规律间隔成簇短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白9(CRISPR-associated protein 9,Cas9)主要元件Cas9蛋白一般采用大肠杆菌表达,但在表达纯化过程中易出现形成包涵体形...规律间隔成簇短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白9(CRISPR-associated protein 9,Cas9)主要元件Cas9蛋白一般采用大肠杆菌表达,但在表达纯化过程中易出现形成包涵体形式的不溶解性蛋白,内毒素含量高、蛋白过大导致蛋白折叠不正确、产量低等问题。为实现化脓性链球菌(Streptococcus pyogenes)Cas9蛋白(SpCas9)在大肠杆菌中的高效可溶表达,进一步促进其应用及编辑技术的推广,本研究应用GB1促溶标签提高Cas9蛋白表达量及溶解度,同时通过使用多重启动子策略进一步提高了Cas9蛋白表达量。两种策略的组合使Cas9蛋白表达量提升了2.52倍。体外酶切分析显示融合GB1标签的Cas9蛋白功能活性不受影响。进一步组装RNP复合物转化黑曲霉宿主成功破坏了pyrG基因。展开更多
Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening pa...Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins(RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3' untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of m RNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.展开更多
Influenza is an acute respiratory infectious disease caused by the influenza virus,affecting people globally and causing significant social and economic losses.Due to the inevitable limitations of vaccines and approve...Influenza is an acute respiratory infectious disease caused by the influenza virus,affecting people globally and causing significant social and economic losses.Due to the inevitable limitations of vaccines and approved drugs,there is an urgent need to discover new anti-influenza drugs with different mechanisms.The viral ribonucleoprotein complex(vRNP)plays an essential role in the life cycle of influenza viruses,representing an attractive target for drug design.In recent years,the functional area of constituent proteins in vRNP are widely used as targets for drug discovery,especially the PA endonuclease active site,the RNA-binding site of PB1,the cap-binding site of PB2 and the nuclear export signal of NP protein.Encouragingly,the PA inhibitor baloxavir has been marketed in Japan and the United States,and several drug candidates have also entered clinical trials,such as favipiravir.This article reviews the compositions and functions of the influenza virus vRNP and the research progress on vRNP inhibitors,and discusses the representative drug discovery and optimization strategies pursued.展开更多
Influenza Avirus is one of the major pathogens that pose a large threat to human health worldwide and has caused pandemics.Influenza A virus is the Orthomyxoviridae prototype,and has 8 segmented negative-sense singles...Influenza Avirus is one of the major pathogens that pose a large threat to human health worldwide and has caused pandemics.Influenza A virus is the Orthomyxoviridae prototype,and has 8 segmented negative-sense singlestranded RNA(vRNA)as its genome.Influenza virus RNA polymerase(RdRp)consists of three subunits PB2,PB1 and PA,and catalyzes both transcription and replication.Recently,intensive biochemical and structural analysis of its RdRp has been performed.In this paper,we review the details from the biochemical analysis of the purified influenza virus RdRp and the classical ribonucleoprotein complex,as well as piece together their structures to form an overall picture.展开更多
文摘规律间隔成簇短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/CRISPR相关蛋白9(CRISPR-associated protein 9,Cas9)主要元件Cas9蛋白一般采用大肠杆菌表达,但在表达纯化过程中易出现形成包涵体形式的不溶解性蛋白,内毒素含量高、蛋白过大导致蛋白折叠不正确、产量低等问题。为实现化脓性链球菌(Streptococcus pyogenes)Cas9蛋白(SpCas9)在大肠杆菌中的高效可溶表达,进一步促进其应用及编辑技术的推广,本研究应用GB1促溶标签提高Cas9蛋白表达量及溶解度,同时通过使用多重启动子策略进一步提高了Cas9蛋白表达量。两种策略的组合使Cas9蛋白表达量提升了2.52倍。体外酶切分析显示融合GB1标签的Cas9蛋白功能活性不受影响。进一步组装RNP复合物转化黑曲霉宿主成功破坏了pyrG基因。
基金Supported by The Agencia Nacional de Promoción Científica y Tecnológica(ANPCyT)to Alejandro Cassola
文摘Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins(RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3' untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of m RNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.
基金Financial support from the National Natural Science Foundation of China (No.81773574, China)Science Foundation for Outstanding Young Scholars of Shandong Province (ZR2020JQ31, China)+9 种基金Foreign cultural and educational experts Project (GXL20200015001, China)Qilu Young Scholars Program of Shandong Universitythe Taishan Scholar Program at Shandong ProvinceShandong Province Key Research and Development Project (No.2019JZZY021011, China)Shandong Modern Agricultural Technology & Industry System (SDAIT-21-06 and SDAIT11-01)Agricultural scientific and technological innovation project of Shandong Academy of Agricultural Sciences (CXGC2021A12, China)Associazione Italiana per la Ricerca sul Cancro, AIRC, grant No.IG18855 (to Arianna Loregian, Italy)British Society for Antimicrobial Chemotherapy, UK, BSAC-2018-0064 (to Arianna Loregian, Italy)Ministero dell’Istruzione, dell’Università e della Ricerca, PRIN 2017-cod.2017KM79NN (to Arianna Loregian, Italy)Fondazione Cassa di Risparmio di Padova e Rovigo-Bando Ricerca COVID-2019 Nr.55777 2020.0162 (to Arianna Loregian, Italy)
文摘Influenza is an acute respiratory infectious disease caused by the influenza virus,affecting people globally and causing significant social and economic losses.Due to the inevitable limitations of vaccines and approved drugs,there is an urgent need to discover new anti-influenza drugs with different mechanisms.The viral ribonucleoprotein complex(vRNP)plays an essential role in the life cycle of influenza viruses,representing an attractive target for drug design.In recent years,the functional area of constituent proteins in vRNP are widely used as targets for drug discovery,especially the PA endonuclease active site,the RNA-binding site of PB1,the cap-binding site of PB2 and the nuclear export signal of NP protein.Encouragingly,the PA inhibitor baloxavir has been marketed in Japan and the United States,and several drug candidates have also entered clinical trials,such as favipiravir.This article reviews the compositions and functions of the influenza virus vRNP and the research progress on vRNP inhibitors,and discusses the representative drug discovery and optimization strategies pursued.
文摘Influenza Avirus is one of the major pathogens that pose a large threat to human health worldwide and has caused pandemics.Influenza A virus is the Orthomyxoviridae prototype,and has 8 segmented negative-sense singlestranded RNA(vRNA)as its genome.Influenza virus RNA polymerase(RdRp)consists of three subunits PB2,PB1 and PA,and catalyzes both transcription and replication.Recently,intensive biochemical and structural analysis of its RdRp has been performed.In this paper,we review the details from the biochemical analysis of the purified influenza virus RdRp and the classical ribonucleoprotein complex,as well as piece together their structures to form an overall picture.
