He mahi nui tā ngā patu pepeke ki te aro atu ki te kore kai o te ao me te whawhai ki ngā mate tangata e kawea mai ana e ngā pepeke. Heoi, ko te raru e tipu haere ana o te ātete ki ngā patu pepeke me tere te kimi i ngā matū hou e aro ana ki ngā ūnga kāore i te tino whakamahia. Ka taea e ngā hongere pūmanawa kaiwhiwhi whakawhiti pepeke (TRPV)—ko Nanzhong (Nan) me te kore mahi (Iav)—te hanga hongere heterologous (Nan-Iav) me te noho ki ngā whekau mechanosensory e whakahaere ana i te geotropism, te whakarongo, me te proprioception i roto i ngā pepeke. Ko ētahi patu pepeke, pērā i te aphidopyrrolidone (AP), ka aro ki a Nan-Iav mā roto i ngā tikanga kāore i te mōhiotia. He whai hua te AP ki ngā pepeke ngote-wero (hemipterans), ka ārai i te kai mā te whakararuraru i te mahi a ngā miro. Ka taea e te AP te here ki a Nan anake, engari ko Nan-Iav anake ka taea te taunekeneke me ngā agonists, tae atu ki te nicotinamide taketake (NAM), ā, ka whakaatu i te mahi hongere. Ahakoa te kaha o Nan-Iav hei ūnga patu pepeke, he iti noa te mōhiotanga mō tōna huihuinga hongere, ngā pae here ture, me te ture Ca2+-dependent, e aukati ana i te whanaketanga atu o ngā patu pepeke. I roto i tēnei rangahau, i whakamahia te karuārai-irahiko-cryo hei whakatau i te hanganga o Nan-Iav i roto i ngā pepeke Hemiptera i te āhua kore-calmodulin-ligand, tae atu ki te AP me te NAM i te rohe o te rohe cytoplasmic ankyrin repeat (ARD). He mea whakamiharo, i kitea e mātou ka taea e te pūmua Nan te hanga i tētahi pentamer, ka whakapūmautia e ngā taunekeneke ARD e whakahaerehia ana e AP. E whakaatu ana tēnei rangahau i ngā taunekeneke ngota i waenga i ngā patu pepeke me ngā agonists me Nan-Iav, e whakaatu ana i te hiranga o te ARD i roto i te mahi me te huihuinga o te hongere, me te tūhura i te tikanga whakahaere Ca2+.
Ahakoa te piki haere o te huringa āhuarangi o te ao, ko te heke haere o te haumarutanga kai o te ao tētahi o ngā wero nui o te rautau 21, me ngā pānga kino ki te hapori.1,2E ai ki te pūrongo a te World Health Organization mō te State of Food Security and Nutrition in the World 2023 (SOFI), tata ki te 2.33 piriona tāngata puta noa i te ao e pāngia ana e te korenga o te kai i roto i te ao, he raruraru roa tēnei.3,4Engari, e tata ana ki te 20% ki te 30%, neke atu rānei, o ngā hua o te whenua ka ngaro i ia tau i ngā pepeke me ngā tukumate, ā, e matapaetia ana ka nui ake te ātete o ngā pepeke me te ngoikore o ngā hua nā te mahana o te ao.4,5,6,7,8He mea nui te whakawhanake i ngā patupatu, ehara i te mea hei tiaki i ngā hua mai i ngā pepeke me te whakaiti i te horapa o ngā mate tuku iho, engari hei whawhai hoki i ngā mate tangata e tukuna ana e ngā patupatu pērā i te kirika dengue, te malaria, me te mate Chagas, e kaha haere ana te ātete ki ngā patupatu.5,9,10,11
I roto i ngā whāinga matua o ngā patu pepeke paitini-pūnaha, ko te hongere TRPV heterotetrameric Nanchung (Nan)-Inactive (Iav) e tohu ana i tētahi akomanga o ngā whāinga patu pepeke i kitea i te tekau tau kua pahure ake nei, tae atu ki ngā patu pepeke arumoni pērā i te imidacloprid me te pyraclostrobin.12,13,14Ko te aphidopyrrolifen (AP) he hua kua whakawhanakehia, kua hokohokohia hoki i ēnei wā tata nei, ko tōna wāhanga matua ko te Inscalis®, he pepeke whakamate hohe, e herea ana te AP i te taumata mahi iti iho i te nanomolar.15He