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Kako mogu izračunati sadržaj alfa heliksa iz molarne eliptičnosti?

Kako mogu izračunati sadržaj alfa heliksa iz molarne eliptičnosti?


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Kako mogu izračunati sadržaj alfa heliksa (tj. postotak) u proteinu iz zadane molarne eliptičnosti od 222 nm, bez korištenja ikakvog softvera. Pokušao sam s Greenfield-Fasmanovom jednadžbom, ali se čini da odgovor nije točan.


Informacije možete koristiti na ovoj stranici, posebno u odjeljku 3. (https://www.photophysics.com/resources/tutorials/circular-dichroism-cd-spectroscopy)

Obratite pažnju na različite spektre CD-a za alfa-helix, beta-listove i nasumične zavojnice. Ako se vaš protein mjeri na 222 nm, trebate usporediti svoje izmjerene vrijednosti sa spektrom na 222 nm, to će vam omogućiti da procijenite sekundarnu strukturu proteina, ali ako želite biti točni, morat ćete mjeriti na nekoliko valnih duljina za određivanje raspodjele alfa spirale, beta-lista i nasumične zavojnice. To je zato što sva tri doprinose mjernom spektru i kao takvi trebate nekoliko mjerenja da shvatite relativne doprinose svake sekundarne strukture.


Usporedba biofizičkih i bioloških svojstava α- Helikalni enantiomerni antimikrobni peptidi

U našoj prethodnoj studiji (Chen et al. J Biol Chem 2005, 280:12316–12329), koristili smo α- spiralni antimikrobni peptid V681 kao okvir za proučavanje učinaka hidrofobnosti, amfipatičnosti i heličnosti peptida na biološke aktivnosti gdje smo dobili nekoliko V681 analozi s dramatičnim poboljšanjem peptidnih terapijskih indeksa protiv gram-negativnih i gram-pozitivnih bakterija. U ovoj studiji, d-enantiomeri tri peptida – V681, V13AD i V13KL sintetizirani kako bi se usporedila biofizička i biološka svojstva s njihovim enantiomernim izomerima. Svaki d-enantiomer je prikazan spektroskopijom kružnog dihroizma kao zrcalna slika odgovarajućeg l-izomera u benignim uvjetima i u prisutnosti 50% trifluoretanola. l- i d-enantiomeri pokazali su ekvivalentno antimikrobno djelovanje protiv raznolike skupine Pseudomonas aeruginosa klinički izolati, razne gram-negativne i gram-pozitivne bakterije i gljivice. Osim toga, l- i d-enantiomerni peptidi bili su jednako aktivni u svojoj sposobnosti da liziraju ljudske crvene krvne stanice. Slična aktivnost l- i d-enantiomernih peptida na prokariotskim ili eukariotskim staničnim membranama sugerira da ne postoje kiralni receptori i da je stanična membrana jedina meta za te peptide. Peptid d -V13KD pokazao značajna poboljšanja u terapijskim indeksima u usporedbi s roditeljskim peptidom V681 za 53 puta protiv P. aeruginosa sojeva, 80 puta protiv gram-negativnih bakterija, 69 puta protiv gram-pozitivnih bakterija i 33 puta protiv Candida albicans. Izvrsna stabilnost d-enantiomera na probavu tripsina (bez proteolize tripsinom) u usporedbi s brzom razgradnjom l-enantiomera naglašava prednost d-enantiomera i njihov potencijal kao kliničkih terapeutika.

Široka uporaba tradicionalnih antibiotika rezultirala je pojavom mnogih sojeva otpornih na antibiotike, što je izazvalo hitnu potrebu za novom klasom antibiotika (1, 2). Kationski antimikrobni peptidi postali su važni kandidati kao potencijalni terapeutski agensi (3-5) i pokazali su se aktivnim u in vivo studije na životinjama (6). Iako točan način djelovanja antimikrobnih peptida nije utvrđen (7-9), predloženo je da je citoplazmatska membrana glavna meta za mnoge od ovih peptida, pri čemu nakupljanje peptida u membrani uzrokuje povećanu propusnost i gubitak barijere funkcija (10, 11). Ne očekuje se razvoj rezistencije na membranski aktivne peptide čija je jedina meta citoplazmatska membrana jer bi to zahtijevalo bitne promjene u lipidnom sastavu staničnih membrana mikroorganizama. Međutim, glavna prepreka upotrebi antimikrobnih peptida kao antibiotika je njihova toksičnost ili sposobnost lize eukariotskih stanica, normalno izražena kao hemolitička aktivnost (toksičnost za ljudske crvene krvne stanice), što je glavni razlog sprječavanja njihove primjene kao injekcijskih terapeutika.

Enantiomerni oblici antimikrobnih peptida sa svim d-aminokiselinama korišteni su za proučavanje mehanizma vezanja na membranu (12-14), budući da se prije smatralo da kiralnost stanične membrane zahtijeva specifičnu kiralnost peptida da bi bila aktivna. Međutim, mnoge studije su pokazale da peptidi all-d-amino kiselina imaju jednaku aktivnost kao i njihovi all-l-enantiomeri (12-19), što sugerira da antimikrobni mehanizam ovih peptida ne uključuje stereoselektivnu interakciju s kiralnim enzimom ili lipidima ili proteinski receptor. Osim toga, all-d-peptidi su otporni na razgradnju proteolitičkih enzima, što povećava njihov potencijal kao kliničkih terapeutika.

U našoj prethodnoj studiji (20) koristili smo a de novo pristup dizajnu za promjenu sekundarne strukture, hidrofobnosti i amfipatičnosti an α-helikalni amfipatski antimikrobni peptid V681 (21, 22) i dobiveni spojevi olova s ​​visokim antimikrobnim djelovanjem i izrazito niskim hemolitičkim djelovanjem. U ovoj studiji uspoređujemo biofizička i biološka svojstva između peptidnih enantiomera sa svim-l- ili all-d-aminokiselinama naših olovnih spojeva, uključujući antimikrobno djelovanje protiv različitih bakterijskih sojeva, posebno raznolike skupine Pseudomonas aeruginosa klinički izolati sa širokim rasponom osjetljivosti na antibiotik ciprofloksacin. Poznato je da respiratorne infekcije po P. aeruginosa su glavni uzrok morbiditeta i mortaliteta u odraslih bolesnika s cističnom fibrozom (23-25). Pseudomonas aeruginosa infekcija je također ozbiljan problem u bolesnika hospitaliziranih s rakom i opeklinama, a smrtnost kod takvih pacijenata iznosi 50% (24, 25). Prema podacima koje su od 1990. do 1996. prikupili američki centri za kontrolu i prevenciju bolesti (CDC), P. aeruginosa bio je drugi najčešći uzrok bolničke pneumonije (17% izolata), treći najčešći uzrok infekcija mokraćnog sustava (11%), četvrti najčešći uzrok infekcija kirurškog mjesta (8%), sedmi najčešći izolirani patogen iz krvotoka (3%), a ukupno je peti najčešći izolat (9%). Vjerujemo da, ovaj komparativ de novo Studija dizajna l- i d-antimikrobnih peptida kritičan je korak prema razvoju novih antimikrobnih terapija i razumijevanju mehanizma djelovanja α-helikalni antimikrobni peptidi.


MATERIJALI I METODE

Plazmidi

Ekspresijski plazmid Pho4p pJ1080, izveden iz pET21d (Novagen), kodira domenu bHLH od 62 aminokiseline Pho4p i dar je D. Wemmera (59). Mutageneza usmjerena na mjesto korištenjem standardnih metoda korištena je za stvaranje plazmida koji kodiraju bHLHKRATAK Pho4p varijante naboja pJ1350, pJ1351 i pJ1352 (kodiraju ostatke SAA, EEE i KKK priložene blizu N-kraja). Peptidi s varijantom dužeg naboja, bHLHDUGO (SAA, EEE, KKK) skup, generirani su iz bHLHKRATAK peptide zamjenom prva 3 N-terminalna ostatka (MDD) s 12 bZIP N-terminalnih ostataka (MASMTGGQQMGRD). Plazmidi pDP-AP-1-21, pDP-AP-1-23, pDP-AP-1-25, pDP-AP-1-26, pDP-AP-1-28 i pDP-AP-1-30 su modificirani uključiti E-box mjesto za vezanje bHLH kako bi se generirale sonde za elektroforetsku faznu analizu (30).

