Proper cell-cell and cell-matrix contacts mediated by integrin adhesion receptors are important for development, immune response, hemostasis and wound healing. Integrins pass trans-membrane signals bidirectionally thr...Proper cell-cell and cell-matrix contacts mediated by integrin adhesion receptors are important for development, immune response, hemostasis and wound healing. Integrins pass trans-membrane signals bidirectionally through their regulated affinities for extracellular ligands and intracellular signaling molecules. Such bidirectional signaling by integrins is enabled by the conformational changes that are often linked among extracellular, transmembrane and cytoplasmic domains. Here, we review how talin-integrin and kindlin-integrin interactions, in cooperation with talin-lipid and kindlin-lipid interactions, regulate integrin affinities and how the progress in these areas helps us understand integrin-related diseases.展开更多
Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme （TACE）, which causes the release of their ectodomains. An ADAM （a disintegrin and metalloprotease domain） family member, TACE co...Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme （TACE）, which causes the release of their ectodomains. An ADAM （a disintegrin and metalloprotease domain） family member, TACE contains several noncatalytic domains whose roles in ectodomain shedding have yet to be fully resolved. Here, we have explored the function of the transmembrane domain （TM） of TACE by coupling molecular engineering and functional analysis. A TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylinositol （GPI）-binding polypeptide failed to restore shedding of transforming growth factor-or （TGF-α）, tumor necrosis factor-α （TNF-α） and L-selectin in cells lacking endogenous TACE activity. Substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor （PDGFR） also resulted in severe loss of TGF-α shedding, but had no effects on the cleavage of TNF-α and L-selectin. Replacement of the TM in TGF-α with that of L-selectin enabled TGF-α shedding by the TACE mutants carrying the TM of prolactin receptor and PDGFR. Taken together, our observations suggest that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates.展开更多
目的对1株在九江地区分离的柯萨奇A6病毒(CoxA6)VP1区域进行克隆,并对其编码蛋白的结构、功能及B细胞表位进行分析和预测,为CoxA6疫苗制备和诊断方法的研究提供理论基础。方法采用RT-PCR法对CoxA6分离株VP1区进行扩增和克隆、序列分析,...目的对1株在九江地区分离的柯萨奇A6病毒(CoxA6)VP1区域进行克隆,并对其编码蛋白的结构、功能及B细胞表位进行分析和预测,为CoxA6疫苗制备和诊断方法的研究提供理论基础。方法采用RT-PCR法对CoxA6分离株VP1区进行扩增和克隆、序列分析,应用SigaIP、TMPRED、TMHMM、Big-PI Predictor、Cell-Ploc、PSORT、NetNES、Netphos、SOPMA、NetNGlyc、MotifScan、InterProscan、SMART、PROSITE、GOR4、Bepipred Linear Epitope Prediction等生物信息学方法预测其VP1基因编码蛋白特性和潜在的B细胞表位。结果该CoxA6分离株VP1基因编码305aa的多肽,分子量为33.5kDa。该蛋白无信号肽、跨膜区,是亲水性蛋白;二级结构主要以无规则卷曲为主,其次为α-螺旋及β-片层。该蛋白共有7个可能的B细胞抗原表位,位于151~173aa区域内的表位分值最高为2.774。结论成功克隆了1株CoxA6的VP1基因,并对其进行序列分析、蛋白质结构和B细胞表位预测,为制备CoxA6疫苗和开发诊断方法提供了分子生物学基础。展开更多
【目的】由于H7N9禽流感病毒能够感染鸡,并且已经变异成了高致病性毒株,因此,鸡群中H7N9禽流感疫苗的免疫是一个趋势,而鸡群免疫后抗体检测方法的建立也十分必要。本研究旨在建立一种灵敏、高效、高通量的鸡群H7N9亚型禽流感病毒抗体间...【目的】由于H7N9禽流感病毒能够感染鸡,并且已经变异成了高致病性毒株,因此,鸡群中H7N9禽流感疫苗的免疫是一个趋势,而鸡群免疫后抗体检测方法的建立也十分必要。本研究旨在建立一种灵敏、高效、高通量的鸡群H7N9亚型禽流感病毒抗体间接酶联免疫吸附试验(ELISA)检测方法。【方法】通过昆虫杆状病毒表达系统分别表达属于W1、W2-A和W2-B分支H7N9流感病毒的3种野生型血凝素(HA)蛋白,以及跨膜区(TM)置换为H3 HA TM的W2-B分支HA蛋白(H7-53TM)。4种HA蛋白经过离子交换层析纯化后作为抗原,通过ELISA检测H7N9禽流感病毒抗体。【结果】ELISA特异性、敏感性和重复性试验结果显示,跨膜区置换主要影响HA蛋白ELISA检测的重复性,以H7-53TM为抗原的ELISA方法具有较好的重复性,其批内和批间变异系数小于10%,然而3种野生型HA蛋白与部分血清反应批内和批间变异系数大于10%,重复性较差,因此选择H7-53TM蛋白作为ELISA包被抗原。通过受试者工作特征曲线(ROC曲线)分析,以H7-53TM为抗原的ELISA能够精准地区分H7N9亚型流感病毒抗体阳性和阴性血清。通过相关性分析,该ELISA方法与134份鸡血清HI试验结果具有显著强相关性(r=0.854 6,P<0.000 1),并且与3个分支疫苗株免疫血清的HI试验结果也具有显著相关性(r>0.5,P<0.05)。【结论】跨膜区置换能够提高HA蛋白抗原检测H7N9禽流感病毒抗体的重复性,并应用跨膜区置换的HA蛋白建立了一种能够检测不同分支疫苗株免疫的H7N9亚型禽流感病毒抗体间接ELISA检测方法。展开更多