iti noa te paitini kino o te AP ki ngā kaiwhakatupu ruirui, ki ngā pepeke whai hua, me ētahi atu rauropi kāore i te whainga, ā, ina whakamahia kia rite ki ngā tohutohu i runga i te tapanga, ka taea te whakaiti i te ātete ki ētahi atu patu pepeke.16,17,18He whānui te horapa o Nan me Iav puta noa i ngā momo pepeke, ka whakaatuhia ngātahitia i roto i ngā neuron kaiwhakawhiwhi totoro chordal o ngā antennae me ngā peka, ā, he mea nui mō te whakarongo, te kite i te kaha ā-papa, me te proprioception.13,16,19,20,21,22Ka whakaohokia e te AP, te imidacloprid, me te pyraclostrobin te matatini Nan-Iav mā roto i tētahi tikanga ahurei, ā, ka mutu, ka aukati i te tuku tohu proprioceptive.13,16,23Mō ngā pepeke ngote wero (hemipterans) pērā i ngā aphids me ngā whiteflies, ko te ngaronga o te proprioception ka whakararu i tō rātou kaha ki te kai, ā, ka mutu, ka mate.13,24He mea whakamīharo, he nui te hononga o AP ki te matatini Nan-Iav, ā, he iti te hononga ki a Nan anake. Mā te here o AP ki a Nan-Iav ka puta he iahiko hiko, engari ko te here ki a Nan anake kāore e whakaoho i te mahi hongere. Kāore a Iav e here ki a AP.16E whakaatu ana tēnei ka herea pea a Nan me Iav ki te hanga i ngā matatini awa Nan-Iav rerekē (hei tauira, me ngā ōwehenga stoichiometric rerekē, ngā whakaritenga rerekē rānei i roto i te ōwehenga stoichiometric kotahi) ka herea rānei a AP ki ngā wāhi maha. Hei tāpiri, ka herea te nicotinamide agonist tūturu (NAM) ki a Drosophila Nan-Iav me te hononga micromolar, e whakaatu ana i ngā pānga rite ki ērā o ngā aphids (AP) i roto i te vitro.16,25ā, ka aukati i te whakaputa uri me te kai a ngā aphid, ka mutu ka mate rātou25,26. He maha ngā pātai e ara ake ana i ēnei raraunga. Hei tauira, kāore anō kia mārama me pēhea te hanganga o te Nan-Iav heterodimer, ko ēhea ngā pae here e whakamahia ana hei whakarerekē i ngā ngota iti, me pēhea te whakahaere a ēnei ngota iti i te mahi hongere mā te pehi i te proprioception. Hei tāpiri, kāore anō kia mārama ngā take kāore a Nan i te mahi, ā, he iti te hononga ki te AP, i te mea he kaha te Nan-Iav heterodimer, ā, he nui ake te hononga ki te AP. Hei whakamutunga, he iti noa ngā mea e mōhiotia ana mō te whakahaere a Ca2+ i te mahi Nan-Iav me pēhea te whakauru ki ngā tukanga tohu neuronal..13,21
I roto i tēnei rangahau, mā te whakakotahi i te karuārai-irahiko-cryo, te electrophysiology, me ngā tikanga here radioligand, i whakamāramahia e mātou te huihuinga o Nan-Iav me te tikanga o tōna herenga ki ngā kaiwhakahaere ngota iti. I tua atu, i kitea e mātou te calmodulin (CaM) e herea pūmau ana ki te Iav me ngā Nan pentamers kua whakapūmautia e te AP. Ka whakaratohia e ēnei hua he māramatanga nui ki te whakahaere i ngā katote konupūmā i roto i ngā hongere, te huihuinga hongere, me ngā āhuatanga e whakatau ana i te hononga here ligand. He mea nui ake, i whakaūhia e mātou he mahi nui tā te ARD i roto i ēnei tukanga. Ko tā mātou rangahau mō ngā hongere pepeke katoa e herea ana ki ngā patupatu ahuwhenua e tika ana.27, 28, 29ka whakatuwhera i ngā huarahi mō te whanaketanga o te umanga patu pepeke, te whakapai ake i te whai huatanga me te motuhaketanga o ngā patu pepeke, me te whakahohe i te tono o ngā matū e aro ana ki te TRPV ki ētahi atu momo hei aro atu ki te haumarutanga kai o te ao me te horapa o ngā mate ka kawea e te kawe pepeke.
