Mankhwala ophera tizilombo amagwira ntchito yofunika kwambiri pothana ndi kusowa kwa chakudya padziko lonse lapansi komanso kuthana ndi matenda opatsirana ndi tizilombo. Komabe, vuto lomwe likukulirakulira la kukana mankhwala ophera tizilombo likufuna kuti pakhale kupezeka kwa mankhwala atsopano omwe amalimbana ndi malo omwe sagwiritsidwa ntchito mokwanira. Njira zolandirira tizilombo zomwe zimakhala ndi mphamvu ya TRPV—Nanzhong (Nan) ndi zosagwira ntchito (Iav)—zitha kupanga njira zosiyanasiyana (Nan-Iav) ndikuziyika m'zigawo zomwe zimathandizira kufalikira kwa tizilombo, kumva, ndi kuzindikira tizilombo. Mankhwala ena ophera tizilombo, monga aphidopyrrolidone (AP), amalimbana ndi Nan-Iav kudzera m'njira zosadziwika. AP imagwira ntchito bwino polimbana ndi tizilombo toyamwa (hemipterans), kuletsa kudya mwa kusokoneza ntchito ya ulusi. AP imatha kumangirira ku Nan yokha, koma Nan-Iav yokha ndi yomwe ingagwirizane ndi agonists, kuphatikiza nicotinamide ya endogenous (NAM), motero ikuwonetsa ntchito ya njira. Ngakhale kuti Nan-Iav ndi yothandiza kwambiri polimbana ndi tizilombo, sizikudziwika zambiri zokhudza kusonkhana kwa njira zake, malo omangira, komanso malamulo odalira Ca2+, zomwe zimalepheretsa kukula kwa tizilombo. Mu kafukufukuyu, cryo-electron microscopy idagwiritsidwa ntchito kudziwa kapangidwe ka Nan-Iav m'tizilombo ta Hemiptera mu mkhalidwe wopanda calmodulin-ligand, komanso ndi AP ndi NAM pamalire a ankyrin repeat cytoplasmic domain (ARD). Chodabwitsa n'chakuti, tapeza kuti puloteni ya Nan yokha imatha kupanga pentamer, yomwe imakhazikika chifukwa cha kuyanjana kwa ARD komwe kumachitika ndi AP. Kafukufukuyu akuwonetsa kuyanjana kwa mamolekyu pakati pa mankhwala ophera tizilombo ndi ma agonists ndi Nan-Iav, kuwonetsa kufunika kwa ARD pakugwira ntchito kwa channel ndi kusonkhana, ndikufufuza momwe Ca2+ imagwirira ntchito.
Poganizira za kusintha kwa nyengo komwe kukukulirakulira padziko lonse lapansi, kutsika kwa chitetezo cha chakudya padziko lonse lapansi ndi chimodzi mwa mavuto akuluakulu a m'zaka za m'ma 2000, zomwe zikubweretsa zotsatirapo zoipa kwa anthu.1,2Lipoti la World Health Organization la State of Food Security and Nutrition in the World 2023 (SOFI) likuyerekeza kuti anthu pafupifupi 2.33 biliyoni padziko lonse lapansi ali ndi vuto la kusowa chakudya chokwanira, vuto lomwe lakhalapo kwa nthawi yayitali.3,4Mwatsoka, pafupifupi 20% mpaka 30% kapena kuposerapo kwa zokolola zimatayika chaka chilichonse chifukwa cha tizilombo toyambitsa matenda, ndipo kutentha kwa dziko lapansi kukuyembekezeka kukulitsa kukana tizilombo komanso kusatetezeka kwa mbewu.4,5,6,7,8Kupanga mankhwala ophera tizilombo ndikofunikira osati kokha kuti titeteze mbewu ku tizilombo toyambitsa matenda komanso kuchepetsa kufalikira kwa tizilombo toyambitsa matenda, komanso kuti tithane ndi matenda opatsirana ndi anthu monga dengue fever, malungo, ndi matenda a Chagas, omwe akuchulukirachulukira kukana mankhwala ophera tizilombo.5,9,10,11
