Magungunan kashe kwari suna taka muhimmiyar rawa wajen magance ƙarancin abinci a duniya da kuma yaƙi da cututtukan da ke ɗauke da ƙwayoyin cuta. Duk da haka, matsalar da ke ƙaruwa ta juriya ga magungunan kashe kwari cikin gaggawa tana buƙatar gano sabbin sinadarai waɗanda ke kai hari ga wuraren da ba a yi amfani da su sosai ba. Tashoshin masu karɓar ƙwayoyin cuta na ɗan lokaci (TRPV) - Nanzhong (Nan) da marasa aiki (Iav) - na iya samar da hanyoyin sadarwa daban-daban (Nan-Iav) kuma su zama wuraren da ke da alaƙa da tsarin ƙasa, ji, da kuma kama-karya a cikin kwari. Wasu magungunan kashe kwari, kamar aphidopyrrolidone (AP), suna kai hari ga Nan-Iav ta hanyar hanyoyin da ba a sani ba. AP yana da tasiri akan kwari masu tsotsa (hemipterans), yana hana ciyarwa ta hanyar katse aikin filaments. AP na iya ɗaurewa ga Nan kawai, amma Nan-Iav ne kawai zai iya hulɗa da agonists, gami da nicotinamide na ciki (NAM), don haka yana nuna ayyukan tashoshi. Duk da yuwuwar Nan-Iav a matsayin abin da ake nufi da maganin kwari, ba a san komai game da haɗuwar hanyoyinsa ba, wuraren ɗaure magunguna, da kuma ƙa'idojin Ca2+-dogara, suna hana ci gaban kwari. A cikin wannan binciken, an yi amfani da na'urar duba ƙwayoyin cuta ta cryo-electron don tantance tsarin Nan-Iav a cikin kwari na Hemiptera a cikin yanayin da ba shi da calmodulin-ligand, da kuma tare da AP da NAM a iyakar yankin cytoplasmic na ankyrin maimaituwa (ARD). Abin mamaki, mun gano cewa furotin Nan da kansa zai iya samar da pentamer, wanda ke daidaita ta hanyar hulɗar ARD da AP ke jagoranta. Wannan binciken ya bayyana hulɗar kwayoyin halitta tsakanin kwari da masu agonists da Nan-Iav, yana nuna mahimmancin ARD a cikin aikin tashar da haɗuwa, da kuma bincika tsarin ƙa'idar Ca2+.
Dangane da yanayin sauyin yanayi da ke ƙara tsananta a duniya, tabarbarewar tsaron abinci a duniya na ɗaya daga cikin manyan ƙalubalen ƙarni na 21, tare da haifar da sakamako mai yawa ga al'umma.1,2Rahoton Hukumar Lafiya ta Duniya kan Tsaron Abinci da Gina Jiki a Duniya na 2023 (SOFI) ya kiyasta cewa kimanin mutane biliyan 2.33 a duk duniya suna fama da matsalar rashin abinci mai tsanani, wata matsala da ta daɗe tana faruwa.3,4Abin takaici, an kiyasta cewa kashi 20% zuwa 30% ko fiye na amfanin gona ana asarar su kowace shekara sakamakon kwari da cututtuka, kuma ana sa ran ɗumamar yanayi zai ƙara ta'azzara juriyar kwari da raunin amfanin gona.4,5,6,7,8Ci gaban magungunan kashe kwari yana da matuƙar muhimmanci ba wai kawai don kare amfanin gona daga kwari da kuma rage yaɗuwar ƙwayoyin cuta da ke ɗauke da ƙwayoyin cuta ba, har ma don yaƙi da cututtukan da ke ɗauke da ƙwayoyin cuta kamar zazzabin dengue, malaria, da cutar Chagas, waɗanda ke ƙara jure wa magungunan kashe kwari.5,9,10,11
Daga cikin manyan abubuwan da ake hari kan magungunan kashe kwari masu guba a kwakwalwa, tashar TRPV mai suna heterotetrameric Nanchung (Nan)-Inactive (Iav) tana wakiltar wani nau'in magungunan kashe kwari da aka gano ne kawai a cikin shekaru goma da suka gabata, ciki har da magungunan kashe kwari da ake samu a kasuwa kamar imidacloprid da pyraclostrobin.12, 13, 14Maganin kwari mai suna aphidopyrrolifen (AP) wani samfuri ne da aka ƙirƙiro kwanan nan kuma aka sayar da shi a kasuwa wanda babban sinadarinsa shine