Oligonukleotidi

Nemodificirani oligonukleotidi sintetizirani su korištenjem standardne kemije fosforamidita. Oligonukleotidi su pročišćeni denaturiranjem PAGE (20% akrilamid 29:1 akrilamid/bisakrilamid, 8 M urea), ekstrahirani iz fragmenata gela (10 mM Tris-HCl, 1 mM EDTA i 300 mM NaOAc), i dezal 5.18 patrone s reverznom fazom (Sep-Pak Waters Corporation). Koncentracije oligonukleotida određene su na 260 nm korištenjem koeficijenata molarne ekstinkcije najbližeg susjeda kao što je prethodno opisano (11). Oligonukleotidi su žareni na ∼50 μM u 250 mM NaCl, zagrijavanjem na 95°C i hlađenjem na sobnu temperaturu preko noći.

Ekspresija i pročišćavanje proteina

bHLH peptidi su eksprimirani u BL21(DE3) stanicama u Luria-Bertani mediju (59). Kulture su uzgajane na 37°C do an A600 od 0,6-0,7 i inducirano s 5 mM izopropil-β-d-tiogalaktopiranozidom. Inducirane kulture su zatim inkubirane 8 sati na 37°C prije centrifugiranja (5000 × g, 20 min). Stanične pelete su smrznute na -80°C, odmrznute i resuspendirane (20 ml/l kulture) u puferu za lizu (50 mM Tris-HCl, pH 6,0, 1 mM EDTA i 20 mM DTT). Početna liza stanica ultrazvukom (svaka 15 s, 10 × 4°C Fisher Sonic Dismembrator 60) je praćena pneumatskim smicanjem pomoću Avestin Emulsiflex-C5 disruptora pokretanog N-om pod tlakom.2 (140 psi, 4°C). Dobiveni sirovi lizati bistreni su centrifugiranjem (20 000 × g45 min, 4°C) i dalje pročišćen na koloni SP sepharose s gravitacijskim protokom (Pharmacia Biotech, Švedska). Nakon punjenja, kationska izmjenjivačka kolona je isprana s 40 ml pufera za lizu dopunjenog povećanjem koncentracije soli (0,5, 1,0, 1,5 i 2,0 M NaCl). Eluirane frakcije koje sadrže bHLH peptide (1,0 M NaCl za bHLHKRATAK i 1,5 M NaCl za bHLHDUGO) koncentrirani su pomoću centrifugalnih koncentratora s 5000 molekularne težine (VivaSpin 20 VivaScience) i zatim pročišćeni HPLC-om [C18 kolona preparativne reverzne faze (250 × 21,2 mm), Beckman 127P System Gold, pufer A: 0,1% trifluoroctena kiselina (TFA), pufer B: 80% CH3CN, 0,1% TFA, gradijent 10-70% pufera B tijekom 50 min]. Čistoća peptida procijenjena je ESI GC-MS na >95%. Pročišćeni peptidi su liofilizirani za skladištenje.

BHLH: Studije afiniteta vezanja DNA

Spektroskopija kružnog dikroizma

Elektroforetski fazni testovi savijanja DNA

DNA sonde za fazne analize generirane su i radioaktivno obilježene PCR-om iz derivata plazmida pDP-AP-1-21, pDP-AP-1-23, pDP-AP-1-25, pDP-AP-1-26, pDP-AP -1-28 i pDP-AP-1-30 (30) koji su modificirani mutagenezom usmjerenom na mjesto izvornog AP-1 veznog mjesta kako bi se stvorila mjesta za vezanje E-kutije prikladna za vezanje bHLH. DNA sonde su inkubirane s dovoljno peptida za ∼50% pomak mobilnosti u reakcijama od 20 μl koje sadrže pufer za vezanje i 100 μg/ml BSA. Reakcije su inkubirane na ledu 30 minuta prije nativnog PAGE (6% akrilamid 29:1 akrilamid/bisakrilamid) u 0,5× TBE puferu tijekom 2000 V-h na 22°C. Osušeni gelovi su analizirani kao gore.

Proračuni fazne analize

Predviđanja energije savijanja DNK

ΔΔG° doprinose cjelokupnom procesu stvaranja kompleksa protein-DNA, definiranom kao ΔG°(varijanta) − ΔG°(SAA), procijenjeni su iz eksperimentalnih podataka ili su predloženi kao razumna nagađanja koja racionaliziraju eksperimentalna opažanja. Pojedinačni ΔΔG° doprinosi koji dolaze iz eksperimenta procijenjeni su na sljedeći način:


Učinak kiselog pH na adsorpciju i litičku aktivnost peptida Polybia-MP1 i njegovog analoga koji sadrži histidin u anionskoj lipidnoj membrani: biofizička studija molekularnom dinamikom i spektroskopijom

Antimikrobni peptidi (AMP) dio su urođenog imunološkog sustava mnogih vrsta. AMP su kratke sekvence bogate nabijenim i nepolarnim ostacima. Djeluju na lipidnu fazu plazma membrane bez potrebe za membranskim receptorima. Polybia-MP1 (MP1), ekstrahiran iz domaće ose, je baktericid širokog spektra, inhibitor proliferacije stanica raka koji nije hemolitički i necitotoksičan. Mehanizam djelovanja MP1 i način njegove adsorpcije još nisu u potpunosti poznati. Njegova adsorpcija na dvosloj lipida i litička aktivnost najvjerojatnije ovise o ionizacijskom stanju njegova dva kisela i tri bazična ostatka i posljedično o pH mase. Ovdje smo istražili učinak rasute kisele (pH 5,5) i neutralne pH (7,4) otopine na adsorpciju, umetanje i litičku aktivnost MP1 i njegovog analoga H-MP1 na anionsku (7POPC:3POPG) membranu modela. H-MP1 je sintetski analog MP1 s lizinima zamijenjenim histidinima. Velike promjene pH vrijednosti mogle bi modulirati učinkovitost ove peptide. Kombinacija različitih eksperimentalnih tehnika i simulacija molekularne dinamike (MD) pokazala je da su adsorpcija, umetanje i litička aktivnost H-MP1 vrlo osjetljivi na pH u masi za razliku od MP1. Atomistički detalji, dobiveni MD simulacijama, pokazali su da peptidi kontaktiraju svoje N-krajeve s dvoslojem prije umetanja, a zatim leže paralelno s dvoslojem. Njihova hidrofobna lica umetnuta u fazu acilnog lanca remete pakiranje lipida.

Ovo je pregled sadržaja pretplate, pristup preko vaše institucije.


Materijali i metode

Modeli

HS sekvenca (kameleon-HS sekvenca kao izolirani fragment) je VLYVKLHN. Nadalje smo razmotrili dvije sekvence, HS-α i HS-β prikazane na Slici 1, kako bismo istražili kako bočni ostaci utječu na tendencije savijanja. Duljina ostataka dodanih HS sekvenci dovoljno je duga da uključi barem jednu sekundarnu strukturnu jedinicu, tj. α-helix, i dva komplementarna β-lanca ili β-list/ukosnicu (vidi sliku 1. ). Osim toga, proučavana je druga kameleonska sekvenca, RVQDNIV dugačka sedam ostataka kameleon-HE (Helix i shEet) sekvenca, kako je opisano u pratećim informacijama).

Molekularno modeliranje

Za poboljšanje uzorkovanja u simulacijama molekularne dinamike koristi se REMD metoda 43. Izvodi se izvođenjem niza neovisnih simulacija na različitim temperaturama i izmjenama određenim kriterijem Metropolisa u svakom vremenskom razdoblju. Poznato je da je REMD učinkovita metoda uzorkovanja 44 i uspješno je primijenjen na α-helike peptide, 34, 45� β peptide β-listove, 47 β-peptide ukosnice 48 i male proteine49 skupština. 20, 50� REMD metoda temeljena na temperaturi, koja omogućuje poboljšano konformacijsko uzorkovanje, prikladan je alat za proučavanje strukturne plastičnosti izoliranog kameleonskog slijeda HS i faktora koji induciraju slijedove kameleona da se presavijaju u konformaciju α-heliksa u HS-α i β-ukosnica u HS-β. Pojedinosti o parametrima i postavkama simulacije mogu se pronaći u odjeljku S1.

