In analyses of protein families that may serve as drug targets,membrane-associated G-protein-coupled receptors(GPCRs)dominate,followed by ion channels,transporters,and—to a lesser extent—membrane-bound enzymes.Howev...In analyses of protein families that may serve as drug targets,membrane-associated G-protein-coupled receptors(GPCRs)dominate,followed by ion channels,transporters,and—to a lesser extent—membrane-bound enzymes.However,various challenges put such membrane proteins among key groups of underutilized opportunities for the application of therapeutic antibodies.Antibodies hold the promise of exquisite specificity,as they are able to target even specific conformations of a particular membrane protein,as well as adaptability through engineering into various antibody formats.However,the ease of raising and isolating specific,effective antibodies targeting membrane proteins depends on many factors.In particular,the generation of specific antibodies is easier when targeting larger,simpler,extracellular domains with greater uniqueness of amino acid sequence.The rareness of such ideal conditions is illustrated by the limited number of approved biologics for targeting GPCRs and other complex membrane proteins.Challenges in developing antibodies to complex membrane proteins such as GPCRs,ion channels,transporters,and membrane-bound enzymes can be addressed by the design of the antigen,antibody-generation strategies,lead optimization technologies,and antibody modalities.A better understanding of the membrane proteins being targeted would facilitate mechanism-based drug discovery.This review describes the advantages and challenges of targeting complex membrane proteins with antibodies and discusses the preparation of membrane protein antigens and antibody generation,illustrated by select examples of success.展开更多
The endoplasmic reticulum(ER)membrane protein complex(EMC)is responsible for monitoring the biogenesis and synthetic quality of membrane proteins with tail-anchored or multiple transmembrane domains.The EMC subunit EM...The endoplasmic reticulum(ER)membrane protein complex(EMC)is responsible for monitoring the biogenesis and synthetic quality of membrane proteins with tail-anchored or multiple transmembrane domains.The EMC subunit EMC6 is one of the core members of EMC and forms an enclosed hydrophilic vestibule in cooperation with EMC3.Despite studies demonstrating that deletion of EMC3 led to rhodopsin mislocalization in rod photoreceptors of mice,the precise mechanism leading to the failure of rhodopsin trafficking remains unclear.Here,we generated the first rod photoreceptor-specific knockout of Emc6(RKO)and cone photoreceptor-specific knockout of Emc6(CKO)mouse models.Deficiency of Emc6 in rod photoreceptors led to progressive shortening of outer segments(OS),impaired visual function,mislocalization and reduced expression of rhodopsin,and increased gliosis in rod photoreceptors.In addition,CKO mice displayed the progressive death of cone photoreceptors and abnormal localization of cone opsin protein.Subsequently,proteomics analysis of the RKO mouse retina illustrated that several cilium-related proteins,particularly anoctamin-2(ANO2)and transmembrane protein 67(TMEM67),were significantly down-regulated prior to OS degeneration.Detrimental rod photoreceptor cilia and mislocalized membrane disc proteins were evident in RKO mice.Our data revealed that in addition to monitoring the synthesis of rhodopsin-dominated membrane disc proteins,EMC6 also impacted rod photoreceptors'ciliogenesis by regulating the synthesis of membrane proteins associated with cilia,contributing to the mislocalization of membrane disc proteins.展开更多
The endoplasmic reticulum(ER) membrane protein complex(EMC) regulates the synthesis and quality control of membrane proteins with multiple transmembrane domains. One of the membrane spanning subunits, EMC3, is a core ...The endoplasmic reticulum(ER) membrane protein complex(EMC) regulates the synthesis and quality control of membrane proteins with multiple transmembrane domains. One of the membrane spanning subunits, EMC3, is a core member of the EMC complex that provides essential hydrophilic vestibule for substrate insertion. Here, we show that the EMC subunit Emc3 plays critical roles in the retinal vascular angiogenesis by regulating Norrin/Wnt signaling. Postnatal endothelial cell(EC)-specific deletion of Emc3 led to retarded retinal vascular development with a hyperpruned vascular network, the appearance of bluntended, aneurysm-like tip endothelial cells(ECs) with reduced numbers of filopodia and leakage of erythrocytes at the vascular front. Diminished tube formation and cell proliferation were also observed in EMC3 depleted human retinal endothelial cells(HRECs). We then discovered a critical role for EMC3 in expression of FZD4 receptor of β-catenin signaling using RNA sequencing, real-time quantitative PCR(RT-q PCR) and luciferase reporter assay. Moreover, augmentation of Wnt activity via lithium chloride(Li Cl) treatment remarkably enhanced β-catenin signaling and cell proliferation of HRECs. Additionally, Li Cl partially reversed the angiogenesis defects in Emc3-c KO mice. Our data reveal that Emc3 plays essential roles in angiogenesis through direct control of FZD4 expression and Norrin/β-catenin signaling.展开更多
基金This work was partly supported by the Cancer Prevention and Research Institute of Texas,USA(PR150551 and RP190561)the Welch Foundation(AU-0042-20030616)+1 种基金The work was also supported by the National Natural Science Foundation of China(31700778 and 31320103918)Jiangsu Province’s Key Laboratory of Medicine(XK201135).