maganin kwari mai aiki Inscalis®, wanda ke ɗaure AP a matakin aikin subnanomolar.15AP yana nuna ƙarancin guba mai tsanani ga masu yin fure, kwari masu amfani, da sauran halittu marasa manufa, kuma idan aka yi amfani da shi bisa ga umarnin lakabin, yana iya rage matsin lamba ga sauran kwari.16, 17, 18Nan da Iav suna yaɗuwa sosai a cikin nau'ikan kwari, ana bayyana su tare a cikin ƙwayoyin jijiyoyi na antennae da gaɓoɓi, kuma suna da mahimmanci ga ji, fahimtar nauyi, da kuma fahimtar dabi'a.13,16,19,20,21,22AP, imidacloprid, da pyraclostrobin suna ƙarfafa hadaddun Nan-Iav ta hanyar wata hanya ta musamman, wanda a ƙarshe ke hana jigilar siginar da ke aiki da kansa.13, 16, 23A cikin kwari masu tsotsa (hemipterans) kamar aphids da whiteflies, rashin iya cin abinci yana lalata ikon cin abincinsu, wanda a ƙarshe ke haifar da mutuwa.13,24Abin sha'awa, AP tana nuna babban sha'awa ga hadaddun Nan-Iav kuma ƙarancin sha'awa ga Nan kaɗai. Haɗa AP zuwa Nan-Iav yana haifar da wutar lantarki, amma ɗaurewa zuwa Nan kaɗai ba ya ƙarfafa ayyukan tashar. Iav da kansa ba ya ɗaurewa da AP kwata-kwata.16Wannan yana nuna cewa Nan da Iav na iya ɗaurewa don samar da hadaddun hanyoyin Nan-Iav daban-daban (misali, tare da rabon stoichiometric daban-daban ko shirye-shirye daban-daban a cikin rabon stoichiometric iri ɗaya) ko kuma AP na iya ɗaurewa zuwa wurare da yawa. Bugu da ƙari, nicotinamide na halitta mai hana ƙwayoyin cuta (NAM) yana ɗaurewa zuwa Drosophila Nan-Iav tare da kusancin micromolar, yana nuna tasirin kama da na aphids (AP) a cikin vitro.16,25da kuma hana haifuwa da ciyar da aphids, wanda a ƙarshe ya kai ga mutuwarsu25,26. Waɗannan bayanai suna tayar da tambayoyi da yawa. Misali, har yanzu ba a fayyace yadda aka samar da heterodimer na Nan-Iav ba, waɗanne wurare ne ake amfani da su don daidaita ƙananan ƙwayoyin halitta, da kuma yadda waɗannan ƙananan ƙwayoyin halitta ke daidaita aikin tashoshi ta hanyar danne proprioception. Bugu da ƙari, dalilan da yasa Nan da kanta ba ta aiki kuma tana da ƙarancin kusanci ga AP, yayin da heterodimer na Nan-Iav ke aiki kuma yana ɗaure AP da babban kusanci, har yanzu ba a fayyace ba. A ƙarshe, ba a san komai game da ƙa'idar Ca2+-dependent na aikin Nan-Iav da kuma yadda aka haɗa shi cikin hanyoyin siginar jijiyoyi ba.. 13,21
A cikin wannan binciken, tare da haɗa fasahar cryo-electron microscopy, electrophysiology, da dabarun ɗaure radioligand, mun bayyana haɗuwar Nan-Iav da tsarin ɗaure shi ga ƙananan masu kula da ƙwayoyin halitta. Bugu da ƙari, mun gano calmodulin (CaM) da aka haɗa a cikin tsari zuwa ga Iav da Nan pentamers masu daidaita AP. Waɗannan sakamakon suna ba da mahimman bayanai game da daidaita ions na calcium a cikin tashoshi, haɗuwar tashoshi, da abubuwan da ke ƙayyade alaƙar ɗaure ligand. Mafi mahimmanci, mun tabbatar da cewa ARD tana taka muhimmiyar rawa a cikin waɗannan hanyoyin. Bincikenmu na cikakkun hanyoyin kwari da aka haɗa da magungunan kashe ƙwari na noma masu dacewa.27, 28, 29yana buɗe damar ci gaban masana'antar magungunan kashe kwari, yana inganta inganci da takamaiman magungunan kashe kwari, da kuma ba da damar amfani da sinadarai masu TRPV ga wasu nau'ikan halittu don magance tsaron abinci a duniya da kuma yaɗuwar cututtukan da ke ɗauke da ƙwayoyin cuta.
