Objective Junctophilin-2(JPH2)is an essential structural protein that maintains junctional membrane complexes(JMCs)in cardiomyocytes by tethering the plasma membrane to the sarcoplasmic reticulum,thereby facilitating ...Objective Junctophilin-2(JPH2)is an essential structural protein that maintains junctional membrane complexes(JMCs)in cardiomyocytes by tethering the plasma membrane to the sarcoplasmic reticulum,thereby facilitating excitationcontraction(E-C)coupling.Mutations in JPH2 have been associated with hypertrophic cardiomyopathy(HCM),but the molecular mechanisms governing its membrane-binding properties and the functional relevance of its membrane occupation and recognition nexus(MORN)repeat motifs remain incompletely understood.This study aimed to elucidate the structural basis of JPH2 membrane association and its implications for HCM pathogenesis.Methods A recombinant N-terminal fragment of mouse JPH2(residues 1-440),encompassing the MORN repeats and an adjacent helical region,was purified under near-physiological buffer conditions.X-ray crystallography was employed to determine the structure of the JPH2 MORN-Helix domain.Sequence conservation analysis across species and junctophilin isoforms was performed to assess the evolutionary conservation of key structural features.Functional membrane-binding assays were conducted using liposome co-sedimentation and cell-based localization studies in COS7 and HeLa cells.In addition,site-directed mutagenesis targeting positively charged residues and known HCM-associated mutations,including R347C,was used to evaluate their effects on membrane interaction and subcellular localization.Results The crystal structure of the mouse JPH2 MORN-Helix domain was resolved at 2.6Å,revealing a compact,elongated architecture consisting of multiple tandem MORN motifs arranged in a curved configuration,forming a continuous hydrophobic core stabilized by alternating aromatic residues.A C-terminalα-helix further reinforced structural integrity.Conservation analysis identified the inner groove of the MORN array as a highly conserved surface,suggesting its role as a protein-binding interface.A flexible linker segment enriched in positively charged residues,located adjacent to the MORN motifs,was found to mediate direct electrostatic interactions with negatively charged phospholipid membranes.Functional assays demonstrated that mutation of these basic residues impaired membrane association,while the HCM-linked R347C mutation completely abolished membrane localization in cellular assays,despite preserving the overall MORN-Helix fold in structural modeling.Conclusion This study provides structural insight into the membrane-binding mechanism of the cardiomyocyte-specific protein JPH2,highlighting the dual roles of its MORN-Helix domain in membrane anchoring and protein interactions.The findings clarify the structural basis for membrane targeting via a positively charged linker and demonstrate that disruption of this interaction—such as that caused by the R347C mutation—likely contributes to HCM pathogenesis.These results not only enhance current understanding of JPH2 function in cardiac E-C coupling but also offer a structural framework for future investigations into the assembly and regulation of JMCs in both physiological and disease contexts.展开更多
2017年上海长征医院史建刚教授团队首次报道颈椎前路椎体-后纵韧带骨化物复合体可控前移融合术(ante-rior controllable antedisplacement and fusion,ACAF)治疗严重颈椎后纵韧带骨化症(ossification of posterior longitu-dinal ligame...2017年上海长征医院史建刚教授团队首次报道颈椎前路椎体-后纵韧带骨化物复合体可控前移融合术(ante-rior controllable antedisplacement and fusion,ACAF)治疗严重颈椎后纵韧带骨化症(ossification of posterior longitu-dinal ligament,OPLL)[1],其可在不切除骨化物的前提下实现脊髓原位减压,已被证明是一种安全有效的治疗严重颈椎OPLL的新型前路减压术式[2-5].然而,目前尚无系统地针对ACAF手术制定的器械,如钛板、螺钉及提拉工具等,尤其是对于提拉工具,仍需要进一步的改进[6].我们团队在早期开展ACAF的过程中,发现使用高速磨钻或超声骨刀开槽后游离椎体-后纵韧带骨化物复合体(vertebrae-OPLL complex,VOC)不彻底,导致不能顺利地完成前移提拉,而在进一步开槽游离椎体的过程中又会增加对神经的干扰,从而导致术后神经根损伤的症状.基于此,我们开发了带线锚钉辅助提拉VOC的方法,在临床应用中取得满意疗效,现对其总结报道.展开更多
