Imiti yica udukoko igira uruhare runini mu gukemura ibura ry'ibiribwa ku isi no kurwanya indwara ziterwa n'udukoko. Ariko, ikibazo cyo kwiyongera k'ubudahangarwa bw'imiti yica udukoko gisaba byihutirwa kuvumbura ibintu bishya byibasira intego zidakoreshwa neza. Imiyoboro ya TRPV—Nanzhong (Nan) na idakora (Iav)—ishobora gukora imiyoboro itandukanye (Nan-Iav) ikagera ku ngingo z'umubiri zikoresha uburyo bwo kumva, kumva no kumenya udukoko. Imiti imwe n'imwe, nka aphidopyrrolidone (AP), yibasira Nan-Iav binyuze mu buryo butazwi. AP ikora neza mu kurwanya udukoko dutobora (hemipterans), ikabuza kurya ibiryo binyuze mu kubangamira imikorere y'udusimba. AP ishobora gufatana na Nan gusa, ariko Nan-Iav yonyine ni yo ishobora gukorana n'imiti irwanya udukoko, harimo na nicotinamide yo mu bwoko bwa endogenous (NAM), bityo ikagaragaza imikorere y'imiyoboro. Nubwo Nan-Iav ifite ubushobozi bwo gutera imiti yica udukoko, nta byinshi bizwi ku bijyanye n'ihuriro ryayo, ahantu hashyirwaho amabwiriza, n'amabwiriza ashingiye kuri Ca2+, bikabangamira iterambere ry'imiti yica udukoko. Muri ubu bushakashatsi, hakoreshejwe mikorosikope ya cryo-electron kugira ngo hamenyekane imiterere ya Nan-Iav mu dukoko twa Hemiptera mu gihe nta ruhushya rwa calmodulin-ligand rurimo, ndetse na AP na NAM ku mupaka wa ankyrin repeat cytoplasmic domain (ARD). Igitangaje ni uko twasanze poroteyine ya Nan ubwayo ishobora gukora pentamer, igatuzwa n'imikoranire ya ARD iterwa na AP. Ubu bushakashatsi bugaragaza imikoranire ya molekile hagati y'imiti yica udukoko n'imiti irwanya udukoko na Nan-Iav, bugaragaza akamaro ka ARD mu mikorere no guteranya imiyoboro, kandi bunasuzuma uburyo Ca2+ ikoreshwa mu kugenzura.
Mu gihe hari imihindagurikire y’ikirere ikomeje kwiyongera ku isi, ihungabana ry’umutekano w’ibiribwa ku isi ni kimwe mu bibazo bikomeye byo mu kinyejana cya 21, bikagira ingaruka mbi ku muryango.1,2Raporo y’Ishami ry’Umuryango w’Abibumbye ryita ku Buzima ku Mirire n’Imirire ku Isi yo mu 2023 (SOFI) ivuga ko abantu bagera kuri miliyari 2.33 ku isi barwaye indwara yo kubura ibiribwa ku rugero rwo hagati kugeza ku rugero rukabije, ikibazo kimaze igihe kirekire.3,4Ikibabaje ni uko umusaruro w’ibihingwa ugabanuka buri mwaka bitewe n’udukoko n’udukoko, kandi ubushyuhe bw’isi bwitezweho kongera imbaraga mu kurwanya udukoko no mu guhangana n’ingaruka z’ibihingwa.4, 5, 6, 7, 8Guteza imbere imiti yica udukoko ni ingenzi cyane, atari ukurinda gusa ibihingwa udukoko no kugabanya ikwirakwira ry’udukoko twanduza inyamaswa, ahubwo no kurwanya indwara ziterwa n’udukoko nk’indwara ya dengue, malariya, n’indwara ya Chagas, zikomeje kurwanywa n’imiti yica udukoko.5, 9, 10, 11
Mu bintu by’ingenzi byibasirwa n’imiti yica udukoko itera ubwonko, umuyoboro wa TRPV witwa heterotetrameric Nanchung (Nan)-Inactive (Iav) uhagarariye ubwoko bw’imiti yica udukoko yavumbuwe mu myaka icumi ishize, harimo n’imiti yica udukoko iboneka ku isoko nka imidacloprid na pyraclostrobin.12, 13, 14Umuti wica udukoko witwa aphidopyrrolifen (AP) uherutse guterwa no kugurishwa, igice cyawo nyamukuru kikaba ari umuti wica udukoko witwa Inscalis®, uhuza AP ku rwego rw’ibikorwa bya subnanomolar.15AP