Protein biogenesis is a complex process,and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles.To direct targeting,organellar proteins carry an organelle...Protein biogenesis is a complex process,and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles.To direct targeting,organellar proteins carry an organelle-specific targeting signal for recognition by organelle-specific import machinery.However,the situation is confusing for transmembrane domain(TMD)-containing signalanchored(SA)proteins of various organelles because TMDs function as an endoplasmic reticulum(ER)targeting signal.Although ER targeting of SA proteins is well understood,how they are targeted to mitochondria and chloroplasts remains elusive.Here,we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts.Mitochondrial targeting requires multiple motifs around and within TMDs:a basic residue and an arginine-rich region flanking the N-and C-termini of TMDs,respectively,and an aromatic residue in the C-terminal side of the TMD that specify mitochondrial targeting in an additive manner.These motifs play a role in slowing down the elongation speed during translation,thereby ensuring mitochondrial targeting in a cotranslational manner.By contrast,the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a post-translational manner.展开更多
Genetics plays an important role in determining the susceptibility of an individual to develop a disease. Complex, multi factorial diseases of modern day(diabetes, cardiovascular disease, hypertension and obesity) are...Genetics plays an important role in determining the susceptibility of an individual to develop a disease. Complex, multi factorial diseases of modern day(diabetes, cardiovascular disease, hypertension and obesity) are a result of disparity between the type of food consumed and genes, suggesting that food which does not match the host genes is probably one of the major reasons for developing life style diseases. Non-alcoholic fatty liver is becoming a global epidemic leading to substantial morbidity. While various genotyping approaches such as whole exome sequencing using next generation sequencers and genome wide association studies have identified susceptibility loci for non-alcoholic fatty liver disease(NAFLD) including variants in patatin-like phospholipase domain containing 3 and transmembrane 6 superfamily member 2 genes apart from others; nutrient based studies emphasized on a combination of vitamin D, E and omega-3 fatty acids to manage fatty liver disease. However majority of the studies were conducted independent of each other and very few studies explored the interactions between the genetic susceptibility and nutrient interactions. Identifying such interactions will aid in optimizing the nutrition tailor made to an individual's genetic makeup, thereby aiding in delaying the onset of the disease and its progression. The present topic focuses on studies that identified the genetic susceptibility for NAFLD, nutritional recommendations, and their interactions for better management of NAFLD.展开更多
Cytochrome P450(CYP)enzymes play crucial roles during the evolution and diversification of ancestral monocel-lular eukaryotes into multicellular eukaryotic organisms due to their essential functionalities including ca...Cytochrome P450(CYP)enzymes play crucial roles during the evolution and diversification of ancestral monocel-lular eukaryotes into multicellular eukaryotic organisms due to their essential functionalities including catalysis of housekeeping biochemical reactions,synthesis of diverse metabolites,detoxification of xenobiotics,and con-tribution to environmental adaptation.Eukaryotic CYPs with versatile functionalities are undeniably regarded as promising biocatalysts with great potential for biotechnological,pharmaceutical and chemical industry applica-tions.Nevertheless,the modes of action and the challenges associated with these membrane-bound proteins have hampered the effective utilization of eukaryotic CYPs in a broader range.This review is focused on comprehen-sive and consolidated approaches to address the core challenges in heterologous expression of membrane-bound eukaryotic CYPs in different surrogate microbial cell factories,aiming to provide key insights for better studies and applications of diverse eukaryotic CYPs in the future.We also highlight the functional significance of the previously underrated cytochrome P450 reductases(CPRs)and provide a rational justification on the progression of CPR from auxiliary redox partner to function modulator in CYP catalysis.