Mun kuma gano cewa Nan-Iav yana ƙarƙashin tsarin Ca2+, kuma tsarin gudanarwa yana ƙarƙashin tsarin CaM mai ɗaurewa. Abu mafi mahimmanci, wannan tsarin Nav mai dogaro da Ca2+ ya bambanta sosai da tsarin daidaita wasu tashoshin ion (misali, tashoshin Na+ mai ƙarfin lantarki da tashoshin TRPV5/6)52,53,54,55,56,57A cikin tashar Nav1.2, yankin C-terminal na CaM yana da alaƙa da yankin C-terminal (CTD), kuma Ca2+ yana haifar da ɗaure yankin N-terminal ɗinsa zuwa ɓangaren nesa na CTD.56A cikin tashar TRPV5/6, yankin C-terminal na CaM yana ɗaurewa da CTH, kuma Ca2+ yana haifar da faɗaɗa yankin N-terminal ɗinsa zuwa cikin rami, ta haka yana toshe damar shiga cation.53,54. Mun gabatar da samfurin aikin da aka tsara na Ca2+ na Nan-Iav-CaM (Hoto na 4h). A cikin wannan samfurin, yankin N-terminal na CaM yana ɗaure zuwa yankin C-terminal (CTH) na Iav. A cikin yanayin hutawa (ƙarancin yawan [Ca2+]), yankin C-terminal na CaM yana hulɗa da Nan, yana daidaita yanayin ARD kuma yana haɓaka buɗewar tashoshi. Ɗaurewar agonist/ƙwari ga tashar yana haifar da buɗewar ramuka, yana haifar da kwararar Ca2+. Sannan Ca2+ yana ɗaure zuwa CaM, yana haifar da rabuwar yankin C-terminal daga ARD na Nan. Saboda toshe haɗin CaM yana kawar da tasirin hana Ca2+, wannan rabuwar yana daidaita motsi na ARD, wanda hakan ke haifar da hanawa ko rage hankali ga Ca2+. Saurin murmurewa na kwararar tashoshi bayan fitar da ion na calcium (Hoto na 4g) yana nuna cewa wannan tsarin yana sauƙaƙe martani cikin sauri ga siginar neuronal na Ca2+. Bugu da ƙari, an ruwaito cewa yankin C-terminal na Iav, wanda har yanzu ba a fahimce shi sosai ba, yana taka wasu rawa a cikin niyya ta hanyoyin sadarwa da kuma ƙa'idojin yanzu.21
A ƙarshe, bincikenmu ya gabatar da babban tsari na wani babban tsari na tashar TRP mai maganin kwari da kwari mai mahimmanci a fannin noma - wani abu da ba mu sani ba a da. Abin lura shi ne, mun bayyana tsari da aikin hanyar kwari a cikin ƙwayoyin ɗan adam (HEK293S GnTi–) maimakon a cikin ƙwayoyin kwari. Dangane da ƙaruwar juriyar kwari da kuma ci gaba da matsin lamba kan tsaron abinci da ƙwayoyin cuta, aikinmu yana ba da muhimman bayanai waɗanda za su sauƙaƙa haɓaka sabbin magungunan kwari don amfanin lafiyar ɗan adam da tsaron abinci na duniya. Nazarin ya nuna cewa magungunan kwari kamar AP suna da tasiri ga wasu kwari idan aka yi amfani da su bisa ga umarnin lakabi kuma suna da ƙarancin guba ga masu amfani da ƙwayoyin cuta, suna nuna amincin muhallinsu.13,16Bugu da ƙari, gwajin wasu abubuwan da aka samo daga AP akan sauro ya nuna cewa daga ƙarshe sun rasa ikon tashi. Fahimtar yadda waɗannan mahaɗan da ke daidaita abubuwa ke haɗuwa da Nan-Iav zai sauƙaƙa gyaggyara mahaɗan da ke akwai ko haɓaka sabbin mahaɗan don ƙarin inganci da inganci.daidaimaganin kwari. Bincikenmu ya nuna cewa hanyar sadarwa ta Nan-Iav ARD tana da matuƙar muhimmanci ba kawai don daidaita ayyukan mahaɗan da ke cikin jiki, magungunan kashe kwari, da Ca2+-CaM ba, har ma don haɗa hanyoyin sadarwa. Muna ba da shawarar cewa wargaza haɗuwar heterodimer tare da ƙananan ƙwayoyin cuta na iya zama hanya ta musamman kuma mai ban sha'awa don haɓaka masu hana tashar ion.