igaragaza uburozi buke ku bitera ubwandu, udukoko twiza, n'ibindi binyabuzima bitari ibyangiritse, kandi iyo ikoreshejwe hakurikijwe amabwiriza ari ku kirango, ishobora kugabanya imbaraga zo kurwanya indi miti yica udukoko.16, 17, 18Nan na Iav bikwirakwira cyane mu moko y'udukoko, bihurira gusa mu mitsi y'inyunganiramirire ya chordal stretch receptor y'antene n'amaguru, kandi ni ingenzi cyane mu kumva, kumva imbaraga zirenze urugero, no kumenya neza aho ibintu biherereye.13, 16, 19, 20, 21, 22AP, imidacloprid, na pyraclostrobin bitera imbaraga Nan-Iav complex binyuze mu buryo budasanzwe, amaherezo bikabuza uburyo bwo gutanga ibimenyetso.13, 16, 23Mu dukoko two mu bwoko bwa hemipterans (hemipterans) nka aphids na whiteflies, kubura uburyo bwo kumenya imiterere y’utundi dukoko bigabanya ubushobozi bwatwo bwo kurya, amaherezo bigatera urupfu.13,24Igishimishije ni uko AP igaragaza ubufatanye bukomeye kuri Nan-Iav complex kandi uburinganire buke kuri Nan gusa. Guhuza AP na Nan-Iav bitera amashanyarazi, ariko guhuza na Nan gusa ntibitera imikorere y'umuyoboro. Iav ubwayo ntabwo ihuza na AP na gato.16Ibi bigaragaza ko Nan na Iav bishobora kwihuza kugira ngo bikore ubwoko butandukanye bwa Nan-Iav channel complexes (urugero, hamwe n’ibipimo bitandukanye bya stoichiometric cyangwa imiterere itandukanye mu gipimo kimwe cya stoichiometric) cyangwa ko AP ishobora kwihuza n’ahantu henshi. Byongeye kandi, nicotinamide karemano ya agonist (NAM) ihuza na Drosophila Nan-Iav ifite ubushobozi bwa micromolar, ikagaragaza ingaruka nk’iza aphids (AP) muri vitro.16,25no kubuza kororoka no kugaburira inda, amaherezo bigatera gupfa kwazo25,26. Aya makuru atera ibibazo byinshi. Urugero, ntibirasobanuka neza uburyo heterodimer ya Nan-Iav ikorwa, ahantu ho guhuza hakoreshwa mu guhindura molekile nto, n'uburyo izi molekile nto zigenzura imikorere y'imiyoboro binyuze mu guhagarika proprioception. Byongeye kandi, impamvu Nan ubwayo idakora kandi ifite affinity nke kuri AP, mu gihe heterodimer ya Nan-Iav ikora kandi igahuza AP na affinity yo hejuru, ntabwo zirasobanuka neza. Amaherezo, nta byinshi bizwi ku bijyanye no kugenzura imikorere ya Nan-Iav bishingiye kuri Ca2+ n'uburyo yinjizwa mu nzira zo gutanga ibimenyetso by'imitsi.. 13,21
Muri ubu bushakashatsi, dukoresheje mikorosikopi ya cryo-electron, electrophysiology, na tekiniki zo gufatanya na radioligand, twasobanuye uburyo Nan-Iav iteranywa n'uburyo ifatana n'udukoko duto. Byongeye kandi, twabonye calmodulin (CaM) ifatanye na Iav na Nan pentamers za AP-stabilized. Ibi bisubizo bitanga ubumenyi bw'ingenzi ku miyoborere ya iyoni za kalisiyumu mu miyoboro, mu guteranya imiyoboro, n'ibintu bigena uburyo ligand ifatana. Icy'ingenzi kurushaho, twemeje ko ARD igira uruhare runini muri izi nzira. Inyigo yacu ku miyoboro yuzuye y'udukoko ifatanye n'imiti yica udukoko mu buhinzi ikoreshwa mu buryo bufatika.27, 28, 29bifungura amahirwe yo guteza imbere inganda zikoresha imiti yica udukoko, binoza imikorere n'uburyo imiti yica udukoko ikoreshwa, kandi bigafasha ikoreshwa ry'imiti ikoreshwa muri TRPV ku zindi nyamaswa kugira ngo habeho umutekano w'ibiribwa ku isi no gukwirakwiza indwara ziterwa n'udukoko.
