Informácie

Ako vedú mutácie PrP k priónovej chorobe?

Ako vedú mutácie PrP k priónovej chorobe?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Moje pochopenie je:

Gén PrP v ľudských bunkách je exprimovaný ako PrP-c (normálny proteín) aj PrP-sc (proteín priónovej choroby). To sa deje posttranskripčne, to znamená, že normálny a chorý proteín sa nerozlišujú na základe genetických mutácií, ale sú syntetizované na základe toho istého génu, ale líšia sa terciárnymi štruktúrami, čím sa líšia aj ich funkcie.

Ak je sekvencia aminokyselín oboch proteínov rovnaká (rovnaký gén), čo potom určuje, či syntetizovaný proteín bude mať chorobu spôsobujúcu terciárnu štruktúru alebo normálnu? Je to kvôli post-translačným úpravám?

A nakoniec (ospravedlňujem sa vopred za viaceré otázky): ako sa prióny, ktoré neobsahujú nukleové kyseliny (ktoré sú proteínmi), integrujú do genómu novo infikovaného hostiteľa reverznou transláciou a stanú sa familiárnym ochorením?


Ak je sekvencia aminokyselín oboch proteínov rovnaká, čo určuje, či syntetizovaný proteín bude mať chorobu spôsobujúcu terciárnu štruktúru alebo normálnu?

PrP-C a PrP-Sc skutočne majú rovnakú primárnu štruktúru. Líšia sa však sekundárnou a terciárnou štruktúrou. Proteín môže mať viac ako jeden tvar (konformáciu), kde pravdepodobné konformačné priestory, ktoré proteín zaberá, sú spôsobené chemickými a fyzikálnymi interakciami. Mnoho proteínov môže zmeniť svoj tvar, často to nie je dobre pochopené v detailoch. Vieme však, že konformačný priestor niektorých proteínov umožňuje niekoľko stabilných konformácií jedného proteínu; faktory, ktoré ho stabilizujú v konformácii alebo spôsobujú jeho zmenu, sú ťažko pochopiteľné in vivo.

PrP-Sc má tendenciu akumulovať sa v kompaktných agregátoch odolných voči proteázam. Kruhový dichroizmus ukazuje, že normálny PrPC mal 43 % alfa helikálneho a 3 % beta listu, zatiaľ čo PrPSc mal len 30 % alfa helixu a 43 % beta listu. Hoci presná 3D štruktúra PrPSc nie je známa, má vyšší podiel štruktúry β-listu namiesto normálnej štruktúry a-helixu. Koniec každého vlákna funguje ako šablóna, na ktorú sa môžu naviazať voľné molekuly proteínu, čo umožňuje vláknu rásť.

Priónové proteíny teda podporujú akumuláciu stále väčšieho množstva priónových proteínov.

Je to kvôli post-translačným úpravám?

Nie

Ako sa prióny, ktoré neobsahujú nukleové kyseliny, integrujú do genómu novo infikovaného hostiteľa reverznou transláciou a stanú sa familiárnym ochorením?

Nevstupujú do genómu reverznou transláciou. Samotná sekvencia DNA je zmutovaná a mutácia sa pohybuje medzi generáciami tak dlho, kým sa prenáša cez zárodočnú líniu.


Aktivita in vitro očkovania priónov s deficitom glykoformy z prionopatie s premenlivou citlivosťou na proteázu a familiárnej CJD spojenej s mutáciou PrP V180I

Zistilo sa, že sporadická variabilne citlivá prionopatia (VPSPr) a familiárna Creutzfeldt-Jakobova choroba spojená s mutáciou priónového proteínu (PrP) V180I (fCJD V180I ) zdieľajú jedinečný patologický priónový proteín (PrP Sc ), ktorý nemá rezistenciu na proteázy. PrP Sc glykozylovaný na zvyšku 181, pretože dve zo štyroch PrP glykoforiem nie sú zjavne konvertované na PrP Sc z ich bunkového PrP (PrPC). Aby sme preskúmali očkovaciu aktivitu týchto jedinečných molekúl PrP Sc, uskutočnili sme in vitro experimenty s konverziou priónov pomocou sériovej cyklickej amplifikácie nesprávneho poskladania proteínov (sPMCA) a testov konverzie vyvolanej trepaním v reálnom čase (RT-QuIC) s rôznymi substrátmi PrPC. Pozorovali sme, že naočkovanie PrP Sc z VPSPr alebo fCJD V180I v sPMCA reakcii obsahujúcej normálne ľudské alebo humanizované transgénne (Tg) myšacie mozgové homogenáty generovalo PrP Sc molekuly, ktoré neočakávane vykazovali dominantnú diglykozylovanú izoformu PrP spolu s PrP monoglykozylovaným na zvyšku 181. účinnosť amplifikácie PrP Sc bola významne vyššia v non-CJDMM ako v non-CJDVV homogenáte ľudského mozgu, zatiaľ čo bola vyššia v normálnom TgVV ako v TgMM myšom mozgovom homogenáte. PrPC zo zmesi normálneho myšacieho mozgu TgMM a Tg exprimujúceho mutáciu PrP V180I (Tg180), ale nie samotný TgV180I, sa premenil na PrP Sc nasadením VPSPr alebo fCJD V180I. Aktivita očkovania RT-QuIC PrP Sc z VPSPr a fCJD V180I bola významne nižšia ako aktivita sCJD. Naše výsledky naznačujú, že tvorba priónov selektívnych pre glykoformy môže byť spojená s neidentifikovaným faktorom v postihnutom mozgu a nedostatok PrP Sc v glykoforme neovplyvňuje glykoformy in vitro novo amplifikovaného PrP Sc.

Kľúčové slová: Humanizované transgénne myši Polymorfizmus Priónová Priónová choroba Konverzia vyvolaná zemetrasením v reálnom čase (RT-QuIC) Cyklická amplifikácia sériového nesprávneho poskladania proteínov (sPMCA) Variabilne citlivá na proteázu prionopatia (VSPPr).