Masena spektrometrija pokretljivosti iona

Masena spektrometrija temeljena na pokretljivosti iona (IM-MS) korištena je za mjerenje presjeka sudara monomera HS-α i HS-β peptida. Eksperimentalne vrijednosti uspoređene su s teorijskim presjecima konstrukcija dobivenim iz simulacija. Ovdje se ioni generiraju nano-elektrosprejom, hvataju kapilarom, prenose se u ionski lijevak, lagano se desolvatiraju, zarobljavaju i pulsiraju u drift ćeliju ispunjenu plinom helija. Ionski paket se provlači kroz ćeliju slabim električnim poljem, izlazi iz stanice, odabire se i detektira. Filter mase može se postaviti tako da odabere određenu vrijednost m/z i izmjerena distribucija vremena dolaska, s t = 0 postavljeno impulsom u ćeliju i t = tA kada ioni stignu do detektora. Promjenom napona na ćeliji pri konstantnom tlaku ćelije vrlo točne vrijednosti smanjene K0, može se dobiti. Ove vrijednosti mogu se transformirati u presjeke sudara koristeći dobro uspostavljen odnos kinetičke teorije. 55 Ako se žele maseni spektri, ioni se kontinuirano prenose iz ionskog lijevka u drift ćeliju. U ovdje opisanim pokusima korištene su dvije odvojene drift ćelije IM-MS, jedna s kratkom (5 cm) drift ćelijom 56 i jedna s mnogo dužom ćelijom (2 m). 57 Potonji instrument ima mnogo veću rezoluciju i zbog dizajna svog izvora vrlo nježno prenosi ione iz kapljica nano-elektrospreja u drift ćeliju. To je važno kako bi se zadržale strukture slične otopini za određivanje presjeka. 58�

Transmisiona elektronska mikroskopija i kružni dikroizam

Slike transmisijske elektronske mikroskopije (TEM) snimljene su pri t = 0 i t = 480 h pomoću transmisionog elektronskog mikroskopa JEOL 1200 EX s ubrzavajućim naponom od 80 kV. Spektri kružnog dikroizma uzeti su pomoću Aviv spektrometra kružnog dikroizma model 202 (Instruments Inc.) i J-810 spektropolarimetra (JASCO, Tokyo, Japan) za proučavanje vremenskog tijeka. Detaljni eksperimentalni postupci detaljno su opisani u odjeljcima Popratne informacije S1.3 i S1.4.


Kružni dikroizam Uredi

Kružni dikroizam (CD) je dikroizam koji uključuje kružno polariziranu svjetlost, tj. diferencijalnu apsorpciju lijevog i desnog svjetla. Lijeva kružna (LHC) i desna kružna (RHC) polarizirana svjetlost predstavljaju dva moguća stanja spin kutnog momenta za foton. Ovaj fenomen se očituje u apsorpcijskim vrpcama optički aktivnih kiralnih molekula. CD spektroskopija ima širok raspon primjena. Najvažnije, UV CD se koristi za istraživanje sekundarne strukture proteina.

CD je usko povezan s optička rotirajuća disperzija (ORD) tehniku, i općenito se smatra naprednijom. CD se mjeri u ili blizu apsorpcijskih vrpci molekule od interesa, dok se ORD može mjeriti daleko od tih vrpca. Prednost CD-a je očita u analizi podataka. Strukturni elementi se jasnije razlikuju jer se njihovi snimljeni pojasevi ne preklapaju u velikoj mjeri na određenim valnim duljinama kao u ORD-u. U načelu se ova dva spektralna mjerenja mogu međusobno pretvoriti kroz integralnu transformaciju, ako su sve apsorpcije uključene u mjerenja.

Fizički principi Uredi

Elektromagnetsko zračenje sastoji se od električnog (E) i magnetskog (B) polja koji osciliraju okomito jedno na drugo i na smjer širenja, poprečni val. Dok se linearno polarizirano svjetlo javlja kada vektor električnog polja oscilira samo u jednoj ravnini, kružno polarizirano svjetlo nastaje kada se smjer vektora električnog polja okreće oko smjera njegovog širenja dok vektor zadržava konstantnu veličinu. U jednoj točki u prostoru, kružno polarizirani vektor će iscrtati krug u jednom periodu frekvencije vala, otuda i naziv. Dva donja dijagrama prikazuju električne vektore linearno i kružno polarizirane svjetlosti, u jednom trenutku vremena, za raspon položaja, dijagram kružno polariziranog električnog vektora tvori spiralu duž smjera širenja (k). Za lijevu kružno polariziranu svjetlost (LCP) s propagacijom prema promatraču, električni vektor rotira suprotno od kazaljke na satu. Za desno kružno polarizirano svjetlo (RCP), električni vektor rotira u smjeru kazaljke na satu.

Kada kružno polarizirana svjetlost prolazi kroz apsorbirajući optički aktivni medij, brzine između desne i lijeve polarizacije razlikuju se kao i njihova valna duljina i opseg u kojem se apsorbiraju (εL≠εR). Kružni dikroizam je razlika Δε ≡ εL- εR.

Jednostavno rečeno, budući da je kružno polarizirana svjetlost sama po sebi "kiralna", ona različito djeluje s kiralnim molekulama. To jest, dvije vrste kružno polarizirane svjetlosti apsorbiraju se u različitoj mjeri. U CD eksperimentu, jednake količine lijeve i desne kružno polarizirane svjetlosti odabrane valne duljine naizmjenično se zrače u (kiralni) uzorak. Jedna od dvije polarizacije apsorbira se više od druge, a ta razlika apsorpcije ovisna o valnoj duljini se mjeri, čime se dobiva CD spektar uzorka. Zbog interakcije s molekulom, vektor električnog polja svjetlosti prati eliptični put nakon prolaska kroz uzorak.

Važno je da kiralnost molekule može biti konformacijska, a ne strukturna. To jest, na primjer, proteinska molekula sa spiralnom sekundarnom strukturom može imati CD koji se mijenja s promjenama u konformaciji.

gdje je ΔA razlika između apsorpcije lijevog kružno polariziranog (LCP) i desnog kružno polariziranog (RCP) svjetlosti (to je ono što se obično mjeri). ΔA je funkcija valne duljine, pa da bi mjerenje imalo smisla mora biti poznata valna duljina na kojoj je provedeno.

Molarni kružni dikroizam definira se kao

gdje su εL i εR molarni koeficijenti ekstinkcije za LCP i RCP svjetlost.

Iako se ΔA obično mjeri, iz povijesnih razloga većina mjerenja se navodi u stupnjevima od eliptičnost. Molarna eliptičnost je kružni dikroizam ispravljen za koncentraciju. Molarni kružni dikroizam i molarna eliptičnost, [θ], lako se međusobno pretvaraju jednadžbom:

U mnogim praktičnim primjenama kružnog dikroizma (CD), izmjereni CD nije samo intrinzično svojstvo molekule, već ovisi o molekularnoj konformaciji. U takvom slučaju CD može također biti funkcija temperature, koncentracije i kemijskog okruženja, uključujući otapala. U ovom slučaju prijavljena vrijednost CD-a također mora specificirati ove druge relevantne čimbenike kako bi bila smislena.

Primjena na biološke molekule Uredi

Daleki UV (ultraljubičasti) CD spektar proteina može otkriti važne karakteristike njihove sekundarne strukture. CD spektri se mogu lako koristiti za procjenu udjela molekule koji je u konformaciji alfa-heliksa, konformaciji beta-lista, konformaciji beta-zavoja ili nekoj drugoj (npr. nasumičnoj zavojnici) konformaciji. Ove frakcijske dodjele postavljaju važna ograničenja na moguće sekundarne konformacije u kojima protein može biti. CD općenito ne može reći gdje se detektirane alfa spirale nalaze unutar molekule ili čak u potpunosti predvidjeti koliko ih ima. Unatoč tome, CD je vrijedan alat, posebno za prikazivanje promjena u konformaciji. Može se, na primjer, koristiti za proučavanje kako se sekundarna struktura molekule mijenja kao funkcija temperature ili koncentracije denaturirajućih sredstava, na pr. Gvanidinijev klorid ili urea. Na taj način može otkriti važne termodinamičke informacije o molekuli (kao što su entalpija i Gibbsova slobodna energija denaturacije) koje se inače ne mogu lako dobiti. Svatko tko pokuša proučavati protein naći će CD vrijedan alat za provjeru da je protein u svojoj prirodnoj konformaciji prije poduzimanja opsežnih i/ili skupih pokusa s njim. Također, postoji niz drugih upotreba za CD spektroskopiju u kemiji proteina koji nisu povezani s procjenom frakcije alfa-heliksa.

