortive SARS-CoV-2 replication, α-syn multimers, and multimer:monomer ratio were not reduced following exposure to the virus and exogenous α-syn, suggesting that only productive viral infection impairs α-syn multimerization and multimer:monomer equilibrium. CONCLUSIONS: Our study provides novel insights into the biology of α-syn, showing that its dynamic conformations are implicated in the innate immune response against SARS-CoV-2 infection in peripheral cells. In particular, our results suggest that promotion of non-toxic α-syn multimers likely occurs as a Type-I IFN-related biological response which partakes in the suppression of viral replication. Further studies are needed to replicate our findings in neuronal cells as well as animal models, and to ascertain the nature of such α-syn conformations.

Expression of BmSPI38 tandem multimers in Escherichia coli and its antifungal activity / 生物工程学报

The aim of this study was to prepare tandem multimeric proteins of BmSPI38, a silkworm protease inhibitor, with better structural homogeneity, higher activity and stronger antifungal ability by protein engineering. The tandem multimeric proteins of BmSPI38 were prepared by prokaryotic expression technology. The effects of tandem multimerization on the structural homogeneity, inhibitory activity and antifungal ability of BmSPI38 were explored by in-gel activity staining of protease inhibitor, protease inhibition assays and fungal growth inhibition experiments. Activity staining showed that the tandem expression based on the peptide flexible linker greatly improved the structural homogeneity of BmSPI38 protein. Protease inhibition experiments showed that the tandem trimerization and tetramerization based on the linker improved the inhibitory ability of BmSPI38 to microbial proteases. Conidial germination assays showed that His6-SPI38L-tetramer had stronger inhibition on conidial germination of Beauveria bassiana than that of His6-SPI38-monomer. Fungal growth inhibition assay showed that the inhibitory ability of BmSPI38 against Saccharomyces cerevisiae and Candida albicans could be enhanced by tandem multimerization. The present study successfully achieved the heterologous active expression of the silkworm protease inhibitor BmSPI38 in Escherichia coli, and confirmed that the structural homogeneity and antifungal ability of BmSPI38 could be enhanced by tandem multimerization. This study provides important theoretical basis and new strategies for cultivating antifungal transgenic silkworm. Moreover, it may promote the exogenous production of BmSPI38 and its application in the medical field.

The construction strategy of gene multimers and their expression modes / 医学研究生学报

The great attempts to clone a gene in the form of multimers were motivated either by the need to produce copious amounts of the particular DNA fragments or by the desire to obtain a large supply of the gene product of interest.The arrangement of the gene multimeric copies is in identical tandem orientation,this head-to-tail arrangement of gene multimers could be constructed by the strategies of tandem repeats,PCR amplification,chemical concatenation and isocaudarners.The expression mode of the gene multimers may be different based on variable construction strategy.

Subtypes of von Willebrand Disease Based on vWF Multimer Analysis in Korea / 대한소아혈액종양학회지

PURPOSE: von Willebrand disease is a common inherited bleeding disorder characterized by high degree of variable clinical presentation due to either quantitative or qualitative defects in von Willebrand factor. Its incidence in Korea is not well studied while that in western countries is extensively studied. METHODS: We classified 16 cases of vWD from 14 unrelated families based on vWF antigen, ristocetin cofactor activity, factor VIII activity and vWF multimeric patterns analysed by agarose gel electrophoresis, according to a revised classification by ISTH. RESULTS: There were 12 cases (75%) of type 1 vWD or 2M/2N with normal multimeric pattern, 3 cases (18.75%) of type 2 vWD lacking high molecular weight multimers and only 1 case of type 3 vWD with no multimers. CONCLUSION: The proportion of each vWD subtype in Korea is similar to that in western countries, however, accurate diagnosis based on ristocetin induced platelet aggregation test, factor VIII binding assay and molecular genetic diagnosis seems to be necessary for a more complete classification of vWD.

Pattern of Von Willebrand Factor Antigen and Large Von Willebrand Factor Multimer in Myeloproliferative Disorder and Reactive Thrombocytosis / 대한혈액학회지

BACKGROUND: Von Willebrand factor (vWf) plays a crucial role in the early phase of hemostasis. Acquired von Willebrand disease (vWD) due to abnormalities of vWf multimers has been reported in patients with myeloproliferative disorders (MPD) who have high platelet counts. We compared the distribution of plasma vWf antigen and large vWf multimers in samples obtained from patients with MPD and reactive thrombocytosis (RT). Furthermore, we tried to find the relationship between the decrease of large vWf multimers in plasma and the bleeding complication. METHODS: Sixteen patients with MPD and twenty-five patients with RT with more than 600x103/microliter of platelets were included in the study. The numbers of platelets and leukocytes, platelet distribution width (PDW), mean platelet volume (MPV), platelet-large cell ratio (P-LCR), vWf : Ag and vWf multimers were measured. RESULTS: The mean values of platelets and leukocytes were 1,091x103/microliter, 82.9x103/ in MPD and 763x103/, 11.4x103/ in RT (P<0.05). Platelet parameters such as MPV,PDW, P-LCR were 11.2 fL, 10.2%, 28.6% in MPD, and 9.6 fL, 9.9%, 19.6% in RT (P<0.05). The levels of vWf : Ag and large vWf multimer were 120.8 U/dL, 13.8% in MPD and 184.3 U/ dL, 20.7% in RT (P<0.05). Large vWf multimers were decreased in 11 MPDs and 2 RTs. Bleeding diathesis appeared only in 2 MPDs with decreased large vWf multimers. Platelet or leukocyte count was inversely correlated with large vWf multimers, but vWf antigen was correlated with large vWf multimer. CONCLUSION: The findings of normal vWf antigen level and decreased percentage of large vWf multimers are more frequent in MPD than in RT, and the measurement of these parameters is useful to differentiate MPD from RT. Bleeding complication in the patients with MPD with decreased large vWf multimers might be prevented by correction of decreased large vWf multimers.