Daga cikin kwayoyin halitta guda takwas na orthologous, an zaɓi cikakkun kwayoyin halitta na ƙwaro mai launin ruwan kasa (Halyomorpha halys) Nanchung da Inactive, wanda ke nuna kyakkyawan daidaito a cikin sabulun wanke-wanke. An inganta kwayoyin halittar da aka haɗa ta hanyar codon don bayyanar ɗan adam kuma an haɗa su cikin vector pBacMam pCMV-DEST (Life Technologies) ta amfani da wuraren ƙuntatawa na XhoI da EcoRI. Wannan ya tabbatar da cewa ƙwayoyin suna cikin firam tare da alamun C-terminal GFP-FLAG-10xHis da mCherry-FLAG-10xHis, waɗanda HRC-3C protease (PPX) suka raba, wanda hakan ya ba da damar zaman kanta.bayyanawa. An yi amfani da firam ɗin da aka yi amfani da su don yin kwafi na Nanchung da Inactive zuwa cikin vector na pBacMam kamar haka:
An samo hotunan ƙwayoyin cuta na ƙwayoyin cuta daban-daban a kan na'urar hangen nesa ta Titan Krios G2 transmission electron microscope (FEI) wacce aka sanya mata kyamarar K3 da matatar makamashi ta Gatan BioQuantum. An yi amfani da na'urar hangen nesa a 300 keV, tare da saitin kuzari na 20 eV, girman samfurin pixel na 1.08 Å/pixel (ƙarfafawa ta musamman ta 81,000x), da kuma matakin defocus wanda ya kama daga -0.8 zuwa -2.2 μm. An yi rikodin bidiyo a firam 40 a kowace daƙiƙa ta amfani da na'urar hangen nesa ta Latitude S (Gatan) tare da ƙimar allurar da ba a ƙayyade ba ta 25 e–px−1 s−1, lokacin fallasa ta 2.4 s, da kuma jimlar allurar da ta kai kusan 60 e–Å−2.
An yi gyaran motsi da kuma auna yawan da aka yi ta hanyar haske a fim ta amfani da MotionCor2 a cikin RELION 4.061. An yi kimanta ma'aunin aikin canja wurin bambanci (CTF) a cikin cryoSPARC ta amfani da hanyar kimantawa ta CTF bisa faci62. An cire hotunan micrographs tare da ƙudurin daidaitawa na CTF ≥4 Å daga binciken da ya biyo baya. Yawanci, an yi amfani da ƙaramin rukuni na hotunan micrographs 500-1000 don zaɓar maki a cikin cryoSPARC, sannan aka biyo baya da zagaye da yawa na rarrabuwa na 2D bayan tacewa don samun hoton tunani mai haske don zaɓar barbashi bisa samfuri. Daga nan aka cire barbashi ta amfani da akwatunan ɗaure pixel 64 da binning na ninki 4. An yi zagaye da yawa na rarrabuwa na 2D don cire nau'ikan barbashi da ba a so. An sake gina samfurin 3D na farko ta amfani da sake gina ab initio kuma an inganta shi ta amfani da gyaran ab initio mara tsari a cikin cryoSPARC. An yi rarrabuwar 3D a cikin cryoSPARC ko RELION bisa ga bambancin ARD. Ba a lura da bambancin da ke tsakanin yankunan membrane ba. An tsaftace ƙwayoyin cuta ta amfani da hanyoyin C1 da C2; an ɗauki ƙwayoyin cuta masu ƙudurin C2 mafi girma a matsayin masu daidaito dangane da C2 kuma an shigo da su cikin RELION don gyaran Bayesian. Daga nan aka mayar da ƙwayoyin zuwa cryoSPARC don gyaran ƙarshe mara tsari da na gida. An nuna ƙudurin ƙarshe da ƙididdigar ƙwayoyin cuta a cikin Tebur 1.
Lokacin da muke sarrafa pentamers na Nan+AP, mun binciki hanyoyi daban-daban don inganta warware yankunan membrane (musamman yankin pore), kamar rage sigina da rufe TMD. Duk da haka, waɗannan yunƙurin ba su yi nasara ba saboda rashin tabbas mai yawa a yankin pore da kuma bambancin TMD gabaɗaya. An ƙididdige ƙudurin ƙarshe ta amfani da abin rufe fuska wanda aka samar ta atomatik ta hanyar sarrafa ba iri ɗaya ba a cikin cryoSPARC, galibi yana mai da hankali kan yankin ARD. Wannan ya sami ƙuduri mafi girma fiye da na yankunan membrane (musamman yankin VSLD).