展开更多
Interferon-inducible transmembrane protein 3(IFITM3)inhibits influenza virus infection by blocking viral membrane fusion,but the exact mechanism remains elusive.Here,we investigated the function and key region of IFIT...Interferon-inducible transmembrane protein 3(IFITM3)inhibits influenza virus infection by blocking viral membrane fusion,but the exact mechanism remains elusive.Here,we investigated the function and key region of IFITM3 in blocking influenza virus entry mediated by hemagglutinin(HA).The restriction of IFITM3 on HAmediated viral entry was confirmed by pseudovirus harboring HA protein from H5 and H7 influenza viruses.Subcellular co-localization and immunocoprecipitation analyses revealed that IFITM3 partially co-located with the full-length HA protein and could directly interact with HA_(2) subunit but not HA_(1) subunit of H5 and H7 virus.Truncated analyses showed that the transmembrane domain of the IFITM3 and HA_(2) subunit might play an important role in their interaction.Finally,this interaction of IFITM3 was also verified with HA_(2) subunits from other subtypes of influenza A virus and influenza B virus.Overall,our data demonstrate for the first time a direct interaction between IFITM3 and influenza HA protein via the transmembrane domain,providing a new perspective for further exploring the biological significance of IFITM3 restriction on influenza virus infection or HA-mediated antagonism or escape.展开更多
文摘Proper cell-cell and cell-matrix contacts mediated by integrin adhesion receptors are important for development, immune response, hemostasis and wound healing. Integrins pass trans-membrane signals bidirectionally through their regulated affinities for extracellular ligands and intracellular signaling molecules. Such bidirectional signaling by integrins is enabled by the conformational changes that are often linked among extracellular, transmembrane and cytoplasmic domains. Here, we review how talin-integrin and kindlin-integrin interactions, in cooperation with talin-lipid and kindlin-lipid interactions, regulate integrin affinities and how the progress in these areas helps us understand integrin-related diseases.
文摘Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme （TACE）, which causes the release of their ectodomains. An ADAM （a disintegrin and metalloprotease domain） family member, TACE contains several noncatalytic domains whose roles in ectodomain shedding have yet to be fully resolved. Here, we have explored the function of the transmembrane domain （TM） of TACE by coupling molecular engineering and functional analysis. A TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylinositol （GPI）-binding polypeptide failed to restore shedding of transforming growth factor-or （TGF-α）, tumor necrosis factor-α （TNF-α） and L-selectin in cells lacking endogenous TACE activity. Substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor （PDGFR） also resulted in severe loss of TGF-α shedding, but had no effects on the cleavage of TNF-α and L-selectin. Replacement of the TM in TGF-α with that of L-selectin enabled TGF-α shedding by the TACE mutants carrying the TM of prolactin receptor and PDGFR. Taken together, our observations suggest that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates.