Pakati pa zolinga zazikulu za mankhwala ophera tizilombo omwe ali ndi poizoni m'mitsempha, njira ya heterotetrameric TRPV Nanchung (Nan)-Inactive (Iav) ikuyimira gulu la zolinga za mankhwala ophera tizilombo zomwe zapezeka m'zaka khumi zapitazi, kuphatikizapo mankhwala ophera tizilombo omwe amapezeka m'masitolo monga imidacloprid ndi pyraclostrobin.12,13,14Mankhwala ophera tizilombo otchedwa aphidopyrrolifen (AP) omwe amapangidwa posachedwapa komanso kugulitsidwa, omwe gawo lake lalikulu ndi mankhwala ophera tizilombo otchedwa Inscalis®, omwe amamanga AP pamlingo wa subnanomolar activity.15AP ili ndi poizoni wochepa kwambiri kwa tizilombo toyamwa mungu, tizilombo tothandiza, ndi zamoyo zina zomwe sizili m'gulu la tizilombo, ndipo ikagwiritsidwa ntchito motsatira malangizo olembedwa pa chizindikiro, imatha kuchepetsa mphamvu yolimbana ndi mankhwala ena ophera tizilombo.16,17,18Nan ndi Iav zimafalikira kwambiri m'mitundu yonse ya tizilombo, zimangopezeka mu chordal stretch receptor neurons ya tinyanga ndi miyendo, ndipo ndizofunikira kwambiri pakumva, kuzindikira mphamvu yokoka, ndi kuzindikira komwe zili.13,16,19,20,21,22AP, imidacloprid, ndi pyraclostrobin zimathandizira Nan-Iav complex kudzera mu njira yapadera, zomwe pamapeto pake zimaletsa kufalikira kwa chizindikiro cha proprioceptive.13,16,23Mu tizilombo toyamwa (hemipterans) monga nsabwe za m'masamba ndi ntchentche zoyera, kutayika kwa chidziwitso cha proprioception kumawononga mphamvu yawo yodyera, zomwe pamapeto pake zimapangitsa kuti afe.13,24Chochititsa chidwi n'chakuti, AP imaonetsa kuyanjana kwakukulu ndi Nan-Iav complex komanso kusagwirizana kochepa kwa Nan yokha. Kulumikizana kwa AP ndi Nan-Iav kumayambitsa magetsi, koma kugwirizana ndi Nan yokha sikulimbikitsa ntchito ya njira. Iav yokha simalumikizana ndi AP konse.16Izi zikusonyeza kuti Nan ndi Iav zimatha kugwirizana kuti zipange ma complexes osiyanasiyana a Nan-Iav channel (monga, ndi ma stoichiometric ratios osiyana kapena makonzedwe osiyanasiyana mkati mwa stoichiometric ratio yomweyo) kapena kuti AP ikhoza kugwirizana ndi malo angapo. Kuphatikiza apo, natural agonist nicotinamide (NAM) imalumikizana ndi Drosophila Nan-Iav ndi micromolar affinity, zomwe zimasonyeza zotsatira zofanana ndi za aphids (AP) mu vitro.16,25ndipo zimaletsa kuberekana ndi kudya nsabwe za m'masamba, zomwe pamapeto pake zimapangitsa kuti zife.25,26. Deta iyi imabweretsa mafunso ambiri. Mwachitsanzo, sizikudziwika bwino momwe Nan-Iav heterodimer imapangidwira, malo omangirira omwe amagwiritsidwa ntchito kusintha mamolekyu ang'onoang'ono, ndi momwe mamolekyu ang'onoang'ono awa amawongolera ntchito ya njira poletsa proprioception. Kuphatikiza apo, zifukwa zomwe Nan yokha siigwira ntchito komanso ili ndi mphamvu yochepa ya AP, pomwe Nan-Iav heterodimer imagwira ntchito ndipo imalumikiza AP ndi mphamvu yapamwamba, sizikudziwika bwino. Pomaliza, zochepa zomwe zimadziwika za Ca2+ -dependent regulation ya Nan-Iav function ndi momwe imaphatikizidwira mu neuronal signaling processes.. 13,21