Blizu UV spektra CD-a (>250 nm) proteina pruža informacije o tercijarnoj strukturi. Signali dobiveni u području 250-300 nm posljedica su apsorpcije, dipolne orijentacije i prirode okolnog okoliša aminokiselina fenilalanina, tirozina, cisteina (ili S-S disulfidnih mostova) i triptofana. Za razliku od dalekog UV CD-a, bliski UV CD spektar ne može se dodijeliti nijednoj određenoj 3D strukturi. Umjesto toga, bliski UV spektri CD-a pružaju strukturnu informaciju o prirodi prostetičkih skupina u proteinima, npr. heme skupine u hemoglobinu i citokromu c.

Vidljiva CD spektroskopija je vrlo moćna tehnika za proučavanje interakcija metal-protein i može razriješiti pojedinačne d-d elektronske prijelaze kao zasebne trake. CD spektri u području vidljive svjetlosti nastaju samo kada je metalni ion u kiralnom okruženju, tako da se slobodni ioni metala u otopini ne detektiraju. Ovo ima prednost u promatranju samo metala vezanog na proteine, pa se ovisnost o pH i stehiometrije lako dobivaju.

CD daje manje specifične strukturne informacije od rendgenske kristalografije i proteinske NMR spektroskopije, na primjer, koje obje daju podatke o atomskoj rezoluciji. Međutim, CD spektroskopija je brza metoda koja ne zahtijeva velike količine proteina ili opsežnu obradu podataka. Stoga se CD može koristiti za ispitivanje velikog broja uvjeta otapala, različitih temperatura, pH, saliniteta i prisutnosti različitih kofaktora.

Eksperimentalna ograničenja Uredi

CD spektroskopija se obično koristi za proučavanje proteina u otopini, te stoga nadopunjuje metode koje proučavaju čvrsto stanje. Ovo je također ograničenje, jer su mnogi proteini ugrađeni u membrane u svom prirodnom stanju, a otopine koje sadrže membranske strukture često se snažno raspršuju.

Mjerenje CD-a je komplicirano činjenicom da tipični vodeni puferski sustavi često apsorbiraju u rasponu u kojem strukturne značajke pokazuju diferencijalnu apsorpciju kružno polarizirane svjetlosti. Fosfatni, sulfatni, karbonatni i acetatni puferi općenito su nekompatibilni s CD-om, osim ako su izuzetno razrijeđeni, npr. u rasponu od 10-50 mM. Tris puferski sustav treba u potpunosti izbjegavati kada izvodite daleko UV CD. Spojevi borata i onija često se koriste za uspostavljanje odgovarajućeg raspona pH za CD eksperimente. Neki su eksperimentatori zamijenili fluorid kloridnim ionom jer fluor upija manje u dalekom UV zraku, a neki su radili u čistoj vodi. Druga, gotovo univerzalna, tehnika je minimiziranje apsorpcije otapala korištenjem stanica kraće duljine puta pri radu u dalekom UV zrakama, duljine puta od 0,1 mm nisu neuobičajene u ovom radu.

Osim mjerenja u vodenim sustavima, CD, posebno daleko UV CD, može se mjeriti u organskim otapalima, npr. etanol, metanol ili trifluoroetanol (TFE). Potonji ima prednost da inducira formiranje strukture proteina, inducirajući beta-limove u nekima i alfa spirale u drugima, koje ne bi pokazali u normalnim vodenim uvjetima. Najčešća organska otapala kao što su acetonitril, THF, kloroform, diklormetan su međutim nekompatibilna s daleko UV zračenjem.

Može biti zanimljivo primijetiti da su proteinski CD spektri korišteni u procjeni sekundarne strukture povezani s π do π* orbitalnim apsorpcijama amidnih veza koje povezuju aminokiseline. Ove apsorpcijske vrpce dijelom leže u takozvanom vakuumskom ultraljubičastom (valne duljine manje od oko 200 nm). Područje valne duljine koje nas zanima zapravo je nedostupno u zraku zbog jake apsorpcije svjetlosti kisikom na tim valnim duljinama. U praksi se ovi spektri ne mjere u vakuumu, već u instrumentu bez kisika (ispunjen čistim plinovitim dušikom).


2. Eksperimentalno

2.1. Instrumenti i reagensi

Matične otopine 5 × 10 −3 mol L −1 NFZ i NFT (Sigma Chemical Co., St. Louis, SAD čistoća – ne manje od 99,0%) pripremljeni su otapanjem njihovih kristala (9,91 × 10 −3 g i 1,19 × 10 −2 g, redom) u 10 mL dimetilformamida (DMF). BSA (2 × 10 −3 mol L −1 ) pripremljen je otapanjem 1,36 g proteina (M = 66 kDa, ≥98%, liofilizirani prah kupljen od Sigma-Aldrich Chemical Co. Ltd., i bez daljnjeg pročišćavanja) u 10,0 mL 5,0 × 10 −2 mol L −1 otopine natrijevog klorida i čuvati na 4 °C. Da bi se potvrdila čistoća pripremljenog BSA, razrijeđen je na 2,0 × 10 −5 mol L −1 , a izmjerena vrijednost apsorbancije bila je (0,896) na 278 nm. Ovo je uspoređeno s apsorbancijom od 0,667 iz referentne količine od 1,0 g L −1 (1.47 × 10 −5 mol L −1 ) čisti BSA. 16 Sve eksperimentalne otopine podešene su puferom Tris-HCl ((hidroksi metil)amino metan-klorovodik) na pH 7,4. Ostale kemikalije bile su reagensi analitičke čistoće, a cijelo vrijeme je korištena dvostruko destilirana voda.

2.2. Studija gašenja fluorescencije

2.3. FT-IR mjerenja

2.4. Studije kružnog dikroizma (CD) i rezonantnog raspršenja svjetlosti (RLS) za interakciju

2.5. Mjerenja mikroskopije atomske sile (AFM).

2.6. Studije molekularnog spajanja


  1. Mjesto cijepanja prohormonske konvertaze unutar predviđene alfa-heliksa posreduje razvrstavanje neuronskog i endokrinog polipeptida VGF u regulirani sekretorni put.
    Garcia i sur.
    J. Biol. Chem., 2005280:41595
  2. Komparativna studija veznog džepa proprotein konvertaza sisavaca i njegove implikacije na dizajn specifičnih inhibitora malih molekula.
    Tian i Jianhua
    Int.J.Biol.Sci., 20106:89
  3. Inhibicija obrade proendotelina-1 povezane s konvertazom.
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    J.Cardiovasc.Pharmacol., 199526:S47
  4. Inhibitori furina blokiraju cijepanje šiljastog proteina SARS-CoV-2 kako bi suzbili proizvodnju virusa i citopatske učinke.
    Cheng i sur.
    Cell Rep., 2020

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Opis i evolucijska analiza mikrostrukturnih varijacija u ljudskom Bcl2l10 proteinu