Priónová patogenéza, diagnostika a terapia: kde sa nachádzame?

Priónové ochorenia, tiež známe ako prenosné spongiformné encefalopatie (TSE), sú vždy fatálne neurodegeneratívne poruchy postihujúce široké spektrum hostiteľských druhov a vznikajú prostredníctvom genetických, infekčných alebo sporadických mechanizmov (tabuľka ​ (tabuľka 1). 1). U ľudí sú priónové choroby výsledkom infekčných spôsobov prenosu (variant Creutzfeldt-Jakobova choroba [vCJD], iatrogénna CJD, Kuru) zdedených spôsobov prenosu, pri ktorých existuje nekonzervatívna mutácia zárodočnej línie PRNP otvorený čítací rámec génu (familiárna CJD, Gerstmann-Sträussler-Scheinkerov syndróm, fatálna familiárna insomnia) (1, 2) a spôsoby prenosu, ktoré zatiaľ neboli stanovené ani pochopené (sporadická CJD [sCJD]). Klinické symptómy spojené s každou z foriem ľudskej priónovej choroby sa dramaticky líšia (2).

Stôl 1

Spektrum priónových chorôb ľudí a zvierat

Nomenklatúra aplikovaná na priónovú biológiu je naďalej zložitá a pre neodborníkov mätúca. Tu používame termín “prión” na označenie pôvodcu priónových chorôb bez toho, aby sme implikovali súvisiace štrukturálne vlastnosti. Hovoríme o priónovom proteíne asociovanom s ochorením (PrP Sc ), chorobe špecifickej izoforme bunkového priónového proteínu kódovaného hostiteľom (PrP C ), ktorý sa akumuluje u jedincov postihnutých väčšinou foriem TSE (obrázok ​ (obrázok 1) 1) (3). Zatiaľ čo PrP Sc je klasicky definovaný ako čiastočne rezistentný na proteázu, agregovaný PrP, nedávno sa ukázalo, že PrPC môže prejsť štrukturálnymi modifikáciami súvisiacimi s ochorením, ktoré neprinášajú vlastnosti vlastnej rezistencie na proteázy (4). Vzhľadom na to sa odporúča, aby bol PrP Sc definovaný skôr na základe štrukturálnych modifikácií spojených s ochorením než na základe vlastností rezistencie na proteázy.

Modely konverzie PrP C na PrP Sc. (AHeterodimérny model navrhuje, že po infekcii vhodnej hostiteľskej bunky prichádzajúca PrP Sc (oranžová) spustí katalytickú kaskádu využívajúcu PrPC (modrá) alebo čiastočne rozvinutý medziprodukt vznikajúci zo stochastických fluktuácií v konformáciách PrPC ako substrátu, ktorý ho premieňa. konformačnou zmenou na nový β-listový– proteín bohatý. Novovytvorený PrP Sc (zeleno-oranžový) zase prevedie nové molekuly PrPC. (B) Model nekatalytickej jadrovej polymerizácie navrhuje, že konformačná zmena PrPC na PrP Sc je termodynamicky riadená: konverzia PrPC na PrP Sc je reverzibilný proces, ale v rovnováhe silne podporuje konformáciu PrPC. Konvertovaný PrPSc sa vytvorí iba vtedy, keď sa pridá na vláknité semeno alebo agregát PrPSc. Akonáhle sú zárodky prítomné, ďalšie pridávanie monoméru sa urýchli.

Priónové choroby sú koncepčne nedávne, prvé prípady Creutzfeldt-Jakobovej choroby boli opísané pred ôsmimi desaťročiami (5, 6), avšak teória priónovej infekcie, ktorá obsahuje iba proteíny, bola pôvodne sformulovaná v roku 1967 (7) a neskôr spresnená a termín “prion& #x0201d vyrazený v roku 1982 (8). Presná fyzikálna povaha priónového činidla je stále predmetom intenzívnych vedeckých sporov. PrP Sc môže alebo nemusí byť v súlade s infekčným agensom. Zostáva formálne dokázať, či infekčná jednotka pozostáva predovšetkým alebo výlučne z: (a) poddruhu PrP Sc (b) strednej formy PrP (PrP*) (9) (c) iných proteínov odvodených od hostiteľa (10 ) alebo (d) neproteínové zlúčeniny (ktoré môžu zahŕňať glykozaminoglykány a možno aj nukleové kyseliny) (11). Stále teda nevieme, či je priónová hypotéza ako celok správna.

Tak ako pri akomkoľvek inom ochorení, dôkladné mechanické pochopenie patogenézy je najlepším základom pre navrhnutie citlivej prediktívnej diagnostiky a účinných terapeutických režimov.

Účelom tohto článku je diskutovať o niektorých aspektoch súčasného stavu techniky v priónovej vede a ich vplyve na diagnostiku priónov, predovšetkým s ohľadom na periférne získané priónové ochorenie. V súčasnosti nie je možné obetiam priónovej choroby ponúknuť žiadnu kauzálnu terapiu. Napriek tomu sme svedkami objavenia sa pôsobivého množstva terapeutických prístupov, z ktorých niektoré si určite zaslúžia otestovať svoju platnosť.


Výsledky

Lokalizácia a povaha mutácií

Mutácie P101L a H186R sú spojené s familiárnym priónovým ochorením u ľudí P101 a H186 sú medzi druhmi cicavcov vysoko konzervované. Tieto mutácie sa vyskytujú vo veľmi odlišných oblastiach proteínu: P101 sa nachádza v neštruktúrovanom chvoste sekvencie PrP 27� (modelované na obr. 1), zatiaľ čo H186 je zbalený do jadra zloženej C-terminálnej domény, medzi & #x003b12, 㬓 a 㬢. Zdá sa pravdepodobné, že dôvody, prečo by tieto mutácie mali uprednostňovať konverziu PrPC na iné formy, vrátane patogénnych foriem, sa môžu líšiť. Dominantné negatívne mutácie Q167R a Q218K sa vyskytujú v oblastiach proteínu, ktoré sú vystavené rozpúšťadlu, v slučke medzi 㬢 a 㬒 pre Q167 a ku koncu špirály 㬓 pre Q218. Ani jeden z týchto zvyškov nie je medzi druhmi cicavcov vysoko konzervovaný: Q167 je nahradený Glu v ľudskej sekvencii, zatiaľ čo Q218 je nahradený Glu v sekvenciách primátov.