I kitea anō hoki e mātou ko te Nan-Iav e whakahaeretia ana e Ca2+, ā, ko te tikanga whakahaere e ārahitia ana e te CaM e herea ana. He mea nui, he rerekē rawa tēnei whakahaere Ca2+-e whakawhirinaki ana ki te Nav e CaM i ngā tikanga whakahaere o ētahi atu hongere katote (hei tauira, ngā hongere Na+ kua āraihia e te ngaohiko me ngā hongere TRPV5/6)52,53,54,55,56,57I roto i te hongere Nav1.2, ka honoa te rohe C-terminal o CaM ki te rohe C-terminal (CTD), ā, ka whakaoho a Ca2+ i te here o tōna rohe N-terminal ki te wāhanga tawhiti o te CTD.56I roto i te hongere TRPV5/6, ka herea te rohe C-terminal o CaM ki te CTH, ā, ka akiakihia e Ca2+ te toronga ake o tōna rohe N-terminal ki roto i te pūwero, ā, ka ārai i te urunga o te cation.53,54. Ka whakaarohia e mātou he tauira mō te mahi whakahaere-Ca2+ o Nan-Iav-CaM (Pikitia 4h). I roto i tēnei tauira, ka herea pūmau te rohe N-terminal o CaM ki te rohe C-terminal (CTH) o Iav. I te āhua okiokinga (he iti te kukū [Ca2+]), ka taunekeneke te rohe C-terminal o CaM ki a Nan, ka whakau i te āhua ARD, ā, ka whakatairanga i te whakatuwheratanga o te awa. Mā te herea o tētahi agonist/insecticide ki te awa ka hua ake te whakatuwheratanga o ngā pore, ka arahi ki te urunga mai o Ca2+. Kātahi ka herea a Ca2+ ki a CaM, ka puta te wehenga o te rohe C-terminal mai i te ARD o Nan. Nā te mea ko te aukati i te herenga CaM ka whakakore i te pānga aukati o Ca2+, ka whakarerekē tēnei wehenga i te nekehanga ARD, ka puta te aukati, te kore rānei e pāngia e te Ca2+. Ko te tere o te whakaoranga o ngā ia awa i muri i te whakakorenga o te katote konupūmā (Pikitia 4g) e kī ana ka āwhina tēnei tikanga i ngā urupare tere ki ngā tohu io e whakahaerehia ana e Ca2+. Hei tāpiri atu ki tēnā, kua pūrongohia te rohe C-terminal o Iav, kāore anō kia tino māramahia, he mahi anō tāna ki te arotahi i te hongere me te whakahaere i tēnei wā.21
Hei whakamutunga, e whakaatu ana tā mātou rangahau i te hanganga teitei o te matatini hongere TRP patu pepeke-patu pepeke he mea nui ki te ahuwhenua—he kitenga kāore i mōhiotia i mua e mātou. He mea nui, i whakaahuahia e mātou te hanganga me te mahi a te hongere pepeke i roto i ngā pūtau tangata (HEK293S GnTi–) kaua i roto i ngā pūtau pepeke. I te aroaro o te tipu haere o te ātete ki ngā patu pepeke me te pēhanga tonu ki te haumarutanga kai me ngā mate pukupuku, ka whakaratohia e tā mātou mahi he mōhiohio nui hei āwhina i te whanaketanga o ngā patu pepeke hou hei painga mō te hauora tangata me te haumarutanga kai o te ao. Kua whakaatuhia e ngā rangahau he whai hua ngā patu pepeke pēnei i te AP ki ētahi pepeke ina whakamahia kia rite ki ngā tohutohu tapanga, ā, he iti te paitini kino ki ngā kaiwhakatipu whai hua, e whakaatu ana i tō rātou haumaru taiao.13,16Hei tāpiri atu, kua whakaatuhia e te whakamātautau i ētahi o ngā hua AP i runga i ngā waeroa ka ngaro te kaha ki te rere i te mutunga. Mā te mārama ki te here o ēnei matū whakarerekētanga ki te Nan-Iav ka āwhina i te whakarerekētanga o ngā matū o nāianei, te whakawhanake rānei i ngā matū hou kia pai ake ai te rere, kia pai ake ai hoki.tino tikate whakahaere i ngā pepeke. E whakaatu ana tā mātou rangahau he mea nui te atanga Nan-Iav ARD ehara i te mea mō te whakahaere i te mahi a ngā matū taketake, ngā patu pepeke, me te Ca2+-CaM anake, engari mō te huihuinga hongere hoki. E kī ana mātou ko te whakakore i te huihuinga heterodimer me ngā ngota iti he huarahi ahurei, he huarahi whai hua hoki mō te whakawhanake i ngā aukati hongere katote.