Detaljne informacije o strukturi i funkciji trenutno su poznate samo za ograničen broj proteina oocita, što znači da je naše osnovno razumijevanje molekularnih aktera koji pokreću sazrijevanje jajašca, oplodnju i razvoj prije implantacije vrlo ograničeno. Osim što je istaknuti majčinski izražen paralog Bcl-2 (Burns et al. 2003 Hamatani et al. 2004 Arnaud et al. 2006 Gallardo et al. 2007 Guillemin et al. 2009 Yoon et al. 2009), Bcl2l10 je ubrzao u volumnu kralježnjaci (Aouacheria i sur. 2001., 2005. Guillemin i sur. 2009.). Kako bismo identificirali specifične aminokiselinske sekvence odgovorne za visoke razine divergencije uočene u ovoj ortološkoj skupini, prvo smo generirali višestruko poravnanje sekvenci Bcl2l10 ortologa od nekoliko sisavaca, ptica i riba, kao i virusa (slika 1). Iz ove usporedbe, čini se da interhelikalni zavoj smješten između predviđenih α5 i α6 spirala nosi dodatnih desetak aminokiselina u ljudskom Bcl2l10 koje nisu prisutne u homolognim proteinima iz kokoši ili zebrice. Štoviše, ljudski Bcl2l10 ima produžetak od 10 aminokiselina na svom N-kraju u usporedbi s ortolognim sekvencama u Mnas muskulus i drugi kralježnjaci koji nisu primati. Predviđeni proizvod kodiran od strane čovjeka Bcl2l10 gen je opisan kao protein od 194- (Aouacheria et al. 2001.) ili 204-amino kiseline (Ke et al. 2001. Zhang i sur. 2001.), ovisno o odsutnosti ili prisutnosti ovog proširenja. Prva regija nazvana je LAAS (za dug A mino A cid S protezanje u prethodnoj publikaciji (Zhang et al. 2001.)), a po analogiji, odlučili smo nazvati drugu SAAS (za kratak A mino A cid S tretch).

Višestruko poravnanje sekvenci ortolognih Bcl2l10 proteina. Korištenih sedam vrsta je kako slijedi: Homo sapiens (NP_065129), Mus musculus (Q9Z0F3), Gallus gallus (Q90ZN1), Coturnix coturnix (CAA59136), Meleagrid herpesvirus 1 (AAG30102), Danio rerio (NP_919379), i Gadus morhua (ACZ62638). Očuvani BH1-BH4 motivi i C-terminalna transmembranska (TM) domena su naznačeni iznad poravnanja. Motivi SAAS i LAAS su u kutiji. The locations of the predicted pore-forming α5 and α6 helices are indicated above the sequence of human Bcl2l10. The presence of a 22-kDa polypeptide corresponding to the form shown in the figure was previously confirmed by western blot analysis of human ovary extracts ( Guillemin et al. 2009).

Multiple sequence alignment of orthologous Bcl2l10 proteins. The seven species used were as follows: Homo sapiens (NP_065129), Mus musculus (Q9Z0F3), Gallus gallus (Q90ZN1), Coturnix coturnix (CAA59136), Meleagrid herpesvirus 1 (AAG30102), Danio rerio (NP_919379), and Gadus morhua (ACZ62638). The conserved BH1–BH4 motifs and the C-terminal transmembrane (TM) domain are indicated above the alignment. The SAAS and LAAS motifs are boxed. The locations of the predicted pore-forming α5 and α6 helices are indicated above the sequence of human Bcl2l10. The presence of a 22-kDa polypeptide corresponding to the form shown in the figure was previously confirmed by western blot analysis of human ovary extracts ( Guillemin et al. 2009).

As a first step in deciphering the evolutionary mechanisms for the appearance of these regions, we have compiled and aligned Bcl2l10 amino acid sequences obtained from publicly available databases ( Blaineau and Aouacheria 2009) or cloned by PCR using genomic DNA from different species, followed by sequencing.

On the one hand, we found that the SAAS region was present in great apes (humans, gorillas, chimps, and orangutans) and absent in gibbons and other monkeys (baboons, macaques, and lemurs) ( fig. 2A) as well as other vertebrates ( supplementary fig. S1 , Supplementary Material online), suggesting that the event giving rise to this N-terminal extension occurred after the Old World monkey–hominoid split and after separation of the lesser apes from the other anthropoid lineages ∼14 to 18 Ma ( Goodman et al. 2005). After careful inspection of the nucleotide alignment ( fig. 2B), it was determined that in anthropoid primates Bcl2l10 contains an internal tandem duplication in which the upstream 30 base pairs are repeated and results in the addition of ten amino acids to the N-terminus (corresponding to the SAAS segment).

Sequence and evolutionary analysis of the SAAS motif of Bcl2l10. (A) Amino acid sequence alignment of the N-terminal region of Bcl2l10 from 11 primates. Right: Schematic phylogeny of the primate species used in the present study (adapted from Goodman et al. 2009). The Bcl2l10 protein found in anthropoid primates (anthropoids) has an extension of ten amino acids in the N-terminal side compared with other primates (others). Arrows denote the first in-frame methionine in each group. (B) Alignment of nucleotide sequences determined for the N-terminal region of primate Bcl2l10. The two tandem duplication units (shown in brackets) are bordered on each side by an “ACC” triplet (dotted lines). Possible tandem duplication history (repeat/ancestral) is indicated above the alignment. Arrows denote the start codon ATG found in Bcl2l10 from anthropoids versus other primates. Species abbreviations refer to full names given in (A). Sequences for Homo sapiens, Pan trogloditi, Pongo pygmaeus, Macaca mulatta, Tarsius syrichta, i Otolemur garnettii were from Ensembl (respective accession numbers: ENSG00000137875, ENSPTRG00000007083, ENSPPYG00000006483, ENSMMUG00000017372, ENSTSYG00000013586, and ENSOGAG00000008687). Sequences for Gorilla gorilla, Callithrix jacchus, Nomascus leucogenys, Papio anubis, i Chlorocebus aethiops were deduced from cloned partial ORFs (see Materials and Methods). (C) Alignments of nucleotide (left) and amino acid (inset) sequence repeats found in human Bcl2l10.

Sequence and evolutionary analysis of the SAAS motif of Bcl2l10. (A) Amino acid sequence alignment of the N-terminal region of Bcl2l10 from 11 primates. Right: Schematic phylogeny of the primate species used in the present study (adapted from Goodman et al. 2009). The Bcl2l10 protein found in anthropoid primates (anthropoids) has an extension of ten amino acids in the N-terminal side compared with other primates (others). Arrows denote the first in-frame methionine in each group. (B) Alignment of nucleotide sequences determined for the N-terminal region of primate Bcl2l10. The two tandem duplication units (shown in brackets) are bordered on each side by an “ACC” triplet (dotted lines). Possible tandem duplication history (repeat/ancestral) is indicated above the alignment. Arrows denote the start codon ATG found in Bcl2l10 from anthropoids versus other primates. Species abbreviations refer to full names given in (A). Sequences for Homo sapiens, Pan trogloditi, Pongo pygmaeus, Macaca mulatta, Tarsius syrichta, i Otolemur garnettii were from Ensembl (respective accession numbers: ENSG00000137875, ENSPTRG00000007083, ENSPPYG00000006483, ENSMMUG00000017372, ENSTSYG00000013586, and ENSOGAG00000008687). Sequences for Gorilla gorilla, Callithrix jacchus, Nomascus leucogenys, Papio anubis, i Chlorocebus aethiops were deduced from cloned partial ORFs (see Materials and Methods). (C) Alignments of nucleotide (left) and amino acid (inset) sequence repeats found in human Bcl2l10.

On the other hand, the α5–α6 interhelical region of Bcl2l10 exhibits variability both in sequence and in length across vertebrate species ( fig. 3A and B). Indeed, the Bcl2l10 protein found in primates, Felis catus and euteleostean fishes, has a long α5–α6 linker, whereas most orthologous proteins present in the other lineages have a shorter interhelical region with unrelated amino acids. Such hypervariability in the LAAS region likely reflects complex patterns of mutation and selection, which perhaps took place after an initial insertion event in the early evolution of the bcl2l10 gene (most probably at the time vertebrates diverged from invertebrates, i.e., when most paralogous genes within the Bcl-2 family were formed— Aouacheria et al. 2005).

The α5–α6 interhelical region of Bcl2l10 has variability in length and in sequence across vertebrate species. (A) Length of the α5–α6 linker of Bcl2l10 in various vertebrates. All sequences were extracted from Ensembl database (version 58). Proposed phylogeny was adapted from Milinkovitch et al. (2010.). Ma, million years ago. (B) Multiple sequence alignment of the region located between the predicted α5 and α6 helices of Bcl2l10. Species abbreviations refer to full names given in (A). The α5–α6 connecting region of Bcl2l10 (named LAAS in the human protein) is highly variable in amino acid sequence between vertebrate species. The difficulty in validly identifying short homologous sequences renders the calculation of an evolutionary rate unreliable. The functionally important aspartate (D) and glutamate (E) residues involved in calcium binding by human Bcl2l10 are marked with asterisks. The locations of the predicted pore-forming α5 and α6 helices are indicated. The LAAS, LAAS*, BCL2L10-α6L, and BCL2L10-α6S peptides used in this study are schematized above the alignment.