Štrukturálne zmeny PrP spôsobené mutáciami a zmenou pH

Poruchy chemického posunu spôsobené pri pH 5,5 mutáciami P101L, Q167R a Q218K sú malé a sú striktne lokalizované na mieste mutácie a bezprostredných susedných zvyškoch (obr. 2A), čo je v súlade s polohami týchto miest mutácií v oblastiach vystavených rozpúšťadlu proteínov, ktoré majú nízku sekundárnu a terciárnu štruktúru (5,6). Naproti tomu mutácia H186R spôsobuje veľké zmeny chemického posunu pre linker medzi 㬑 a 㬢 (Y156, Q159, Y162), 㬒 (Q185, T191, G194) a 㬓 (F20697,). a drobné zmeny chemického posunu okolo 㬡. Relatívna veľkosť týchto zmien je v súlade s pozíciou zvyšku 186 v strede 㬒 a usporiadaním bočného reťazca do jadra molekuly. Napriek tomu, dokonca aj pre H186R, väčšina chemických posunov bola prakticky neovplyvnená mutáciami, čo naznačuje, že celková štruktúra myšieho PrP nebola podstatne zmenená v žiadnej z mutácií. Variácie v chemickom posune pri meniacich sa podmienkach, ako je pH (3,5 − 5,5), KCl (0 − 150 mM), močovina (0 − 1,5 M) a teplota (25 − 40ଌ) ukázali, že štrukturálne poruchy boli najvýznamnejšie pri zmene pH (doplnkový obrázok S2). Rozdiely v chemickom posune medzi mutantnými proteínmi a proteínmi divokého typu pri pH 3,5 (obr. 2B) vykazovali podobné trendy ako tie pri pH 5,5 Pretože rozdiely v chemickom posune medzi mutantmi a divokým typom pri pH 5,5 a pH 3,5 boli podobné, usúdili sme, že všetky proteíny boli ovplyvnené kyslým pH podobným spôsobom.

Poruchy spôsobené každou mutáciou na chemických posunoch 15N a 1H. Každý panel zobrazuje absolútnu hodnotu priemerného rozdielu chemických posunov (Δδ) vypočítaného spriemerovaním rozdielov chemických posunov amidu 15N a 1H pomocou empirických rovnica, Δδpriem = [Δδ( 1 H) 2 +Δδ( 15 N) 2 ] ½ , vynesené ako funkcia primárnej sekvencie a zahŕňa stĺpce predstavujúce polohy α-helixov ( červená) a β-vlákna (modrá) v 3D štruktúre proteínu divokého typu. A. pH 5,5 B. pH 3,5. Údaje Δδ nie sú dostupné pre zvyšky 168�, pre ktoré neboli pozorované žiadne rezonancie hlavného reťazca (5,26).

Pretože kyslé pH zvyšuje pravdepodobnosť konverzie na patogénnu formu (10-15), predpokladali sme, že zdedené patogénne mutanty PrP môžu po okyslení vykazovať rôzne štrukturálne poruchy ako dominantný negatívny mutant PrP alebo proteíny divokého typu. Avšak rozdiely v chemickom posune medzi pH 5,5 a pH 3,5 boli rovnaké pre divoký typ, P101L, Q167R a Q218K, pričom všetky vykazovali podobné a veľké zmeny chemického posunu pre zvyšky z K184 na T198 (obr. 3). Táto oblasť zahŕňa C-koncovú polovicu 㬒 a spojovaciu slučku medzi špirálami 㬒 a 㬓. Spomedzi ovplyvnených zvyškov H186, T191 a K193 podliehajú najväčším zmenám chemických posunov, čo naznačuje, že C-terminálna časť 㬒 hrá dominantnú úlohu v štrukturálnych zmenách, ktoré sa vyskytujú pri kyslom pH. Táto oblasť má tiež najväčšie rozdiely v chemickom posune medzi pH 7,0 a pH 4,5 v ľudskom PrP (121�) (44). V ostrom kontraste, mutant H186R vykazuje len malé rozdiely v chemickom posune pre helix 㬒 (obr. 3), čo znamená, že tento mutant nepodlieha štrukturálnym poruchám závislým od pH. Tieto pozorovania identifikujú histidín 186 ako zodpovedný za zmeny závislé od pH v NMR spektrách divého typu, mutantných proteínov P101L, Q167R a Q218K. Okrem toho divoký typ a všetky mutanty zdieľajú významné zmeny chemického posunu závislé od pH pre zvyšky 141� a 156� na N- a C-koncoch 㬒 a 㬢 a zvyšky 205𢮄 x003b13 (obr. 3), čo znamená druhú titrovateľnú skupinu.

Perturbácie spôsobené zmenou pH z 5,5 na 3,5 pre divoký typ a každý z mutantných proteínov. Δδpriem = [Δδ(1H)2 +Δδ(15N)2] ½.

Ďalšie zníženie pH na ≈ 2,1 viedlo k výraznému rozšíreniu čiar všetkých rezonancií s výnimkou N-koncovej rozvinutej oblasti (údaje nie sú uvedené), toto správanie bolo podobné správaniu β-oligomérnej formy ľudského PrP pod miernym denaturačné podmienky (1 M močovina, 0,2 M NaCl, 20 mM octan sodný pH 3,6) (45).