O ngā ira orthologous e waru, ko ngā ira roa-katoa o te pīrere parauri (Halyomorpha halys) Nanchung me Inactive i tīpakohia, e whakaatu ana i te pumau pai i roto i ngā hopi horoi. Ko ngā ira i hangaia i arotauhia ki te codon mō te whakaaturanga tangata, ā, i tāruatia ki roto i te pBacMam pCMV-DEST vector (Life Technologies) mā te whakamahi i ngā pae here XhoI me EcoRI. Nā tēnei i whakarite kia noho ngā tārua ki roto i te anga me ngā tohu C-terminal GFP-FLAG-10xHis me mCherry-FLAG-10xHis, e tapahia ana e te HRC-3C protease (PPX), e āhei ai te motuhakete whakapuakitangaKo ngā paraime i whakamahia hei tārua i a Nanchung me Inactive ki roto i te irahiko pBacMam koia ēnei:
I tangohia ngā whakaahua karuārai o ngā matūriki takitahi i runga i te karuārai hiko tuku Titan Krios G2 (FEI) he kāmera K3 me te tātari pūngao Gatan BioQuantum. I whakahaerehia te karuārai i te 300 keV, me te tautuhinga pūngao o te 20 eV, te rahi o te pika tauira o te 1.08 Å/pika (te whakanui ingoa o te 81,000x), me te rōnaki defocus mai i te -0.8 ki te -2.2 μm. I mahia te hopu ataata i te 40 anga ia hekona mā te whakamahi i te karuārai Latitude S (Gatan) me te tere horopeta ingoa o te 25 e–px−1 s−1, te wā whakaatu o te 2.4 s, me te horopeta katoa o te 60 e–Å−2.
I whakahaerea te whakatikatika nekehanga i puta mai i te hihi me te taumaha horopeta ki runga i te kiriata mā te whakamahi i te MotionCor2 i roto i te RELION 4.061. I whakahaerea te aromatawai tawhā mahi whakawhiti rerekētanga (CTF) i roto i te cryoSPARC mā te whakamahi i te tikanga aromatawai CTF e hangai ana ki te papaki62. I tangohia ngā whakaahua iti me te taumira whakauru CTF ≥4 Å i roto i te tātaritanga i muri mai. I te nuinga o te wā, i whakamahia he huinga iti o te 500–1000 whakaahua iti mō te kōwhiringa ira i roto i te cryoSPARC, ā, i whai ake ko ētahi rauna o te whakarōpūtanga 2D i muri i te tātari hei whiwhi i tētahi whakaahua tohutoro mārama mō te kōwhiringa matūriki e hangai ana ki te tauira. I tangohia ngā matūriki mā te whakamahi i ngā pouaka here 64-pika me te whakarōpūtanga 4-fold. He maha ngā rauna o te whakarōpūtanga 2D i whakahaerea hei tango i ngā kāwai matūriki kāore e hiahiatia. I hangaia anō te tauira 3D tuatahi mā te whakamahi i te hanganga ab initio me te whakapai ake mā te whakamahi i te whakapai ake kore-taurite i roto i te cryoSPARC. I whakahaerea te whakarōpūtanga 3D i roto i te cryoSPARC, i te RELION rānei i runga i te rerekētanga ARD. Kāore i kitea he rerekētanga nui o ngā rohe membrane. I whakatikatikaina ngā matūriki mā te whakamahi i ngā tikanga C1 me C2; i kiia ngā matūriki he teitei ake te taumira C2 he ōrite ki a C2, ā, i kawemai ki RELION mō te whakatikatika Bayesian. Kātahi ka whakahokia ngā matūriki ki cryoSPARC mō te whakatikatika whakamutunga kāore i te ōrite me te rohe. Kei te Ripanga 1 te taumira whakamutunga me te maha o ngā matūriki.
I te tukatuka i ngā pentamers Nan+AP, i tūhuratia e mātou ngā tikanga maha hei whakapai ake i te whakataunga o ngā rohe membrane (ina koa te rohe pore), pērā i te tango tohu me te huna TMD. Heoi, kāore i angitu ēnei nganatanga nā te raruraru nui pea i roto i te rohe pore me te rerekētanga whānui o te TMD. I tatauhia te whakataunga whakamutunga mā te whakamahi i tētahi uhi i hangaia aunoatia e te tikanga tukatuka kore-taurite i roto i te cryoSPARC, e aro nui ana ki te rohe ARD. I tutuki i tēnei he whakataunga teitei ake i tērā o ngā rohe membrane (ina koa te rohe VSLD).