Mu kafukufukuyu, kuphatikiza ma microscopy a cryo-electron, electrophysiology, ndi njira zomangira ma radioligand, tafotokoza bwino za kusonkhana kwa Nan-Iav ndi momwe imagwirira ntchito yomangira mamolekyu ang'onoang'ono. Kuphatikiza apo, tapeza calmodulin (CaM) yolumikizidwa ndi Iav ndi AP-stabilized Nan pentamers. Zotsatirazi zimapereka chidziwitso chofunikira pakuwongolera ma calcium ions mu njira, kusonkhana kwa njira, ndi zinthu zomwe zimatsimikiza kugwirizana kwa ligand. Chofunika kwambiri, tatsimikiza kuti ARD imagwira ntchito yofunika kwambiri munjira izi. Kafukufuku wathu wa njira zonse za tizilombo zomwe zimagwirizanitsidwa ndi mankhwala ophera tizilombo a zaulimi.27, 28, 29Zimatsegula mwayi woti makampani opanga mankhwala ophera tizilombo azikula, kupititsa patsogolo luso la mankhwala ophera tizilombo, komanso kulola kugwiritsa ntchito mankhwala opangidwa ndi TRPV ku mitundu ina ya zomera kuti athetse vuto la chakudya padziko lonse komanso kufalikira kwa matenda opatsirana ndi tizilombo.
Tinapezanso kuti Nan-Iav imayendetsedwa ndi Ca2+, ndipo njira yoyendetsera imayendetsedwa ndi CaM yolumikizidwa. Chofunika kwambiri, njira yoyendetsera iyi ya Ca2+ ya Nav ndi CaM imasiyana kwambiri ndi njira zoyendetsera njira zina za ion (monga, njira za Na+ zotetezedwa ndi magetsi ndi njira za TRPV5/6)52,53,54,55,56,57Mu njira ya Nav1.2, gawo la C-terminal la CaM limagwirizana ndi gawo la C-terminal (CTD), ndipo Ca2+ imayambitsa kumangirira gawo lake la N-terminal ku gawo lakutali la CTD.56Mu njira ya TRPV5/6, gawo la C-terminal la CaM limalumikizana ndi CTH, ndipo Ca2+ imapangitsa kuti gawo lake la N-terminal likwere kupita mmwamba mu pore, motero imaletsa kutseguka kwa cation.53,54. Tikupereka chitsanzo cha ntchito yolamulidwa ndi Ca2+ ya Nan-Iav-CaM (Chithunzi 4h). Mu chitsanzo ichi, dera la N-terminal la CaM limalumikizana ndi dera la C-terminal (CTH) la Iav. Mu mkhalidwe wopumula (kuchuluka kochepa kwa [Ca2+]), dera la C-terminal la CaM limalumikizana ndi Nan, kukhazikika kwa mawonekedwe a ARD ndikulimbikitsa kutsegulidwa kwa njira. Kumangirira kwa agonist/tizilombo ku njira kumayambitsa kutseguka kwa ma pore, zomwe zimapangitsa kuti Ca2+ ilowe. Kenako Ca2+ imalumikizana ndi CaM, zomwe zimapangitsa kuti dera la C-terminal ligawikane kuchokera ku ARD ya Nan. Chifukwa choletsa kumangidwa kwa CaM kwenikweni chimathetsa mphamvu yoletsa ya Ca2+, kugawikana kumeneku kumawongolera kuyenda kwa ARD, motero kumayambitsa kuletsa kapena kuletsa kumva kwa Ca2+. Kubwezeretsa mwachangu kwa ma channel currents pambuyo pa calcium ion elution (Chithunzi 4g) kukuwonetsa kuti njira iyi imathandizira kuyankha mwachangu ku zizindikiro za neuronal zomwe zimayendetsedwa ndi Ca2+. Kuphatikiza apo, dera la C-terminal la Iav, lomwe silikumveka bwino, lanenedwa kuti likuchita ntchito zina mu njira zowunikira komanso malamulo omwe alipo.21