The α5–α6 interhelical region of Bcl2l10 has variability in length and in sequence across vertebrate species. (A) Length of the α5–α6 linker of Bcl2l10 in various vertebrates. All sequences were extracted from Ensembl database (version 58). Proposed phylogeny was adapted from Milinkovitch et al. (2010.). Ma, million years ago. (B) Multiple sequence alignment of the region located between the predicted α5 and α6 helices of Bcl2l10. Species abbreviations refer to full names given in (A). The α5–α6 connecting region of Bcl2l10 (named LAAS in the human protein) is highly variable in amino acid sequence between vertebrate species. The difficulty in validly identifying short homologous sequences renders the calculation of an evolutionary rate unreliable. The functionally important aspartate (D) and glutamate (E) residues involved in calcium binding by human Bcl2l10 are marked with asterisks. The locations of the predicted pore-forming α5 and α6 helices are indicated. The LAAS, LAAS*, BCL2L10-α6L, and BCL2L10-α6S peptides used in this study are schematized above the alignment.

Bcl2l10 Microstructural Changes Distinctly Impact Cellular Function Toward Apoptosis Regulation

At the structural level, neither the SAAS, at the N-terminus, nor the LAAS additional residues, located between two elements of secondary structure (5th and 6th helices), are expected to modify the global protein fold (see model in fig. 4), as such regions are known to be quite permissive with regards to indels. However, secondary structure predictions using the [email protected] server ( Combet et al. 2000) suggest the possibility of some subtle structural changes ( supplementary fig. S2 , Supplementary Material online). Because the actual structure of membrane-bound or complexed human Bcl2l10 is unknown, making any structural or mechanistic predictions difficult, we sought to identify specific residues in the SAAS and LAAS sequences that may have functional importance. We first asked whether deletion of the SAAS and LAAS regions could impact human Bcl2l10 protein function in apoptosis regulation. For this purpose, recombinant Bcl2l10 constructs harboring a deletion of the first ten amino acids (Nrh, Aouacheria et al. 2001) or missing the segment from amino acids 118 to 133 ( fig. 5A) were assayed by scoring either condensed Hoescht-stained nuclei ( fig. 5B) or Annexin V-positive HeLa cells ( fig. 5C) after treatment with classical apoptosis inducing agents such as thapsigargin or staurosporin. While Bcl2l10 mutants lacking the N-terminal ten amino acids (Δ1–10) retained prosurvival activity at a similar level to the full-length protein ( fig. 5B and C), the mutant lacking the LAAS region lost most of its antiapoptotic ability, irrespective of whether apoptosis is triggered by thapsigargin ( fig. 5C) or by other cell death inducers such as taxol, staurosporin, and tunicamycin (not shown). Although this latter mutant (Δ118–133) did not show reduced mitochondrial localization ( fig. 6), cells expressing pEGFPC1-Bcl2l10Δ118–133 frequently had aggregated GFP fluorescence in the cytoplasm, which may be indicative of structural defects. A more detailed mutational analysis was conducted using Bcl2l10 mutants that included deletions of residues 117–129 and 129–133. As shown in figure 6B, the mutant (▵129–133) deleted for residues 129–133, but not the one lacking amino acids 117–129 (Δ117–129), was deficient in antiapoptotic activity. Furthermore, point mutations involving the negatively charged residues located in this region (E129, E131, D133), or in its close vicinity (D137), decreased the prosurvival effect of human Bcl2l10 ( fig. 5C). Subcellular distribution of the wild-type Bcl2l10 protein and of these mutants fused to GFP was virtually identical ( fig. 6A and not shown). The staining pattern shows a partially diffuse and partially mitochondrial and perinuclear localization consistent with previously published in vitro ( Aouacheria et al. 2001 Ke et al. 2001 Zhang et al. 2001) and in vivo ( Guillemin et al. 2009) results. Taken together, these results reveal that the LAAS region contains a cluster of acidic amino acids that are important for human Bcl2l10 function.

Homology-driven model of human Bcl2l10. The human Bcl2l10 protein (A) has been modeled using M4T ( Fernandez-Fuentes et al. 2007) on the known 3D solution structure of mouse Diva/Boo (B) (pdb code: 2kua, Rautureau et al. 2010). The positions of α5–α6 helices are indicated and BH domains are colored as follows: BH1, orange BH2, purple BH3, yellow BH4, red. Presence of the SAAS region is predicted to extend the BH4-containing first helix (A). In the structural model of human Bcl2l10 (A), a potential calcium-binding domain is localized in the α5–α6 interhelical loop (in cyan) that protrudes from the surface of the more tightly packed portion of the Bcl-2-like globular fold. The negatively charged Asp/Glu residues expected to be involved in calcium binding are colored in deep blue, with side chains shown as sticks and residue numbers indicated. Proposed structure of the SAAS (in green) is consistent with a disordered N-terminal tail. For the published molecular conformation of the mouse Diva/Boo protein (B), the first five residues (GPLGS) were deleted from the original PDB file. Despite the absence of SAAS motif, the N-terminus also appears unstructured and solvent exposed. The interhelical α5–α6 linker is colored cyan.

Homology-driven model of human Bcl2l10. The human Bcl2l10 protein (A) has been modeled using M4T ( Fernandez-Fuentes et al. 2007) on the known 3D solution structure of mouse Diva/Boo (B) (pdb code: 2kua, Rautureau et al. 2010). The positions of α5–α6 helices are indicated and BH domains are colored as follows: BH1, orange BH2, purple BH3, yellow BH4, red. Presence of the SAAS region is predicted to extend the BH4-containing first helix (A). In the structural model of human Bcl2l10 (A), a potential calcium-binding domain is localized in the α5–α6 interhelical loop (in cyan) that protrudes from the surface of the more tightly packed portion of the Bcl-2-like globular fold. The negatively charged Asp/Glu residues expected to be involved in calcium binding are colored in deep blue, with side chains shown as sticks and residue numbers indicated. Proposed structure of the SAAS (in green) is consistent with a disordered N-terminal tail. For the published molecular conformation of the mouse Diva/Boo protein (B), the first five residues (GPLGS) were deleted from the original PDB file. Despite the absence of SAAS motif, the N-terminus also appears unstructured and solvent exposed. The interhelical α5–α6 linker is colored cyan.

Functional evaluation of mutant Bcl2l10 proteins. (A) Schematic representation and expression of the recombinant Bcl2l10 constructs in which deletions or point mutations were introduced. The various Flag- or GFP-tagged fusion proteins are depicted. Western Blot analyses were performed 24-h posttransfection on transiently transfected HeLa cells. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblot with anti-Flag or anti-GFP antibody. Nitrocellulose membranes were rehybridized with anti-α-vinculin antibody. Bottom: quantification for relative band intensities of Flag- or GFP-tagged products to vinculin. Quantifications were carried out by densitometric analysis using the Kodak EDA 290 Digital Imaging System and 1D Software Package. (B) Cell death was determined 24-h posttransfection by analyzing anti-Flag immunoreactive HeLa cells (∼250 cells in each experiment) under a fluorescence microscope. Data were compiled from three different fields. Cells were left untreated (−) or were treated for 24 h with dimethyl sulfoxide (control), etoposide (ETO, 10 μM), staurosporin (STS, 1 μM), or thapsigargin (THAP, 10 μM). The mode of cell death, necrosis versus apoptosis, was determined by the cellular permeability to propidium iodide (necrosis) and the morphology of the nuclei after staining with Hoechst 33342 (apoptosis). Propidium iodide–negative cell with condensed or fragmented nuclei were counted as apoptotic. Data are represented as mean ± standard deviation (SD). Results represent the means from three independent experiments. (C) FACS assays of Annexin V staining in HeLa cells transfected with the mutant constructs. Transfected cells were stained for phosphatidylserine exposure 24 h after transfection using Cy3-conjugated Annexin V, and the percentage of apoptotic GFP-expressing cells was determined by FACS. Cells were left untreated (−) or were treated with thapsigargin (10 μM, 24 h). GFP-NRZ and GFP-BFL-1 were used for comparison. Assays were performed in triplicate. Cell death levels are expressed as mean ± SD. *Significant to Δ129–133 P < 0,05. Treatment with taxol, tunicamycin, or staurosporin yielded similar results (not shown).