I hangaia tuatahitia ngā tauira hou o ngā momo apo o ngā pepeke Nanchung me Inactive mā te whakamahi i a Coot63, ā, i hangaia ngā tauira o ngā pepeke Nan me Iav mā te whakamahi i a AlphaFold264 hei tautuhi i ngā rohe iti-whakawhirinaki. I ahu mai te whakatauira Calmodulin i ngā taunga tinana pakari o ngā tauira here-Ca2+ me te kore-Ca2+ i roto i ngā whakaurunga PDB 4JPZ56 me 1CFD65. I whakatikatikaina ngā tauira mā te whakamahi i te whakatikatika porowhita hei whakarite i te stereochemistry tika me te āhua pai. Kātahi ka whakatauirahia te Phosphatidylcholine, te phosphatidylethanolamine, me te phosphatidylserine hei kiato lipid kua tautuhia pai, ā, i whakanohoia ngā ligands NAM me AP ki ngā kiato e rite ana i roto i ngā hononga kikī. I hangaia ngā kōnae here mai i te aho SMILES o ngā isoform mā te whakamahi i te eLBOW i PHENIX66. Hei whakamutunga, i whakatikatikaina ngā tauira i te wāhi tūturu i PHENIX mā te whakamahi i te rapu whatunga ā-rohe me te whakaiti ā-ao me ngā here hanganga tuarua. I whakamahia te tūmau MolProbity mō te whakatikatika tauira me te tātari hanganga, ā, i mahia ngā whakaahua mā te whakamahi i te PyMOL me te UCSF Chimera X. 67,68,69 I mahia te tātari whakatuwheratanga mā te whakamahi i te tūmau HOLE,70 ā, i mahia te mahere tiaki raupapatanga mā te whakamahi i te tūmau Consurf.71
I whakahaerehia te tātari tatauranga mā te whakamahi i te Igor Pro 6.2, Excel Office 365, me te GraphPad Prism 7.0. Ka whakaatuhia ngā raraunga tauanga katoa hei toharite ± hapa paerewa (SEM). I whakamahia te whakamātautau-t a te ākonga (rua-hiku, kāore i te takirua) hei whakatairite i ngā rōpū e rua. I whakamahia te tātari rerekētanga kotahi-ara (ANOVA) ā, i whai ake ko te whakamātautau muri-hoc a Dunnett hei whakatairite i ngā rōpū maha. *P< 0.05, **P< 0.01, me ***PI kiia te < 0.001 he hiranga ā-tatauranga i runga i te tohatoha raraunga. I tatauhia ngā uara Kd, Ki, me ō rātou āputa whakawhirinaki 95% kore taurite mā te whakamahi i te GraphPad Prism 10.
Mō ētahi atu kōrero mō te tikanga rangahau, tirohia te Whakarāpopototanga Pūrongo Pūkete Taiao e hono ana ki tēnei tuhinga.
I hangaia te tauira tuatahi mā te whakamahi i ngā tauira calmodulin mai i ngā pātengi raraunga PDB 4JPZ me 1CFD. Kua whakatakotoria ngā taunga ki te Pūmua Raraunga PDB (PDB) i raro i ngā tau urunga 9NVN (Nan-Iav-CaM kāore he ligand), 9NVO (Nan-Iav-CaM e herea ana ki te nicotinamide), 9NVP (Nan-Iav-CaM e herea ana ki te nicotinamide me te EDTA), 9NVQ (Nan-Iav-CaM e herea ana ki te aphenidolpyrrolline me te konupūmā), 9NVR (Nan-Iav-CaM e herea ana ki te aphenidolpyrrolline me te EDTA), me te 9NVS (Nan pentamer e herea ana ki te aphenidolpyrrolline). Ko ngā whakaahua o te karuārai-irahiko e rite ana kua whakatakotoria ki roto i te Pūtake Raraunga Karuārai-irahiko (EMDB) i raro i ngā tau whakauru e whai ake nei: EMD-49844 (Nan-Iav-CaM kāore he ligand), EMD-49845 (matatini Nan-Iav-CaM me te nicotinamide), EMD-49846 (matatini Nan-Iav-CaM me te nicotinamide me te EDTA), EMD-49847 (matatini Nan-Iav-CaM me te aphidopyrrolline me te konupūmā), EMD-49848 (matatini Nan-Iav-CaM me te aphidopyrrolline me te EDTA), me EMD-49849 (matatini Nan pentamer me te aphidopyrrolline). Kei roto i tēnei pepa ngā raraunga mata mō te tātari mahi.
Wā tuku: Hanuere-28-2026