文摘In analyses of protein families that may serve as drug targets,membrane-associated G-protein-coupled receptors(GPCRs)dominate,followed by ion channels,transporters,and—to a lesser extent—membrane-bound enzymes.However,various challenges put such membrane proteins among key groups of underutilized opportunities for the application of therapeutic antibodies.Antibodies hold the promise of exquisite specificity,as they are able to target even specific conformations of a particular membrane protein,as well as adaptability through engineering into various antibody formats.However,the ease of raising and isolating specific,effective antibodies targeting membrane proteins depends on many factors.In particular,the generation of specific antibodies is easier when targeting larger,simpler,extracellular domains with greater uniqueness of amino acid sequence.The rareness of such ideal conditions is illustrated by the limited number of approved biologics for targeting GPCRs and other complex membrane proteins.Challenges in developing antibodies to complex membrane proteins such as GPCRs,ion channels,transporters,and membrane-bound enzymes can be addressed by the design of the antigen,antibody-generation strategies,lead optimization technologies,and antibody modalities.A better understanding of the membrane proteins being targeted would facilitate mechanism-based drug discovery.This review describes the advantages and challenges of targeting complex membrane proteins with antibodies and discusses the preparation of membrane protein antigens and antibody generation,illustrated by select examples of success.
基金supported by The National Natural Science Foundation of China(No.82121003,81970841,82101160)the program of Science and Technology International Cooperation Project of Qinghai province(China)(No.2022-HZ-814)+2 种基金the CAMS Innovation Fund for Medical Sciences(No.2019-12M-5-032)Sichuan Intellectual Property Office(China)(No.2022-ZS-0070)the Department of Chengdu Science and Technology(Sichuan,China)(No.2021-YF05-01316-SN).
文摘The endoplasmic reticulum(ER)membrane protein complex(EMC)is responsible for monitoring the biogenesis and synthetic quality of membrane proteins with tail-anchored or multiple transmembrane domains.The EMC subunit EMC6 is one of the core members of EMC and forms an enclosed hydrophilic vestibule in cooperation with EMC3.Despite studies demonstrating that deletion of EMC3 led to rhodopsin mislocalization in rod photoreceptors of mice,the precise mechanism leading to the failure of rhodopsin trafficking remains unclear.Here,we generated the first rod photoreceptor-specific knockout of Emc6(RKO)and cone photoreceptor-specific knockout of Emc6(CKO)mouse models.Deficiency of Emc6 in rod photoreceptors led to progressive shortening of outer segments(OS),impaired visual function,mislocalization and reduced expression of rhodopsin,and increased gliosis in rod photoreceptors.In addition,CKO mice displayed the progressive death of cone photoreceptors and abnormal localization of cone opsin protein.Subsequently,proteomics analysis of the RKO mouse retina illustrated that several cilium-related proteins,particularly anoctamin-2(ANO2)and transmembrane protein 67(TMEM67),were significantly down-regulated prior to OS degeneration.Detrimental rod photoreceptor cilia and mislocalized membrane disc proteins were evident in RKO mice.Our data revealed that in addition to monitoring the synthesis of rhodopsin-dominated membrane disc proteins,EMC6 also impacted rod photoreceptors'ciliogenesis by regulating the synthesis of membrane proteins associated with cilia,contributing to the mislocalization of membrane disc proteins.
基金This work was supported by the National Natural Science Foundation of China(81970841,81770950,81790643,82000913,and 82071009)the Department of Science and Technology of Sichuan Province(2020JDZH0027 and 2020ZYD037)+2 种基金the CAMS Innovation Fund for Medical Sciences(2019-12M-5-032)the fund for Sichuan Provincial People’s Hospital(2021QN01)the Chengdu Science and Technology Bureau(2019-YF05-00572-SN).
文摘The endoplasmic reticulum(ER) membrane protein complex(EMC) regulates the synthesis and quality control of membrane proteins with multiple transmembrane domains. One of the membrane spanning subunits, EMC3, is a core member of the EMC complex that provides essential hydrophilic vestibule for substrate insertion. Here, we show that the EMC subunit Emc3 plays critical roles in the retinal vascular angiogenesis by regulating Norrin/Wnt signaling. Postnatal endothelial cell(EC)-specific deletion of Emc3 led to retarded retinal vascular development with a hyperpruned vascular network, the appearance of bluntended, aneurysm-like tip endothelial cells(ECs) with reduced numbers of filopodia and leakage of erythrocytes at the vascular front. Diminished tube formation and cell proliferation were also observed in EMC3 depleted human retinal endothelial cells(HRECs). We then discovered a critical role for EMC3 in expression of FZD4 receptor of β-catenin signaling using RNA sequencing, real-time quantitative PCR(RT-q PCR) and luciferase reporter assay. Moreover, augmentation of Wnt activity via lithium chloride(Li Cl) treatment remarkably enhanced β-catenin signaling and cell proliferation of HRECs. Additionally, Li Cl partially reversed the angiogenesis defects in Emc3-c KO mice. Our data reveal that Emc3 plays essential roles in angiogenesis through direct control of FZD4 expression and Norrin/β-catenin signaling.