Pomaliza, kafukufuku wathu akuwonetsa kapangidwe kabwino kwambiri ka njira ya TRP yophera tizilombo yomwe ndi yofunika kwambiri paulimi—chomwe sitinkachidziwa kale. Chodziwika bwino n'chakuti, tinafotokoza kapangidwe ndi ntchito ya njira ya tizilombo m'maselo a anthu (HEK293S GnTi–) osati m'maselo a tizilombo. Poyang'anizana ndi kukana kwa tizilombo toyambitsa matenda komanso kukakamizidwa kosalekeza pa chitetezo cha chakudya ndi tizilombo toyambitsa matenda, ntchito yathu imapereka chidziwitso chofunikira chomwe chingathandize kupanga mankhwala atsopano ophera tizilombo kuti athandize thanzi la anthu komanso chitetezo cha chakudya padziko lonse lapansi. Kafukufuku wasonyeza kuti mankhwala ophera tizilombo monga AP ndi othandiza polimbana ndi tizilombo tina akagwiritsidwa ntchito motsatira malangizo olembedwa ndipo ali ndi poizoni wochepa kwambiri kwa tizilombo topindulitsa, zomwe zimasonyeza chitetezo chawo pa chilengedwe.13,16Kuphatikiza apo, kuyesa kwa mankhwala ena a AP pa udzudzu kwawonetsa kuti pamapeto pake amataya mphamvu zawo zouluka. Kumvetsetsa momwe mankhwala osinthira awa amamangirirana ndi Nan-Iav kungathandize kusintha kwa mankhwala omwe alipo kapena kupanga mankhwala atsopano kuti agwire bwino ntchito komanso moyenera.molondolaKuwongolera tizilombo. Kafukufuku wathu akuwonetsa kuti mawonekedwe a Nan-Iav ARD ndi ofunikira osati kokha pakulamulira ntchito ya mankhwala achilengedwe, mankhwala ophera tizilombo, ndi Ca2+-CaM, komanso pakusonkhanitsa njira. Tikupereka lingaliro lakuti kusokoneza kusonkhana kwa heterodimer ndi mamolekyu ang'onoang'ono kungakhale njira yapadera komanso yodalirika yopangira zoletsa njira za ion.
Mwa majini asanu ndi atatu a orthologous, majini athunthu a brown beetle (Halyomorpha halys) Nanchung ndi Inactive adasankhidwa, zomwe zikuwonetsa kukhazikika bwino mu sopo. Majini opangidwawo adakonzedwa bwino kuti azitha kuwonetsa mawonekedwe a anthu ndipo adapangidwa mu pBacMam pCMV-DEST vector (Life Technologies) pogwiritsa ntchito malo oletsa XhoI ndi EcoRI. Izi zidatsimikizira kuti ma clones anali mu chimango ndi ma C-terminal GFP-FLAG-10xHis ndi mCherry-FLAG-10xHis tag, omwe amadulidwa ndi HRC-3C protease (PPX), zomwe zimapangitsa kuti pakhale kudziyimira pawokha.mawonekedweMa primer omwe adagwiritsidwa ntchito pophatikiza Nanchung ndi Inactive mu pBacMam vector anali awa:
Zithunzi za microscopic za tinthu tating'onoting'ono ta tinthu tating'onoting'ono ta Titan Krios G2 transmission electron microscope (FEI) yokhala ndi kamera ya K3 ndi fyuluta ya mphamvu ya Gatan BioQuantum. Maikulosikopuyo idagwiritsidwa ntchito pa 300 keV, yokhala ndi mphamvu ya 20 eV, kukula kwa pixel ya chitsanzo cha 1.08 Å/pixel (kukulitsa kwa nominal kwa 81,000x), ndi gradient ya defocus kuyambira -0.8 mpaka -2.2 μm. Kujambula kanema kunachitika pa mafelemu 40 pa sekondi pogwiritsa ntchito maikulosikopu ya Latitude S (Gatan) yokhala ndi mlingo wocheperako wa 25 e–px−1 s−1, nthawi yowonekera ya 2.4 s, ndi mlingo wonse wa pafupifupi 60 e–Å−2.