Functional evaluation of mutant Bcl2l10 proteins. (A) Schematic representation and expression of the recombinant Bcl2l10 constructs in which deletions or point mutations were introduced. The various Flag- or GFP-tagged fusion proteins are depicted. Western Blot analyses were performed 24-h posttransfection on transiently transfected HeLa cells. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblot with anti-Flag or anti-GFP antibody. Nitrocellulose membranes were rehybridized with anti-α-vinculin antibody. Bottom: quantification for relative band intensities of Flag- or GFP-tagged products to vinculin. Quantifications were carried out by densitometric analysis using the Kodak EDA 290 Digital Imaging System and 1D Software Package. (B) Cell death was determined 24-h posttransfection by analyzing anti-Flag immunoreactive HeLa cells (∼250 cells in each experiment) under a fluorescence microscope. Data were compiled from three different fields. Cells were left untreated (−) or were treated for 24 h with dimethyl sulfoxide (control), etoposide (ETO, 10 μM), staurosporin (STS, 1 μM), or thapsigargin (THAP, 10 μM). The mode of cell death, necrosis versus apoptosis, was determined by the cellular permeability to propidium iodide (necrosis) and the morphology of the nuclei after staining with Hoechst 33342 (apoptosis). Propidium iodide–negative cell with condensed or fragmented nuclei were counted as apoptotic. Data are represented as mean ± standard deviation (SD). Results represent the means from three independent experiments. (C) FACS assays of Annexin V staining in HeLa cells transfected with the mutant constructs. Transfected cells were stained for phosphatidylserine exposure 24 h after transfection using Cy3-conjugated Annexin V, and the percentage of apoptotic GFP-expressing cells was determined by FACS. Cells were left untreated (−) or were treated with thapsigargin (10 μM, 24 h). GFP-NRZ and GFP-BFL-1 were used for comparison. Assays were performed in triplicate. Cell death levels are expressed as mean ± SD. *Significant to Δ129–133 P < 0,05. Treatment with taxol, tunicamycin, or staurosporin yielded similar results (not shown).

Subcellular localization of wild-type and mutant Bcl2l10 proteins. HeLa cells were cotransfected with mito-DsRed plasmid (encoding DsRed2 fused to the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase) and the Flag- or GFP-tagged constructs. Subcellular distribution was analyzed by confocal microscopy 24 h after transfection. DNA dye Topro-3 (blue) was used to visualize nuclei. In merge images (overlay), yellow color shows the colocalization of GFP or Flag fluorescence (green) with mitochondria (red).

Subcellular localization of wild-type and mutant Bcl2l10 proteins. HeLa cells were cotransfected with mito-DsRed plasmid (encoding DsRed2 fused to the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase) and the Flag- or GFP-tagged constructs. Subcellular distribution was analyzed by confocal microscopy 24 h after transfection. DNA dye Topro-3 (blue) was used to visualize nuclei. In merge images (overlay), yellow color shows the colocalization of GFP or Flag fluorescence (green) with mitochondria (red).

Interestingly, these negative residues, which are not predicted to be part of the BH3-binding hydrophobic groove of Bcl2l10 ( fig. 4), appear to be strictly conserved in primates ( fig. 3B), whereas being absent (except D137) in other mammals. Given the phyletic distribution of these Asp/Glu residues, we reasoned that positive selection should have operated on the interhelical region to drive their acquisition at the base of the primate lineage, before they became fixed by purifying selection. In order to determine if the negative charge at these positions was involved in conferring some biochemical peculiarity to human Bcl2l10, that is, a favorable characteristic that could be selected for, we carried out extensive similarity searches and sequence motif analysis. We detected that the acidic region EQEGDVARD was bearing resemblance to reported calcium-binding sequences ( Chattopadhyaya et al. 1992 Siedlecka et al. 1999 Shashoua et al. 2003, 2004), and it was thus hypothesized that these residues could be involved in calcium chelation.

Human Bcl2l10 Has Evolved a Novel Calcium-Binding Site

To test the hypothesis that human Bcl2l10 binds to Ca 2+ , standard dot blot 45 Ca–overlay assays were performed. Radiolabeled Ca 2+ was not bound by GST or BSA, was bound robustly by the established Ca 2+ -binding protein calreticulin, and was reproducibly bound by GST-Bcl2l10 ( fig. 7, left, middle, and right panels). Moreover, using the same system, we demonstrated that Nrz-His6 and GST-Nr-13, which are Bcl2l10 orthologous proteins devoid of the LAAS region, were not able to bind 45 Ca 2+ ( fig. 7, left panel). To map the region responsible for the calcium-binding activity of human Bcl2l10, we constructed two mutant genes: a first with an internal deletion whose product was missing the region from residues 118 to 133 and a second encoding amino acid substitutions of selected negatively charged residues in this region (E 131 R/D 133 N). The mutant GST-Bcl2l10 proteins where the LAAS region was deleted (GST-Bcl2l10Δ118–133) or mutated (GST-Bcl2l10[E 131 R/D 133 N]) were successfully produced and purified. Full-length GST-Bcl2l10 and calreticulin, but not GST-BCl2l10Δ118–133 ( fig. 7, left panel) nor GST-Bcl2l10[E 131 R/D 133 N] ( fig. 7, middle panel), bound 45 Ca 2+ . Moreover, while Ca 2+ -binding was detectable for a 26-mer peptide corresponding to the “wild-type” LAAS region of human Bcl2l10 (residues from 115 to 140), a peptide (LAAS*) having the identical point mutations E 131 R/D 133 N, or a control peptide (Bax-BH3) of similar length and containing more Asp and Glu residues (20.6% vs. 15.4% for the LAAS), did not bind Ca 2+ ( fig. 7, middle panel). From this, it is possible to conclude that residues within the LAAS region are crucial for Bcl2l10 binding to Ca 2+ . Moreover, the negatively charged residues important for Bcl2l10 protective activity appear to be required for calcium binding in vitro.

Recombinant Bcl2l10 protein and LAAS peptide bind calcium-45 in vitro. The left and middle panels represent the autoradiographs showing calcium-45 binding and the Ponceau-S-stained blots as sample loading controls. Right panel: Calcium binding was quantified by densitometric analysis followed by normalization based on Ponceau-S staining. Representative data from three individual experiments are presented. Relative binding is indicated (arbitrary units). The indicated polypeptides were directly transferred to nitrocellulose membranes, which were in turn incubated with 45 Ca 2+ , washed, and exposed to film for autoradiography. Approximately 30 μg of recombinant purified GST-wild-type Bcl2l10, GST-Nr-13, Nrz-His6, GST-Bcl2l10Δ118–133, and GST-Bcl2l10[E 131 R/D 133 N] proteins were used along with GST, BSA, and calreticulin (a known calcium-binding protein) as controls. The calcium-45 overlay dot blots were also performed with a synthetic peptide (LAAS) derived from the hypervariable loop of human Bcl2l10, a mutated peptide (LAAS*) incorporating the double amino acid substitution E 131 R/D 133 N, and a peptide that contains the BH3 domain of Bax (BAX-BH3). As expected, the positive control calreticulin (CALR) strongly bound calcium, whereas BSA did not (left and middle panels). The GST-human Bcl2l10 fusion protein reproducibly bound calcium in vitro (left, middle, and left panels), whereas the GST-Bcl2l10Δ118–133 and GST-Bcl2l10[E 131 R/D 133 N] mutant proteins, as well as the homologous chicken GST-Nr-13 and zebrafish Nrz-His6 proteins, which are Bcl2l10 orthologous proteins devoid of LAAS region, did not. Contrary to the BAX-BH3 control peptide, which was defective in calcium binding (middle panel), the LAAS peptide displayed calcium-binding activity (left panel). Amino acid substitutions E 131 R/D 133 N (LAAS*) abolished calcium-binding capacity of this peptide (middle panel).