Dynamika chrbtice divokého typu a mutantných PrPs

15 N T1, 15 N T2a súbory údajov [1H]-15N NOE sa získali pri 298 K v 20 mM octane sodnom (pH 5,5 a pH 3,5) (doplnkový obrázok S3). Vzhľadom na prítomnosť ≈ 35 neštruktúrovaných zvyškov na N-konci v PrP(89�), ktoré ovplyvňujú prevracanie zloženej domény, nebolo možné tenzor rotačnej difúzie odvodiť priamo zo štruktúry, ale bol získaný z prispôsobenie súborov relaxačných údajov štruktúre myšieho PrP(121�) divokého typu (PDB id: 1xyx) (26) za predpokladu, že celkové štruktúry PrP divokého typu a mutantných PrP sa podstatne nelíšia. Rotačné korelačné časy vypočítané z relaxačných údajov boli 9,8�,9 ns, oveľa dlhšie ako tie, ktoré sa očakávali z empirického Stokes-Einsteinovho odhadu (≈ 8,4 ns) (46), a odrážajú vplyv rozvinutia N-konca oblasti na rotačnom prevracaní C-koncovej zloženej domény (38). Molekulárne omieľanie divokého typu a mutantných PrPs je mierne anizotropné (DD ≈ 1.4𢄡.7) tak, že osovo symetrické modely difúzie zodpovedajú experimentálnym údajom oveľa lepšie ako model izotropnej difúzie (doplnková tabuľka S1). V armatúre sa os najdlhšej špirály 㬓 zhoduje s hlavnou hlavnou osou rotačného difúzneho tenzora. Dominantný účinok 㬓 na anizotropiu myšieho PrP(89�) je v súlade s predchádzajúcimi pozorovaniami na proteínoch PrP (23� a 90�) sýrskeho škrečka (47). Pomocou prispôsobených rotačných difúznych tenzorov sa dynamika hlavného reťazca každého zvyšku stanovila bezmodelovou analýzou (35-37). Predchádzajúce pokusy o bezmodelovú analýzu PrP sýrskeho škrečka (23� a 90�) viedli k neplatným parametrom poradia (S 2 > 1) pre mnohé zvyšky (25, 47). Súčasná analýza používa neizotropný rotačný difúzny tenzor (48) a Bayesovské informačné kritérium (39,40) na výber modelu v spojení s elimináciou nerealistických modelov, ktoré poskytujú fyzikálne zmysluplné hodnoty S2 pre všetky prispôsobené zvyšky vo voľnej prírode. typu a mutantov. Je pozoruhodné, že nedávna bezmodelová analýza skrátenej formy myši PrP (113�) pomocou izotropného rotačného difúzneho tenzora tiež viedla k platným hodnotám S2 (49).

Pohyby N-koncových nezložených zvyškov sa analyzovali pomocou modelu lokálnej rotačnej difúzie, pretože ich rotačné prevracanie by malo byť nezávislé od C-koncovej zloženej domény proteínu. Hodnoty S2 v tejto oblasti sú ≈ 0,4, čo je v súlade s vysoko flexibilnou povahou N-koncovej oblasti (6). Avšak pre všetky PrP existuje zhluk zvyškov okolo H95, pre ktorý sa S2 (≈ 0,6𢄠,8) pohybuje nad zvyškom N-koncovej rozvinutej oblasti (obr. 4).

Parametre poradia (S 2 ) pri pH 5,5 (zelená) a pH 3,5 (červená) pre proteíny divokého typu a mutantné proteíny, vypočítané z bezmodelovej analýzy 500 a 600 MHz 15 N T1, 15 N T2a súbory údajov [1H]-15N NOE merané pri 298 K (údaje zobrazené na doplnkovom obrázku S3). Pre N-koncovú rozvinutú oblasť (89�) sa použil model lokálnej rotačnej difúzie. Pre zloženú oblasť (127�) bol optimalizovaný globálny axiálne symetrický rotačný difúzny model pomocou dvojpoľových relaxačných dátových súborov a myšacej PrP štruktúry (PDB ID: 1xyx) (26). Hodnoty S2 nie sú dostupné pre zvyšky 168�, pre ktoré neboli pozorované žiadne rezonancie hlavného reťazca (5,26).

Pri pH 5,5 (zelené stĺpce na obr. 4) má C-terminálna zložená oblasť všetkých proteínov S2 väčšiu ako 0,85, čo svedčí o obmedzenom pohybe kostry. Avšak dve široké oblasti od 㬡 po 㬑 (zvyšky ≈ 134�) a od C-koncovej polovice 㬒 po začiatok 㬓 (zvyšky ¶x0224) nižšie hodnoty S2, čo svedčí o flexibilite chrbtice. Všetky proteíny, divokého typu aj mutanty, zdieľajú podobný vzor S2 okrem toho, že krátky segment v mutante H186R po zvyšku 186 (≈ 187�) vykazuje prudký pokles v S2, zatiaľ čo v ostatných proteínoch, pokles bol pozvoľnejší (obr. 4). Pri pH 3,5 (červené stĺpce na obr. 4) všetky proteíny okrem H186R vykazovali podstatné zníženie S2 v rovnakej oblasti (zvyšky 187�), zatiaľ čo zvyšok proteínu nebol v podstate ovplyvnený znížením pH. S2 pre mutant H186R bol podobný hodnotám pozorovaným pre neusporiadaný N-koncový chvost. Zmeny v S2 medzi pH 5,5 a 3,5 pre divoký typ PrP sú znázornené na obr. 5A, B a porovnané s variáciami pre mutant H186R na obr. 5C. Tieto pozorovania naznačujú, že zvyšky 187� sú v mutantnom proteíne H186R neusporiadané aj pri neutrálnom pH.