文摘目的对1株在九江地区分离的柯萨奇A6病毒(CoxA6)VP1区域进行克隆,并对其编码蛋白的结构、功能及B细胞表位进行分析和预测,为CoxA6疫苗制备和诊断方法的研究提供理论基础。方法采用RT-PCR法对CoxA6分离株VP1区进行扩增和克隆、序列分析,应用SigaIP、TMPRED、TMHMM、Big-PI Predictor、Cell-Ploc、PSORT、NetNES、Netphos、SOPMA、NetNGlyc、MotifScan、InterProscan、SMART、PROSITE、GOR4、Bepipred Linear Epitope Prediction等生物信息学方法预测其VP1基因编码蛋白特性和潜在的B细胞表位。结果该CoxA6分离株VP1基因编码305aa的多肽,分子量为33.5kDa。该蛋白无信号肽、跨膜区,是亲水性蛋白;二级结构主要以无规则卷曲为主,其次为α-螺旋及β-片层。该蛋白共有7个可能的B细胞抗原表位,位于151~173aa区域内的表位分值最高为2.774。结论成功克隆了1株CoxA6的VP1基因,并对其进行序列分析、蛋白质结构和B细胞表位预测,为制备CoxA6疫苗和开发诊断方法提供了分子生物学基础。
文摘【目的】由于H7N9禽流感病毒能够感染鸡,并且已经变异成了高致病性毒株,因此,鸡群中H7N9禽流感疫苗的免疫是一个趋势,而鸡群免疫后抗体检测方法的建立也十分必要。本研究旨在建立一种灵敏、高效、高通量的鸡群H7N9亚型禽流感病毒抗体间接酶联免疫吸附试验(ELISA)检测方法。【方法】通过昆虫杆状病毒表达系统分别表达属于W1、W2-A和W2-B分支H7N9流感病毒的3种野生型血凝素(HA)蛋白,以及跨膜区(TM)置换为H3 HA TM的W2-B分支HA蛋白(H7-53TM)。4种HA蛋白经过离子交换层析纯化后作为抗原,通过ELISA检测H7N9禽流感病毒抗体。【结果】ELISA特异性、敏感性和重复性试验结果显示,跨膜区置换主要影响HA蛋白ELISA检测的重复性,以H7-53TM为抗原的ELISA方法具有较好的重复性,其批内和批间变异系数小于10%,然而3种野生型HA蛋白与部分血清反应批内和批间变异系数大于10%,重复性较差,因此选择H7-53TM蛋白作为ELISA包被抗原。通过受试者工作特征曲线(ROC曲线)分析,以H7-53TM为抗原的ELISA能够精准地区分H7N9亚型流感病毒抗体阳性和阴性血清。通过相关性分析,该ELISA方法与134份鸡血清HI试验结果具有显著强相关性(r=0.854 6,P<0.000 1),并且与3个分支疫苗株免疫血清的HI试验结果也具有显著相关性(r>0.5,P<0.05)。【结论】跨膜区置换能够提高HA蛋白抗原检测H7N9禽流感病毒抗体的重复性,并应用跨膜区置换的HA蛋白建立了一种能够检测不同分支疫苗株免疫的H7N9亚型禽流感病毒抗体间接ELISA检测方法。
基金supported by the National Research Foundation of Korea(NRF)grant funded by the Korea government(MSIT)(No.2022R1A2C1091844)。
文摘Protein biogenesis is a complex process,and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles.To direct targeting,organellar proteins carry an organelle-specific targeting signal for recognition by organelle-specific import machinery.However,the situation is confusing for transmembrane domain(TMD)-containing signalanchored(SA)proteins of various organelles because TMDs function as an endoplasmic reticulum(ER)targeting signal.Although ER targeting of SA proteins is well understood,how they are targeted to mitochondria and chloroplasts remains elusive.Here,we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts.Mitochondrial targeting requires multiple motifs around and within TMDs:a basic residue and an arginine-rich region flanking the N-and C-termini of TMDs,respectively,and an aromatic residue in the C-terminal side of the TMD that specify mitochondrial targeting in an additive manner.These motifs play a role in slowing down the elongation speed during translation,thereby ensuring mitochondrial targeting in a cotranslational manner.By contrast,the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a post-translational manner.