Recombinant Bcl2l10 protein and LAAS peptide bind calcium-45 in vitro. The left and middle panels represent the autoradiographs showing calcium-45 binding and the Ponceau-S-stained blots as sample loading controls. Right panel: Calcium binding was quantified by densitometric analysis followed by normalization based on Ponceau-S staining. Representative data from three individual experiments are presented. Relative binding is indicated (arbitrary units). The indicated polypeptides were directly transferred to nitrocellulose membranes, which were in turn incubated with 45 Ca 2+ , washed, and exposed to film for autoradiography. Approximately 30 μg of recombinant purified GST-wild-type Bcl2l10, GST-Nr-13, Nrz-His6, GST-Bcl2l10Δ118–133, and GST-Bcl2l10[E 131 R/D 133 N] proteins were used along with GST, BSA, and calreticulin (a known calcium-binding protein) as controls. The calcium-45 overlay dot blots were also performed with a synthetic peptide (LAAS) derived from the hypervariable loop of human Bcl2l10, a mutated peptide (LAAS*) incorporating the double amino acid substitution E 131 R/D 133 N, and a peptide that contains the BH3 domain of Bax (BAX-BH3). As expected, the positive control calreticulin (CALR) strongly bound calcium, whereas BSA did not (left and middle panels). The GST-human Bcl2l10 fusion protein reproducibly bound calcium in vitro (left, middle, and left panels), whereas the GST-Bcl2l10Δ118–133 and GST-Bcl2l10[E 131 R/D 133 N] mutant proteins, as well as the homologous chicken GST-Nr-13 and zebrafish Nrz-His6 proteins, which are Bcl2l10 orthologous proteins devoid of LAAS region, did not. Contrary to the BAX-BH3 control peptide, which was defective in calcium binding (middle panel), the LAAS peptide displayed calcium-binding activity (left panel). Amino acid substitutions E 131 R/D 133 N (LAAS*) abolished calcium-binding capacity of this peptide (middle panel).

Last, we asked whether a peptide corresponding to helix α6 of human Bcl2l10, and which extends a few residues beyond the predicted helical region to incorporate the putative calcium-binding residues, could change its secondary structure in the presence of Ca 2+ . Our CD spectra ( fig. 8A–E) indicate that the folding of this peptide is limited in buffer (small negative shoulder around 220 nm, fig. 8A), and the large negative band around 202 nm indicates a predominance of unfolding. The large intensity decrease of this band upon addition of Ca 2+ indicates some increase of folding. As expected for helix α6, in the presence of sodium dodecyl sulfate (SDS) or trifluoroethanol (TFE) used to probe the conformational preferences of peptides ( Buck 1998 Montserret et al. 2000), the CD spectra were typical of α-helical folding with two minima at 208 and 222 nm ( fig. 8A and B). Addition of Ca 2+ , but not Mg 2+ ( fig. 8D), caused an increase in α-helical content of about one helix turn in the presence of SDS ( fig. 8A) or 50% TFE ( fig. 8B) at physiological pH. In contrast, calcium had no effect at acidic pH (where Asp/Glu residues are expected to be protonated and uncharged) ( fig. 8A) or on a truncated peptide (α6S) lacking the Asp/Glu residues located at the C-terminal end of the LAAS region ( fig. 8E). In addition, intrinsic tryptophan fluorescence of this BCL2L10-α6L peptide was quenched by calcium ions (see supplementary fig. S3 , Supplementary Material online). Thus, a peptide containing the negatively charged residues of the α5–α6 connecting region is structurally responsive to the presence of calcium ions. Moreover, these findings suggest that calcium could induce or stabilize the structure of this BCL2L10 region.

Circular dichroism analysis of a peptide corresponding to helix α6 of Bcl2l10 as a function of TFE and calcium concentration. The conformation in solution of a peptide corresponding to the α6 helix of human Bcl2l10 (BCL2L10-α6L: RLKEQEGDVARDGQRLVALLSSRLMGQHRAWLQA, amino acids 126–159) and comprising the potential calcium-binding residues and of a truncated peptide variant lacking this site (BCL2L10-α6S: RDGQRLVALLSSRLMGQHRAWLQA, amino acids 136–159) was determined by CD spectroscopy. (A) Percent of estimated α-helicity of BCL2L10-α6L in the presence or absence of Ca 2+ and in the membrane-mimicking solvent SDS, at neutral or acidic pH. (B) Effect of calcium ion on the CD spectra. Addition of saturating amounts of CaCl2 enhances helicity of the BCL2L10-α6L peptide in 50% (v/v) TFE. (C) Percent of estimated α-helicity of BCL2L10-α6L in different concentrations of TFE. The addition of TFE increases the α-helicity of the peptide, reaching a plateau at ∼40% TFE. (D) Percent of estimated α-helicity of BCL2L10-α6L in presence of excess amounts of CaCl2 or MgCl2. The α-helicity of the peptide increases by more than 10% in the presence of Ca 2+ (but not Mg 2+ ). (E) Percent of estimated α-helicity of BCL2L10-α6S in presence of excess amounts of CaCl2. Estimated α-helicity of BCL2L10-α6S does not vary in presence of excess amounts of CaCl2.

Circular dichroism analysis of a peptide corresponding to helix α6 of Bcl2l10 as a function of TFE and calcium concentration. The conformation in solution of a peptide corresponding to the α6 helix of human Bcl2l10 (BCL2L10-α6L: RLKEQEGDVARDGQRLVALLSSRLMGQHRAWLQA, amino acids 126–159) and comprising the potential calcium-binding residues and of a truncated peptide variant lacking this site (BCL2L10-α6S: RDGQRLVALLSSRLMGQHRAWLQA, amino acids 136–159) was determined by CD spectroscopy. (A) Percent of estimated α-helicity of BCL2L10-α6L in the presence or absence of Ca 2+ and in the membrane-mimicking solvent SDS, at neutral or acidic pH. (B) Effect of calcium ion on the CD spectra. Addition of saturating amounts of CaCl2 enhances helicity of the BCL2L10-α6L peptide in 50% (v/v) TFE. (C) Percent of estimated α-helicity of BCL2L10-α6L in different concentrations of TFE. The addition of TFE increases the α-helicity of the peptide, reaching a plateau at ∼40% TFE. (D) Percent of estimated α-helicity of BCL2L10-α6L in presence of excess amounts of CaCl2 or MgCl2. The α-helicity of the peptide increases by more than 10% in the presence of Ca 2+ (but not Mg 2+ ). (E) Percent of estimated α-helicity of BCL2L10-α6S in presence of excess amounts of CaCl2. Estimated α-helicity of BCL2L10-α6S does not vary in presence of excess amounts of CaCl2.


Acceleration of protein glycation by oxidative stress and comparative role of antioxidant and protein glycation inhibitor

Hyperglycemia in diabetes causes protein glycation that leads to oxidative stress, release of cytokines, and establishment of secondary complications such as neuropathy, retinopathy, and nephropathy. Several other metabolic disorders, stress, and inflammation generate free radicals and oxidative stress. It is essential to study whether oxidative stress independently enhances protein glycation leading to rapid establishment of secondary complications. Oxidative stress was experimentally induced using rotenone and Fenton reagent for in vivo and in vitro studies, respectively. Results showed significant increase in the rate of modification of BSA in the form of fructosamine and protein-bound carbonyls in the presence of fenton reagent. Circular dichroism studies revealed gross structural changes in the reduction of alpha helix structure and decreased protein surface charge was confirmed by zeta potential studies. Use of rotenone demonstrated enhanced AGE formation, ROS generation, and liver and kidney tissue glycation through fluorescence measurement. Similar findings were also observed in cell culture studies. Use of aminoguanidine, a protein glycation inhibitor, demonstrated reduction in these changes however, a combination of aminoguanidine along with vitamin E demonstrated better amelioration. Thus, oxidative stress accelerates the process of protein glycation causing gross structural changes and tissue glycation in insulin-independent tissues. Use of antioxidants and protein glycation inhibitors in combination are more effective in preventing such changes and could be an effective therapeutic option for preventing establishment of secondary complications of diabetes.

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Komentari:

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  6. JoJomuro

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