Hodnoty S 2 sú zobrazené mapované na myšacej PrP štruktúre (PDB ID: 1xyx) (26) ako súvislá farebná škála: červená pre S 2 < 0,6, červená až žltá pre 0,6 ≤ S 2 < 0,8, a žltá až modrá pre 0,8 ≤ S 2 < 1,0. Prolíny a zvyšky, pre ktoré sa S2 neurčuje v dôsledku spektrálneho prekrytia, absencie údajov alebo zlyhania pri montáži, sú zobrazené sivou farbou. A. PrP divokého typu pri pH 5,5. B. PrP divokého typu pri pH 3,5. C. H186R mutant PrP pri pH 5,5. Vedľajší reťazec H186 je znázornený zelenou farbou.

Vnútorné pohyby na časovej škále ns sa objavujú v oboch flexibilných segmentoch (zvyšky ≈ 134� a ≈ 187�) (obr. 6). Vnútorné pohyby ns v prvom segmente sa šíria smerom k N-koncu až po A116. Znížené spektrálne hustoty J(ωN) a J(0,89ωH) by sa mohli použiť bez toho, aby boli potrebné predpoklady v analýze bez modelu, a poskytnúť ďalšie dôkazy o významnej flexibilite v týchto regiónoch (doplnkový obrázok S4). Časové škály vnútorných pohybov sú prakticky neovplyvnené pH s výnimkou divokého typu a P101L. Je zaujímavé, že iba proteíny divokého typu a proteíny P101L majú rozsiahle vnútorné pohyby ns v helixoch 㬑-㬓 pri pH 5,5 (obr. 6), aj keď vysoké hodnoty S2 naznačujú obmedzený pohyb chrbtice v čase ps-ns stupnica. Mnohé z týchto ns vnútorných pohybov zmiznú pri pH 3,5 (obr. 6). Kombinácia nízkych amplitúd pohybu chrbtice ps-ns s rozsiahlym vnútorným pohybom ns v tej istej oblasti bola hlásená v mnohých prípadoch (50). Čiastočná agregácia divokého typu a mutantných proteínov P101L by mohla byť v zásade zodpovedná za tieto zjavne anomálne vnútorné pohyby (51). Na základe meraní translačnej difúzie by sme však mohli túto možnosť vylúčiť: pri rovnakej koncentrácii ako merania relaxácie NMR (0,55 mM), divoký typ, P101L a Q218K majú rovnaké koeficienty translačnej difúzie v rámci experimentálnej chyby (1,06 ± 0,01, 1,08 ± 0,01 a 1,08 ± 0,02 × 10 𢄦 cm2 s 𢄡 pri 298 K).

Korelačný čas vnútorného pohybu (τe) pri pH 5,5 (zelená) a pH 3,5 (červená). τe bol vypočítaný z bezmodelovej analýzy 500 a 600 MHz 15N T1, 15 N T2, [ 1 H] - 15 N NOE súbory údajov merané pri 298 K. τe údaje nie sú dostupné pre zvyšky 168�, pre ktoré neboli pozorované žiadne rezonancie hlavného reťazca (5,26).

Všetky proteíny divokého typu a mutantné proteíny vykazujú prudké zvýšenie R2 relaxačná miera blízko 㬡 a 㬢, pri G130, V165 a D166 (doplnkový obrázok S3) sa takéto zvýšenie zvyčajne interpretuje ako dôkaz konformačnej výmeny na časovej škále μs-ms. Aby sa oddelil výmenný príspevok od R2, bez výmeny R2 (R20) bola stanovená z priečnej krížovej korelácie (ηxy) sadzby (52). Naše zistenia ukazujú, že tieto miesta skutočne podliehajú významnej konformačnej výmene (obr. 7A). Carr-Purcell-Meiboom-Gill (CPMG) 15 N R2 údaje o relaxačnej disperzii G130, V165 a D166 ukazujú zreteľné rozdiely medzi R2 relaxačné frekvencie pri 500 a 800 MHz, aj keď každá má malé R2 disperzia (υs𢄡) (obr. 7B). Výmenný kurz a počet obyvateľov štátov boli odhadnuté zo súčasného prispôsobenia 15 N R2 disperzné dáta G130, V165 a D166 s použitím nameraných R20 a model výmeny na dvoch miestach. Tieto zvyšky podliehajú rýchlej výmene (7000 ± 2000 s 𢄡 ), kde populácia menej priaznivej konformácie je ≈ 0,4 %. Rýchla konformačná výmena G130, V165 a D166 môže súvisieť s prechodným časovým rozsahom (μs až ms) konformačnou fluktuáciou v slučke spájajúcej 㬢 a 㬒 (zvyšky 168�), ktoré sú s najväčšou pravdepodobnosťou zodpovedné za závažné rozšírenie rezonancií hlavného reťazca týchto zvyškov u divokého typu a všetkých štyroch mutantných PrP (5, 26). Slučka medzi zvyškami 170� sa podieľa na chorobe (53,54), nadmerná expresia PrP(170N, 174T) spôsobuje ochorenie spongiformnej encefalopatie u myší (55). Je zaujímavé, že táto slučková oblasť ľudského, kravského, myšieho, psa a mačacieho PrP je flexibilná, zatiaľ čo oblasť losa, sýrskeho škrečka a hraboša je nepružná (6,26,54,56-58), čo poskytuje prehľad o druhových bariérach pre priónová choroba.