文摘Objective Junctophilin-2(JPH2)is an essential structural protein that maintains junctional membrane complexes(JMCs)in cardiomyocytes by tethering the plasma membrane to the sarcoplasmic reticulum,thereby facilitating excitationcontraction(E-C)coupling.Mutations in JPH2 have been associated with hypertrophic cardiomyopathy(HCM),but the molecular mechanisms governing its membrane-binding properties and the functional relevance of its membrane occupation and recognition nexus(MORN)repeat motifs remain incompletely understood.This study aimed to elucidate the structural basis of JPH2 membrane association and its implications for HCM pathogenesis.Methods A recombinant N-terminal fragment of mouse JPH2(residues 1-440),encompassing the MORN repeats and an adjacent helical region,was purified under near-physiological buffer conditions.X-ray crystallography was employed to determine the structure of the JPH2 MORN-Helix domain.Sequence conservation analysis across species and junctophilin isoforms was performed to assess the evolutionary conservation of key structural features.Functional membrane-binding assays were conducted using liposome co-sedimentation and cell-based localization studies in COS7 and HeLa cells.In addition,site-directed mutagenesis targeting positively charged residues and known HCM-associated mutations,including R347C,was used to evaluate their effects on membrane interaction and subcellular localization.Results The crystal structure of the mouse JPH2 MORN-Helix domain was resolved at 2.6Å,revealing a compact,elongated architecture consisting of multiple tandem MORN motifs arranged in a curved configuration,forming a continuous hydrophobic core stabilized by alternating aromatic residues.A C-terminalα-helix further reinforced structural integrity.Conservation analysis identified the inner groove of the MORN array as a highly conserved surface,suggesting its role as a protein-binding interface.A flexible linker segment enriched in positively charged residues,located adjacent to the MORN motifs,was found to mediate direct electrostatic interactions with negatively charged phospholipid membranes.Functional assays demonstrated that mutation of these basic residues impaired membrane association,while the HCM-linked R347C mutation completely abolished membrane localization in cellular assays,despite preserving the overall MORN-Helix fold in structural modeling.Conclusion This study provides structural insight into the membrane-binding mechanism of the cardiomyocyte-specific protein JPH2,highlighting the dual roles of its MORN-Helix domain in membrane anchoring and protein interactions.The findings clarify the structural basis for membrane targeting via a positively charged linker and demonstrate that disruption of this interaction—such as that caused by the R347C mutation—likely contributes to HCM pathogenesis.These results not only enhance current understanding of JPH2 function in cardiac E-C coupling but also offer a structural framework for future investigations into the assembly and regulation of JMCs in both physiological and disease contexts.
文摘2017年上海长征医院史建刚教授团队首次报道颈椎前路椎体-后纵韧带骨化物复合体可控前移融合术(ante-rior controllable antedisplacement and fusion,ACAF)治疗严重颈椎后纵韧带骨化症(ossification of posterior longitu-dinal ligament,OPLL)[1],其可在不切除骨化物的前提下实现脊髓原位减压,已被证明是一种安全有效的治疗严重颈椎OPLL的新型前路减压术式[2-5].然而,目前尚无系统地针对ACAF手术制定的器械,如钛板、螺钉及提拉工具等,尤其是对于提拉工具,仍需要进一步的改进[6].我们团队在早期开展ACAF的过程中,发现使用高速磨钻或超声骨刀开槽后游离椎体-后纵韧带骨化物复合体(vertebrae-OPLL complex,VOC)不彻底,导致不能顺利地完成前移提拉,而在进一步开槽游离椎体的过程中又会增加对神经的干扰,从而导致术后神经根损伤的症状.基于此,我们开发了带线锚钉辅助提拉VOC的方法,在临床应用中取得满意疗效,现对其总结报道.