Kukonza kayendedwe koyendetsedwa ndi beam-induced ndi kulemera kwa mlingo kunachitidwa pa filimu pogwiritsa ntchito MotionCor2 mu RELION 4.061. Kuyerekeza kwa magawo a ntchito yosinthira kusiyana (CTF) kunachitidwa mu cryoSPARC pogwiritsa ntchito njira yowerengera ya CTF yochokera ku patch62. Ma Photomicrograph okhala ndi resolution yoyenerera ya CTF ≥4 Å sanaphatikizidwe mu kusanthula kotsatira. Kawirikawiri, gawo la ma photomicrograph 500–1000 linagwiritsidwa ntchito posankha mfundo mu cryoSPARC, kutsatiridwa ndi ma round angapo a 2D classification pambuyo posefa kuti apeze chithunzi chowonekera bwino cha kusankha tinthu tomwe timachokera ku template. Kenako tinthu tinachotsedwa pogwiritsa ntchito mabokosi ozungulira a 64-pixel ndi ma binning anayi. Ma round angapo a 2D classification adachitidwa kuti achotse magulu osafunikira a tinthu. Chitsanzo choyamba cha 3D chinamangidwanso pogwiritsa ntchito ab initio reconstruction ndikukonzedwanso pogwiritsa ntchito nonuniform refinement mu cryoSPARC. Ma 3D classification adachitika mu cryoSPARC kapena RELION kutengera ARD heterogeneity. Palibe kusiyana kwakukulu kwa madera a membrane komwe kunawonedwa. Tinthu tinakonzedwa pogwiritsa ntchito njira za C1 ndi C2; tinthu tomwe tinali ndi resolution yapamwamba ya C2 tinaonedwa kuti ndi tofanana poyerekeza ndi C2 ndipo tinalowetsedwa mu RELION kuti tikonzedwenso ku Bayesian. Tinthu tinasinthidwanso ku cryoSPARC kuti tikonzedwenso komaliza kosakhala kofanana komanso koyambirira. Resolution yomaliza ndi kuwerengera kwa tinthu tating'onoting'ono kwawonetsedwa mu Table 1.
Pokonza ma pentamers a Nan+AP, tinafufuza njira zosiyanasiyana zowongolera kutsimikizika kwa madera a nembanemba (makamaka madera a pore), monga kuchotsa ma signal ndi TMD masking. Komabe, kuyesaku sikunapambane chifukwa cha kusokonezeka kwakukulu komwe kulipo m'dera la pore komanso kusiyana konse kwa TMD. Kutsimikizika komaliza kunawerengedwa pogwiritsa ntchito chigoba chopangidwa chokha ndi njira yosagwiritsidwa ntchito mofanana mu cryoSPARC, makamaka kuyang'ana madera a ARD. Izi zinapeza kutsimikizika kwakukulu kuposa kwa madera a nembanemba (makamaka madera a VSLD).