Konformačná výmena. A. Prínos konformačnej výmeny R2 relaxácia. Miera priečnej vzájomnej korelácie, ηxymedzi 15N-1H dipól-dipólovou interakciou a 15N anizotropiou chemického posunu (CSA) sa merala pri 500 MHz pre divoký typ (čierna) a H186R (červená) PrP(89�) pri pH 5,5 pri 298 K Bez výmeny R2 (R20) vypočítal R20 = -ηxy𢆣[(4c 2 +3d 2 )]/[12cdP2(cos(β))] (52), v ktorom c = (ωN/𢆣)Δσ, d = [μ0hγNγH/(8π 2 )](1/r 3 NH), P2(x) = (3x 2 -1)/2 je Legendreov polynóm druhej kategórie, h je Planckova konštanta, μ0je priepustnost volneho priestoru, Δσ je 15 N CSA, r.NH je dĺžka amidovej NH väzby a β je uhol medzi hlavnou osou 15N CSA tenzora a amidovým NH väzbovým vektorom. 15 N ČSA, rNH a β sa považovali za � ppm, 1,02 Å a 20°. B. Carr-Purcell-Meiboom-Gill (CPMG) na báze 15 N R2 relaxačná disperzia divokého typu PrP (89�) pri pH 5,5 a 298 K. 15 N R2 relaxačné disperzné dáta G130, V165 a D166 pri 500 a 800 MHz boli súčasne prispôsobené všeobecnej rovnici pre výmenu na dvoch miestach (knapr=kA𡤫+kB𡤪, sA(=1- strB), strB, Δω) (42) pomocou nameraného R20. Údaje (plný kruh) a preložené krivky (plná čiara) pri 500 MHz (čierna) a 800 MHz (červená) sú zobrazené pre G130, V165 a D166.

Na rozdiel od divokého typu a iných mutantov má mutant H186R dve sady rezonancií na zvyškoch Y162 a R163 v 㬢 pri pH 5,5, pričom jedna sada má chemické posuny podobné tým, ktoré majú ostatné mutanty a proteíny divokého typu (doplnkový obrázok S1 ). M128, L129 a G130 v 㬡 nevykazujú túto pomalú konformačnú výmenu, hoci majú o niečo širšie rezonancie ako iné zvyšky. Tieto pozorovania pripisujeme pomalej výmene medzi dvoma konformáciami (rýchlosť výmeny je príliš pomalá na to, aby ju bolo možné detegovať v experimentoch s 15 N longitudinálnou výmenou 2-spin-order (zz-výmena), ktoré nie sú uvedené). V konformácii s chemickými posunmi odlišnými od divokého typu a iných mutantov Y162 vykazuje výrazne väčšiu flexibilitu: ([1H]-15N NOE sú 0,19 ± 0,02 a 0,29 ± 0,04 pri 500 a 600 MHz, v tomto poradí (obr. S3G) a S2 je 0,34 ± 0,01) ako konformácia s chemickými posunmi podobnými divokému typu a ostatným mutantom: ([1H]-15N NOE sú 0,72 ± 0,09 a 0,61 ± 5000b1 0. a 600 MHz a S2 je 0,92 ± 0,01). Zdá sa, že mutácia H186R destabilizuje β2, čo vedie k rovnováhe medzi usporiadanými a neusporiadanými stavmi. Je zaujímavé, že vrchol Y162 pochádzajúci z pevnejšej konformácie zmizne pri pH 3,5, zatiaľ čo druhý vrchol Y162 pochádzajúci z flexibilnejšej konformácie zostáva (S2 = 0,30 ± 0,01), čo naznačuje, že konformačná rovnováha v oblasti 㬢 H186R sa posúva smerom k neusporiadanému stavu pri nižšom pH.

Protonácia H186

Keďže naše výsledky ukázali, že H186 sa podieľa na destabilizácii PrP, ku ktorej dochádza pri znižovaní pH, protonačné stavy H186 sa skúmali pri pH 5,5 a 3,5 pomocou 15 chemických posunov N㭁 a 15 N㭒 (priradenia pre stranu reťazcové rezonancie 5 histidínov sú znázornené na doplnkovom obrázku S5). Rezonančné frekvencie 15 N㭁 a 15 N㭒 sú pri ≈ 168 a ≈ 250 ppm v neutrálnom imidazole a obe rezonujú pri ≈ 177 ppm (29, keď sú úplne protónované). Stredné hodnoty chemického posunu svedčia o rýchlej výmene medzi protónovanými a deprotonovanými stavmi. Spomedzi piatich histidínov v myšiach divokého typu PrP (89�) sú na povrchu exponované H95, H110, H139 a H176 a H186 je čiastočne pochovaný a obklopený hydrofóbnymi zvyškami (5). Chemické posuny 15 N㭁 a 15 N㭒 naznačujú, že H110 a H176 sú protónované pri pH 5,5 H95, H139 a H186 sú čiastočne deprotonizované pri pH 5,5, ale stávajú sa protonizované pri pH 3,5 (doplnková tabuľka S2) že pKas H95, H139 a H186 imidazolového bočného reťazca sú podstatne nižšie ako typické pKa hodnota (6,6 ± 1) významne znížila pKa hodnoty sú často pozorované pre histidíny nachádzajúce sa vo vnútri proteínov (59-61). Teoretické pKa výpočty tiež odhadujú trvalo nízke pKa pre H186 (62), čo podporuje názor, že nízke pKa H186 je spôsobený čiastočne zakopaným prostredím. Okrem toho široké čiary rezonancií imidazolu H186 v spektre HMQC pri pH 5,5 aj pH 3,5 (obr. 8) naznačujú prítomnosť chemickej výmeny na časovej škále μs-ms.

1H-15N HMQC spektrá s dlhým dosahom histidínových zvyškov divokého typu PrP (89�) pri pH 5,5 (čierna) a pH 3,5 (červená). Protonačné stavy histidínov myšieho PrP divokého typu (89�) v pH 5,5 a 3,5 pri 292 K sú odvodené z 15 chemických posunov N㭁 a 15 N㭒. Polohy krížových píkov očakávaných medzi jadrami histidínového kruhu, keď je kruh úplne protónovaný, sú mapované v ľavom hornom rohu. 15N㭒 rezonancia H186 nebola pozorovaná pri žiadnom pH. Priradenia piatich histidínov sú vyznačené červenou (H95), oranžovou (H110), zelenou (H139), modrou (H176) a čiernou (H186).