Twasanze kandi ko Nan-Iav igenzurwa na Ca2+, kandi uburyo bwo kuyigenga bugenzurwa na CaM ifatanye. Icy'ingenzi ni uko iyi gahunda ya Ca2+ ishingiye kuri Nav na CaM itandukanye cyane n'uburyo bwo kuyigenga bw'indi miyoboro ya ion (urugero, imiyoboro ya Na+ igenzurwa na voltage na TRPV5/6)52,53,54,55,56,57Muri Nav1.2 channel, domaine ya C-terminal ya CaM ifitanye isano n'domaine ya C-terminal (CTD), naho Ca2+ igatuma domaine yayo ya N-terminal ifatana n'igice cya kure cya CTD.56Muri TRPV5/6 channel, domaine ya C-terminal ya CaM ifatana na CTH, maze Ca2+ itera kwiyongera kwa domaine yayo ya N-terminal mu mwobo, bityo ikabuza cation permeability53,54. Turatanga icyitegererezo cy'imikorere ya Ca2+ ya Nan-Iav-CaM (Ishusho ya 4h). Muri iki cyitegererezo, urwego rwa N-terminal rwa CaM ruhuza buri gihe na C-terminal domain (CTH) ya Iav. Mu gihe cyo kuruhuka (ubucucike buke bwa [Ca2+], urwego rwa C-terminal rwa CaM rukorana na Nan, bigatuza imiterere ya ARD bityo bigatera imbere gufungura umuyoboro. Gufata umuti wica udukoko ku muyoboro bitera gufungura utwenge, bigatera Ca2+ kwinjira. Ca2+ hanyuma ifata CaM, bigatera kwitandukanya kwa C-terminal na ARD ya Nan. Kubera ko guhagarika kwihambira kwa CaM ahanini bikuraho ingaruka zo gukumira za Ca2+, uku kwitandukanya guhindura imikorere ya ARD, bityo bigatera kubuzwa cyangwa kudasobanukirwa na Ca2+. Kugaruka vuba kw'imiyoboro y'imiyoboro nyuma yo gukuraho iyoni ya kalisiyumu (Ishusho ya 4g) bigaragaza ko ubu buryo bworohereza gusubiza vuba ibimenyetso by'ubwonko bya Ca2+. Byongeye kandi, agace ka C-terminal ka Iav, katarasobanuka neza, byatangajwe ko gafite izindi nshingano mu gushakira hamwe uburyo bwo kugenzura no kugenzura ubu.21
Amaherezo, ubushakashatsi bwacu bugaragaza imiterere y’umuyoboro wa TRP w’udukoko duto duto ufite akamaro mu buhinzi—ikintu twari tutazi mbere. Cyane cyane, twagaragaje imiterere n’imikorere y’umuyoboro w’udukoko mu turemangingo tw’abantu (HEK293S GnTi–) aho kuba mu turemangingo tw’udukoko. Mu gihe cy’ubudahangarwa bw’udukoko buri kwiyongera ndetse n’igitutu gikomeje ku mutekano w’ibiribwa n’udukoko, akazi kacu gatanga amakuru y’ingenzi azafasha mu iterambere ry’udukoko duto duto ku bw’inyungu z’ubuzima bw’abantu no mu mutekano w’ibiribwa ku isi. Ubushakashatsi bwagaragaje ko udukoko nka AP tugira ingaruka nziza ku dukoko tumwe na tumwe iyo dukoreshejwe hakurikijwe amabwiriza ari ku kirango kandi dufite uburozi buke ku bitera ubwandu by’ingirakamaro, bigaragaza umutekano wabyo mu bidukikije.13,16Byongeye kandi, igeragezwa rya zimwe mu nganda zikomoka kuri AP ku mibu ryagaragaje ko amaherezo zitakaza ubushobozi bwo kuguruka. Gusobanukirwa uburyo izi nganda zihindura imiterere yazo zifatanya na Nan-Iav bizafasha guhindura imvange isanzwe cyangwa guteza imbere imvange nshya kugira ngo zirusheho kugira akamaro no gukora neza.nezakurwanya udukoko. Ubushakashatsi bwacu bwerekanye ko uburyo bwa Nan-Iav ARD bukoreshwa atari ingenzi gusa mu kugenzura imikorere y'ibintu bikomoka ku bimera, imiti yica udukoko, na Ca2+-CaM, ahubwo no mu guteranya imiyoboro. Turagaragaza ko guhagarika guteranya heterodimer hakoreshejwe molekile nto bishobora kuba uburyo bwihariye kandi butanga icyizere mu guteza imbere uburyo bwo gukumira imiyoboro ya iyoni.