文摘Genetics plays an important role in determining the susceptibility of an individual to develop a disease. Complex, multi factorial diseases of modern day(diabetes, cardiovascular disease, hypertension and obesity) are a result of disparity between the type of food consumed and genes, suggesting that food which does not match the host genes is probably one of the major reasons for developing life style diseases. Non-alcoholic fatty liver is becoming a global epidemic leading to substantial morbidity. While various genotyping approaches such as whole exome sequencing using next generation sequencers and genome wide association studies have identified susceptibility loci for non-alcoholic fatty liver disease(NAFLD) including variants in patatin-like phospholipase domain containing 3 and transmembrane 6 superfamily member 2 genes apart from others; nutrient based studies emphasized on a combination of vitamin D, E and omega-3 fatty acids to manage fatty liver disease. However majority of the studies were conducted independent of each other and very few studies explored the interactions between the genetic susceptibility and nutrient interactions. Identifying such interactions will aid in optimizing the nutrition tailor made to an individual's genetic makeup, thereby aiding in delaying the onset of the disease and its progression. The present topic focuses on studies that identified the genetic susceptibility for NAFLD, nutritional recommendations, and their interactions for better management of NAFLD.
基金supported by the National Key Research and Develop-ment Program of China(2019YFA0706900)the National Natural Sci-ence Foundation of China(32025001 and 21472204)the Shandong Provincial Natural Science Foundation(ZR2019ZD20).
文摘Cytochrome P450(CYP)enzymes play crucial roles during the evolution and diversification of ancestral monocel-lular eukaryotes into multicellular eukaryotic organisms due to their essential functionalities including catalysis of housekeeping biochemical reactions,synthesis of diverse metabolites,detoxification of xenobiotics,and con-tribution to environmental adaptation.Eukaryotic CYPs with versatile functionalities are undeniably regarded as promising biocatalysts with great potential for biotechnological,pharmaceutical and chemical industry applica-tions.Nevertheless,the modes of action and the challenges associated with these membrane-bound proteins have hampered the effective utilization of eukaryotic CYPs in a broader range.This review is focused on comprehen-sive and consolidated approaches to address the core challenges in heterologous expression of membrane-bound eukaryotic CYPs in different surrogate microbial cell factories,aiming to provide key insights for better studies and applications of diverse eukaryotic CYPs in the future.We also highlight the functional significance of the previously underrated cytochrome P450 reductases(CPRs)and provide a rational justification on the progression of CPR from auxiliary redox partner to function modulator in CYP catalysis.
基金supported by the National Natural Science Foundation of China (31702210, 31972719)the CAMS Innovation Fund for Medical Sciences (2020–12M-5-001)
文摘Interferon-inducible transmembrane protein 3(IFITM3)inhibits influenza virus infection by blocking viral membrane fusion,but the exact mechanism remains elusive.Here,we investigated the function and key region of IFITM3 in blocking influenza virus entry mediated by hemagglutinin(HA).The restriction of IFITM3 on HAmediated viral entry was confirmed by pseudovirus harboring HA protein from H5 and H7 influenza viruses.Subcellular co-localization and immunocoprecipitation analyses revealed that IFITM3 partially co-located with the full-length HA protein and could directly interact with HA_(2) subunit but not HA_(1) subunit of H5 and H7 virus.Truncated analyses showed that the transmembrane domain of the IFITM3 and HA_(2) subunit might play an important role in their interaction.Finally,this interaction of IFITM3 was also verified with HA_(2) subunits from other subtypes of influenza A virus and influenza B virus.Overall,our data demonstrate for the first time a direct interaction between IFITM3 and influenza HA protein via the transmembrane domain,providing a new perspective for further exploring the biological significance of IFITM3 restriction on influenza virus infection or HA-mediated antagonism or escape.