An fara samar da samfuran farko na novo na nau'ikan apo na kwaro na Nanchung da Inactive ta amfani da Coot63, kuma an samar da samfuran kwaro na Nan da Iav ta amfani da AlphaFold264 don gano yankunan da ba su da ƙarfin gwiwa. Tsarin Calmodulin ya dogara ne akan daidaiton jiki mai tauri na samfuran Ca2+-binding da Ca2+-free a cikin haɗin PDB 4JPZ56 da 1CFD65, bi da bi. An inganta samfuran ta amfani da gyaran fuska don tabbatar da daidaiton stereochemistry da kyakkyawan lissafi. Daga nan aka tsara Phosphatidylcholine, phosphatidylethanolamine, da phosphatidylserine a matsayin ƙayyadaddun yawan lipid, kuma an sanya NAM da AP ligands a cikin yawan da suka dace a cikin mahadar matsewa. An samar da fayilolin ƙuntatawa daga layin SMILES na isoforms ta amfani da eLBOW a cikin PHENIX66. A ƙarshe, an inganta samfuran a cikin sararin samaniya na gaske a cikin PHENIX ta amfani da binciken grid na gida da rage girman duniya tare da ƙuntatawa na tsarin sakandare. An yi amfani da uwar garken MolProbity don gyaran samfura da nazarin tsari, kuma an yi zane-zane ta amfani da PyMOL da UCSF Chimera X. An gudanar da nazarin Aperture na 67,68,69 ta amfani da uwar garken HOLE,70 kuma an gudanar da taswirar kiyaye jerin abubuwa ta amfani da uwar garken Consurf.71
An gudanar da nazarin ƙididdiga ta amfani da Igor Pro 6.2, Excel Office 365, da GraphPad Prism 7.0. Duk bayanan adadi an gabatar da su azaman matsakaicin ± kuskuren daidaitattun (SEM). An yi amfani da gwajin t-test na ɗalibi (mai wutsiya biyu, mara haɗin kai) don kwatanta ƙungiyoyi biyu. An yi amfani da nazarin bambance-bambancen hanya ɗaya (ANOVA) sannan aka biyo bayan gwajin Dunnett na bayan hoc don kwatanta ƙungiyoyi da yawa. *P<0.05, **P<0.01, da kuma ***PAn yi la'akari da <0.001 a matsayin muhimmiyar kididdiga dangane da rarraba bayanai. An ƙididdige ƙimar Kd, Ki, da tazara amincewa mara daidaituwa ta 95% ta amfani da GraphPad Prism 10.
Don ƙarin bayani kan hanyar binciken, da fatan za a duba Takaitaccen Rahoton Fayilolin Yanayi wanda aka haɗa a cikin wannan labarin.
An gina samfurin farko ta amfani da samfuran calmodulin daga bayanan PDB 4JPZ da 1CFD. An ajiye daidaitattun a cikin Bankin Bayanan Protein (PDB) a ƙarƙashin lambobin shiga 9NVN (Nan-Iav-CaM ba tare da ligand ba), 9NVO (Nan-Iav-CaM da aka haɗa zuwa nicotinamide), 9NVP (Nan-Iav-CaM da aka haɗa zuwa nicotinamide da EDTA), 9NVQ (Nan-Iav-CaM da aka haɗa zuwa aphenidolpyrrolline da calcium), 9NVR (Nan-Iav-CaM da aka haɗa zuwa aphenidolpyrrolline da EDTA), da 9NVS (Nan pentamer da aka haɗa zuwa aphenidolpyrrolline). An ajiye hotunan na'urar gano ƙwayoyin cuta ta cryo-electron a cikin Bayanan Ma'ajiyar Bayanai na Electron (EMDB) a ƙarƙashin lambobin shiga masu zuwa: EMD-49844 (Nan-Iav-CaM ba tare da ligand ba), EMD-49845 (Nan-Iav-CaM hadaddun tare da nicotinamide), EMD-49846 (Nan-Iav-CaM hadaddun tare da nicotinamide da EDTA), EMD-49847 (Nan-Iav-CaM hadaddun tare da aphidopyrrolline da calcium), EMD-49848 (Nan-Iav-CaM hadaddun tare da aphidopyrrolline da EDTA), da EMD-49849 (Nan pentamer hadaddun tare da aphidopyrrolline). An gabatar da bayanai na asali don nazarin aiki a cikin wannan takarda.
Lokacin Saƙo: Janairu-28-2026