Je prekvapujúce, že H95 a H139 zobrazujú pKas tak nízke ako H186, napriek ich zjavnej povrchovej expozícii. Nezdá sa, že by existovali podstatné rozdiely v chemickom posune v blízkosti týchto zvyškov pri zmene pH, na rozdiel od H186 (obr. 3). Nízke pKas imidazolov H95 a H139 môžu byť spôsobené dynamickými účinkami, ako sú prechodné hydrofóbne alebo elektrostatické interakcie.


Prvý celohumánny myšací model dedičnej priónovej choroby

Ľudské priónové choroby zahŕňajú Creutzfeldt-Jakobovu chorobu (CJD) a Gerstmann-Sträussler-Scheinkerovu chorobu (GSS). Nová štúdia v časopise PLOS Biology s otvoreným prístupom uvádza významný pokrok vo vývoji myších modelov ľudských priónových chorôb. Štúdia Emmanuela Asanteho a kolegov z Medical Research Council Prion Unit na University College London demonštruje spontánnu tvorbu prenosných priónových proteínových zostáv u myší, ktoré nesú len ľudské formy priónového proteínu.

Priónové ochorenia sú spôsobené chybným poskladaním a prenosom priónových proteínov z bunky do bunky, ktoré ďalej vyvolávajú chybné poskladanie v bunke príjemcu. Významnou črtou priónových chorôb je, že rôzne mutácie vedú k chorobám s nápadne odlišnými klinickými prejavmi. Pri štúdiu týchto chorôb bola verná tvorba a propagácia odlišných kmeňov špecifických pre chorobu nevyhnutná na pochopenie prenosu a patogenézy.

Priónové ochorenia boli do značnej miery modelované u myší zavedením génu pre priónový proteín nesúci mutáciu spôsobujúcu ochorenie. V predchádzajúcich štúdiách sa mutácie spôsobujúce ochorenie neštudovali priamo na géne ľudského priónového proteínu, ale namiesto toho sa ekvivalentné mutácie zaviedli do génu myšieho priónového proteínu. This complication can cause formation and propagation of a strain of misfolded protein that is not found in human disease, thereby limiting our understanding of the human prion disease.

To overcome this problem, the research team introduced a mutant human prion gene into mice carrying no mouse prion gene. As the mice aged over a year and a half, they spontaneously developed clusters of misfolded prion protein, something never observed before. When those clusters were used to inoculate younger mice carrying the same mutation, those mice developed misfolded prion protein clusters as well, directly demonstrating infectivity of the mutant protein, and mimicking the infectivity of patient-derived clusters of the same mutant protein. This is the first time that a spontaneous infection due entirely to mutant human prion protein has been shown in mice.

"This new model of an inherited prion disease is likely to provide important insights into human disease that we have previously been unable to study in the mouse," Dr Asante said, including events of disease initiation and spread that may inform development of therapies.


How do PrP mutations lead to prion disease? - Biológia

Healthy proteins misfold into potentially deadly prions

Prion diseases eat away at the brain. At top, a normal neuron, and below, an infected neuron with an accumulation of the abnormal, scrapie form of the prion protein. Scrapie is a fatal disease affecting sheep.

Humans get prion disorders from inherited mutations, through contami-nation during a medical procedure or, in very rare instances, from consumption of infected animals.

Like any genuinely new area of research, prions seem to have a habit of throwing unexpected surprises at scientists.

Connecting prion disease to other more common disorders, such as Alzheimer’s
or other neuro-degenerative disorders, may expand the number of investigators interested in conducting prion research.

FOR RESEARCHERS LIKE David A. Harris, MD, PhD, the long, slow exit from the twilight zone is all but over. Harris has been studying prions, a new kind of infectious agent thought to be at the heart of several rare neurodegenerative disorders that devastate the brains of humans, cows and sheep.

Prions are weird — unlike any other infectious agent ever identified before. Harris, professor of cell biology and physiology, remembers a time when describing his research sometimes gave him the impression other scientists thought he had “gone to outer space” or was working on “black magic.”

Seven years after the Nobel Prize went to a prion researcher, Harris admits that an ironclad proof of prion theory has yet to be produced. But the skeptics are finding it harder and harder to make their case, and Harris now has a colleague in prion research in his own department, Heather L. True-Krob, PhD, assistant professor of cell biology and physiology.

Harris and True-Krob are gathering new insights into how prions form and cause disease, and as they do, tantalizing hints are starting to emerge that prions may be connected to a much wider range of biological phenomena than the rare brain disorders that first led to their discovery.

Heather L. True-Krob, PhD, and David A. Harris, MD, PhD

Until prions came along, infectious agents always contained some type of genetic material. That material carried the linchpin of the infection cycle: instructions for hijacking host cells to produce new copies of the infectious agent and begin the cycle anew.

Not so the prion — it consists entirely of a misfolded protein. The prion perpetuates itself by influencing nearby normal copies of the same protein, somehow increasing the chances they will misfold and become prions. In cows with mad cow disease, sheep with scrapie, and humans with Creutzfeldt-Jakob disease, this causes a chain reaction that leaves the brain a spongy, hole-filled mess.

Humans get prion disorders from inherited mutations, through contamination during a medical procedure or, in very rare instances, from consumption of infected animals. In addition, some “spontaneous” cases of human prion disease currently can’t be tracked to any genetic or environmental cause. The disorders have no treatment and are fatal in months to several years.

The first part of prion theory, the idea that a change in folding can radically change a protein’s properties, is well-established biological fact. Proteins are long, complex chains, and as those chains fold up, they form specialized structures that can perform various functions. Rearranging the way a protein folds can eliminate those structures, create new structures, or change their accessibility.

The process is roughly comparable to a Swiss Army knife: fold the protein in one configuration, and the can opener sticks out and can be used fold it into another, and the can opener vanishes, a screwdriver sticks out, and the protein has suddenly become a tool used for a very different purposes.

Much of the resistance among scientists to accepting prions springs from the second part of prion theory: the idea that interaction with a misfolded protein can cause another copy of the same protein to become badly folded. The details of how this unprecedented conversion takes place are still a mystery.