Muri gene umunani z’imiterere y’umubiri, gene z’uburebure bwose bw’ikinyabuzima cy’umukara (Halyomorpha halys) Nanchung na Inactive zatoranijwe, zigaragaza ko zihamye cyane mu byuma bisukura. gene zakozwe zahinduwe kodon kugira ngo zigaragaze neza imiterere y’umuntu maze zishyirwa muri pBacMam pCMV-DEST vector (Life Technologies) hakoreshejwe ahantu habuza XhoI na EcoRI. Ibi byatumye clones zigaragara hamwe na C-terminal GFP-FLAG-10xHis na mCherry-FLAG-10xHis tags, zigacibwamo HRC-3C protease (PPX), bigatuma zigenga.imiterere. Imashini zakoreshejwe mu gushushanya Nanchung na Inactive muri vektori ya pBacMam zari izi zikurikira:
Amashusho ya mikorosikopi y'uduce duto twabonetse kuri mikorosikopi ya Titan Krios G2 electron transmission (FEI) ifite kamera ya K3 na Gatan BioQuantum energy filter. Mikorosikopi yakoreshejwe kuri 300 keV, ifite ingufu za 20 eV, ingano ya pikseli ya 1.08 Å/pixel (nominal magnification ya 81.000x), na gradient ya defocus iri hagati ya -0.8 na -2.2 μm. Amashusho yafashwe kuri frame 40 ku isegonda hakoreshejwe mikorosikopi ya Latitude S (Gatan) ifite igipimo cya dose cya 25 e–px−1 s−1, igihe cyo kwigaragaza cya 2.4 s, na dose yose ya 60 e–Å−2.
Gukosora ingendo hakoreshejwe imirasire no gupima uburemere bw'umubare byakozwe kuri firime hakoreshejwe MotionCor2 muri RELION 4.061. Isuzuma ry'imiterere y'impinduka (CTF) ryakozwe muri cryoSPARC hakoreshejwe uburyo bwa CTF bushingiye ku gipimo62. Fotografi zifite ubushobozi bwo guhuza CTF ≥4 Å ntizakuwe mu isesengura ryakurikiyeho. Ubusanzwe, itsinda rya fotografi 500–1000 ryakoreshejwe mu guhitamo ingingo muri cryoSPARC, hagakurikiraho ibyiciro byinshi byo gushyira mu byiciro bya 2D nyuma yo kuyungurura kugira ngo haboneke ishusho isobanutse yo guhitamo ibyiciro bishingiye ku gishushanyo. Uduce twakuwemo hakoreshejwe udusanduku twa pixel 64 no gufunga inshuro 4. Ibyiciro byinshi byo gushyira mu byiciro bya 2D byakozwe kugira ngo hakurweho ibyiciro by'uduce tudakenewe. Icyitegererezo cya mbere cya 3D cyongeye kubakwa hakoreshejwe kongera kubaka ab initio no kunoza hakoreshejwe gutunganya non uniform muri cryoSPARC. Ibyiciro bya 3D byakozwe muri cryoSPARC cyangwa RELION bishingiye ku itandukaniro rya ARD. Nta tandukaniro rikomeye ry'uturere twa membrane ryagaragaye. Uduce twatunganyijwe hakoreshejwe uburyo bwa C1 na C2; utwo duce dufite ubushobozi bwo hejuru bwa C2 twafatwaga nk'uduhuje ugereranije na C2 maze twinjizwa muri RELION kugira ngo twongere uburyo bwa Bayesian. Utwo duce twasubijwe muri cryoSPARC kugira ngo twongere uburyo bwa nyuma butari bumwe kandi busanzwe. Ubushobozi bwa nyuma n'ibarwa ry'uduce bigaragara muri Imbonerahamwe ya 1.
Ubwo twatunganyaga Nan + AP pentamers, twashakishije uburyo butandukanye bwo kunoza uburyo membrane domains zigaragaramo (cyane cyane agace k'imyenge), nko gukuraho ibimenyetso no gupfuka TMD. Ariko, ibi byagerageje ntibyagenze neza bitewe n'ikibazo gikomeye gishobora kubaho mu gace k'imyenge ndetse n'ubusumbane muri rusange bwa TMD. Igisubizo cya nyuma cyabazwe hakoreshejwe agapfukamunwa gakozwe mu buryo butazwi muri cryoSPARC, cyane cyane mu gace ka ARD. Ibi byageze ku busumbane buruta ubw'agace k'imyenge (cyane cyane agace ka VSLD).