Ma model atsopano oyamba a apoforms a Nanchung ndi Inactive bugs adapangidwa koyamba pogwiritsa ntchito Coot63, ndipo ma model a Nan ndi Iav bugs adapangidwa pogwiritsa ntchito AlphaFold264 kuti adziwe madera omwe ali ndi chidaliro chochepa. Calmodulin modeling idakhazikitsidwa pa rigid-body fit of Ca2+-binding and Ca2+-free models in PDB accessions 4JPZ56 and 1CFD65, motsatana. Ma model adakonzedwa pogwiritsa ntchito spherical refinement kuti atsimikizire stereochemistry yolondola komanso geometry yabwino. Kenako Phosphatidylcholine, phosphatidylethanolamine, ndi phosphatidylserine adapangidwa ngati lipid densities yodziwika bwino, ndipo NAM ndi AP ligands adayikidwa mu densities yofanana mu tight junctions. Mafayilo oletsa adapangidwa kuchokera ku SMILES string ya isoforms pogwiritsa ntchito eLBOW mu PHENIX66. Pomaliza, ma model adakonzedwanso mu malo enieni mu PHENIX pogwiritsa ntchito local grid search ndi global reduction with secondary structure constrictions. Seva ya MolProbity idagwiritsidwa ntchito pokonza chitsanzo ndi kusanthula kapangidwe kake, ndipo zithunzi zidachitika pogwiritsa ntchito PyMOL ndi UCSF Chimera X. 67,68,69 Kusanthula kwa Aperture kudachitika pogwiritsa ntchito seva ya HOLE,70 ndipo mapu osungira zinthu motsatira ndondomeko adachitika pogwiritsa ntchito seva ya Consurf.71
Kusanthula ziwerengero kunachitika pogwiritsa ntchito Igor Pro 6.2, Excel Office 365, ndi GraphPad Prism 7.0. Deta yonse yowerengera imaperekedwa ngati cholakwika chapakati ± standard (SEM). Mayeso a t a wophunzira (okhala ndi michira iwiri, osaphatikizidwa) adagwiritsidwa ntchito poyerekeza magulu awiri. Kusanthula kwa kusiyana kwa njira imodzi (ANOVA) kotsatiridwa ndi mayeso a post hoc a Dunnett adagwiritsidwa ntchito poyerekeza magulu angapo. *P< 0.05, **P< 0.01, ndi ***P<0.001 adawonedwa kuti ndi ofunika kwambiri kutengera kugawa kwa deta. Ma Kd, Ki values, ndi ma asymmetric 95% confidence intervals awo adawerengedwa pogwiritsa ntchito GraphPad Prism 10.
Kuti mudziwe zambiri za njira yophunzirira, chonde onani Chidule cha Nature Portfolio Report chomwe chili munkhaniyi.
Chitsanzo choyamba chinapangidwa pogwiritsa ntchito ma calmodulin model ochokera ku PDB 4JPZ ndi 1CFD databases. Ma coordinates aikidwa mu Protein Data Bank (PDB) pansi pa manambala olowa 9NVN (Nan-Iav-CaM yopanda ligand), 9NVO (Nan-Iav-CaM yolumikizidwa ku nicotinamide), 9NVP (Nan-Iav-CaM yolumikizidwa ku nicotinamide ndi EDTA), 9NVQ (Nan-Iav-CaM yolumikizidwa ku aphenidolpyrrolline ndi calcium), 9NVR (Nan-Iav-CaM yolumikizidwa ku aphenidolpyrrolline ndi EDTA), ndi 9NVS (Nan pentamer yolumikizidwa ku aphenidolpyrrolline). Zithunzi zofanana za cryo-electron microscopy zimayikidwa mu Electron Microscopy Database (EMDB) pansi pa manambala otsatirawa: EMD-49844 (Nan-Iav-CaM yopanda ligand), EMD-49845 (Nan-Iav-CaM complex yokhala ndi nicotinamide), EMD-49846 (Nan-Iav-CaM complex yokhala ndi nicotinamide ndi EDTA), EMD-49847 (Nan-Iav-CaM complex yokhala ndi aphidopyrrolline ndi calcium), EMD-49848 (Nan-Iav-CaM complex yokhala ndi aphidopyrrolline ndi EDTA), ndi EMD-49849 (Nan pentamer complex yokhala ndi aphidopyrrolline). Deta yosafunikira ya kusanthula kwa ntchito ikuwonetsedwa mu pepalali.
Nthawi yotumizira: Januwale-28-2026