Ingero nshya za mbere z’imiterere y’udukoko twa Nanchung na Inactive zabanje gukorwa hakoreshejwe Coot63, naho ingero z’udukoko twa Nan na Iav zakozwe hakoreshejwe AlphaFold264 kugira ngo hamenyekane uturere tudakomeye. Ingero za Calmodulin zashingiye ku miterere ihamye y’imiterere ya Ca2+ na Ca2+ muri PDB accessions 4JPZ56 na 1CFD65, uko bikurikirana. Ingero zatunganyijwe hakoreshejwe uburyo bwo gutunganya imiterere kugira ngo hamenyekane stereochemistry ikwiye n’imiterere myiza. Phosphatidylcholine, phosphatidylethanolamine, na phosphatidylserine byaje gushushanywa nk’ubucucike bwa lipide bugenwe neza, maze ligande za NAM na AP zishyirwa mu bucucike bujyanye n’aho zihurira. Dosiye z’inzitizi zakozwe uhereye ku murongo wa SMILES wa isoforms hakoreshejwe eLBOW muri PHENIX66. Amaherezo, ingero zatunganyijwe mu mwanya nyawo muri PHENIX hakoreshejwe ishakisha ry’aho uherereye no kugabanya imiterere y’isi yose hamwe n’imbogamizi z’imiterere ya kabiri. Seriveri ya MolProbity yakoreshejwe mu kunoza icyitegererezo no gusesengura imiterere, kandi amashusho yakozwe hakoreshejwe PyMOL na UCSF Chimera X. 67,68,69 Isesengura ry'ahabonetse ryakozwe hakoreshejwe seriveri ya HOLE,70 naho igenamigambi ryo kubungabunga imiterere ryakozwe hakoreshejwe seriveri ya Consurf.71
Isesengura ry'imibare ryakozwe hakoreshejwe Igor Pro 6.2, Excel Office 365, na GraphPad Prism 7.0. Amakuru yose y'imibare agaragazwa nk'ikosa risanzwe rya ± (SEM). Ikizamini cya t cy'umunyeshuri (cuzuye imirizo ibiri, kidahujwe) cyakoreshejwe mu kugereranya amatsinda abiri. Isesengura ry'uburyo bumwe bw'impinduka (ANOVA) rikurikirwa n'ikizamini cya nyuma cya Dunnett cyakoreshejwe mu kugereranya amatsinda menshi. *P< 0.05, **P< 0.01, na ***P<0.001 byafashwe nk'ibifite agaciro gakomeye mu mibare bitewe n'ikwirakwizwa ry'amakuru. Agaciro ka Kd, Ki, hamwe n'intera z'icyizere zabyo za 95% zidafite ingano byabazwe hakoreshejwe GraphPad Prism 10.
Kugira ngo umenye byinshi ku buryo bwo kwiga, reba Incamake ya Raporo y'Uburyo bw'Ibintu Bihari ihujwe muri iyi nkuru.
Icyitegererezo cya mbere cyakozwe hakoreshejwe moderi za calmodulin zo muri database za PDB 4JPZ na 1CFD. Imirongo yashyizwe muri Protein Data Bank (PDB) munsi y'imibare ya 9NVN (Nan-Iav-CaM idafite ligand), 9NVO (Nan-Iav-CaM ihambiriye kuri nicotinamide), 9NVP (Nan-Iav-CaM ihambiriye kuri nicotinamide na EDTA), 9NVQ (Nan-Iav-CaM ihambiriye kuri aphenidolpyrrolline na calcium), 9NVR (Nan-Iav-CaM ihambiriye kuri aphenidolpyrrolline na EDTA), na 9NVS (Nan pentamer ihambiriye kuri aphenidolpyrrolline). Amashusho ya mikorosikopi ya cryo-electron ajyanye nayo abikwa muri Database ya Electron Microscopy (EMDB) munsi y'imibare ikurikira: EMD-49844 (Nan-Iav-CaM idafite ligand), EMD-49845 (Nan-Iav-CaM complex ifite nicotinamide), EMD-49846 (Nan-Iav-CaM complex ifite nicotinamide na EDTA), EMD-49847 (Nan-Iav-CaM complex ifite aphidopyrrolline na calcium), EMD-49848 (Nan-Iav-CaM complex ifite aphidopyrrolline na EDTA), na EMD-49849 (Nan pentamer complex ifite aphidopyrrolline). Amakuru y'ibanze y'isesengura ry'imikorere agaragazwa muri iyi nyandiko.
Igihe cyo kohereza: Mutarama-28-2026