“ The problem is that no one knows the exact three-dimensional structure of the prion,” Harris explains. “We know the normal structure of the protein that becomes a prion, but not the structure of the prion itself, and that’s left the process by which prions spread a kind of black box.”

The normal function of the protein that becomes a prion also remains a mystery. Scientists named the protein PrP: The normally folded copy is referred to as PrPC (for cellular PrP), while the prion form is known as PrPSc (for scrapie PrP).

Recent evidence even has scientists questioning one of their most basic assumptions about prions: the idea that PrPSc is the form of the prion protein that kills brain cells. Studies by Harris have shown that transgenic mice with a mutant form of PrP prone to forming prions will get symptoms like those in human prion disorders, but the disease is not infectious to other animals.

“ In terms of the different forms of PrP, we have early evidence that what’s needed to kill a neuron may be different from what’s needed to pass on an infection,” Harris notes.

Like any genuinely new area of research, prions seem to have a habit of throwing unexpected surprises at scientists. One such surprise has actually boosted acceptance of prions among the research community: the identification of prions in yeast cells.

True-Krob specializes in the study of yeast prions, which don’t affect humans and other mammals but have similar structural elements. Yeast prions spread the same way as mammalian prions, with proximity to misfolded copies of the yeast prion protein, Sup35, somehow increasing the chances that normal copies of the same protein will become prions.

During her postdoctoral studies, True-Krob led a project that uncovered another major prion surprise: a positive role for yeast prions. Sup35 normally helps yeast read protein-building instructions from its DNA, a process called translation. True-Krob showed that the prion form of Sup35 disrupted this process. As a result, new material was added to proteins.

The switch to prion-prone Sup35, which occurs spontaneously about once in every million generations of yeast, often has harmful effects. But in about 20 percent of test cases, the disruptions gave the yeast a survival advantage in an environment in which temperature, the availability of food or other factors had changed.

“This system is advantageous for the yeast because they have a way of turning prions on and off,” True-Krob notes. “And that gives us hope that what we learn from yeast may help us find a way to turn prions off in humans.”

Working with prions in yeast lets True-Krob conduct studies that would be prohibitively complex or even impossible in mammalian cells. She can simultaneously expose many different yeast cell lines to a wide range of environmental conditions and genetic variables and see how these factors influence the likelihood that prions will form.

True-Krob is active in the search for additional yeast prions, which has netted a second yeast prion also linked to the translation of information in DNA.

“ People have speculated that there may be up to 100 different prions in yeast,” True-Krob says. “What we learn in yeast will help us search for prions in other systems.”

Harris, who studies mammalian prions, describes his lab’s interests as the molecular and cellular biology of the prion protein: What do both forms of PrP do in the nerve cell, where do they do it, and what do they interact with?

Harris conducts the bulk of his research in approximately 50 lines of mice genetically modified to produce prions and symptoms similar to human prion diseases. In recent years, they’ve produced important clues about what PrPC and PrPSc may be doing in the brain.

Work in mouse models has shown that PrP scrapie builds up in clumps in the brain similar to those seen in more common neurodegenerative disorders like Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease.

Another similarity to these disorders emerged in a recent study, led by Harris, of a cellular suicide switch known as Bax. Harris had read about experiments from other researchers linking Bax to nerve cell suicide in other neurodegenerative disorders, so he decided to see what would happen in one of his mouse models if the Bax gene was knocked out.

As he had hoped, the alteration saved a class of mouse brain cells normally killed off in dramatic fashion in the mouse model of the prion disorder. But the mice still developed movement disorders and other symptoms that were characteristic of their condition when they had a functioning Bax gene.

Further investigation revealed extensive damage to the synapses, areas where branches of two brain cells come together to communicate.

“ This connects prion diseases to other more common disorders because it shows nerve cell death isn’t the only thing we have to worry about in these conditions,” Harris explains. “We have to be concerned about damage to the synapse too, and there’s increasing evidence that is the case in other disorders like Alzheimer’s disease.”

That may make a big difference for therapeutics currently in development, Harris notes.

“ Our results suggest that if we just prevent cell death without doing something to maintain the functionality of the synapse, patients may still get sick,” he says.

Although they work on very different aspects of prion research, True-Krob and Harris collaborate on projects, have a monthly joint lab meeting, and interact frequently.

Harris jokes that he and True-Krob make up “the largest center of prion research within 1,000 miles or so.” True-Krob notes when she was looking for her first faculty position, the possibility of coming to a department with another faculty member studying prions had “definite appeal.”

Their field may soon be getting much less lonely. Connections to more common neurodegenerative disorders are increasing, and other researchers (including True-Krob’s postdoctoral mentor) recently proposed that prions may help store memories in brain cells.

“ That theory’s got a long way to go,” True-Krob says, “but it’s indicative of a new willingness to think about the possibility that prions could have a beneficial role in other systems besides yeast. More and more people are becoming aware of the prion and considering it as a possible explanation for puzzling results.”


PrP Sc

The abnormal, disease-producing protein

  • sa volá PrP Sc (for scrapie)
  • has the same amino acid sequence as the normal protein that is, their primary structures are identical ale
  • its secondary structure is dominated by beta conformation
  • is insoluble in all but the strongest solvents
  • is highly resistant to digestion by proteases
  • When PrP Sc comes in contact with PrP C , it converts the PrP C into more of itself (even in the test tube).
  • These molecules bind to each other forming aggregates.
  • It is not yet clear if these aggregates are themselves the cause of the cell damage or are simply a side effect of the underlying disease process.

Inherited Prion Diseases

Creutzfeldt-Jakob Disease (CJD)

10&ndash15% of the cases of CJD are inherited that is, the patient comes from a family in which the disease has appeared before. The disease is inherited as an autosomal dominantný. The patients have inherited at least one copy of a mutated PRNP gén. Some of the most common mutations are:

  • a change in codon 200 converting glutamic acid (E) at that position to lysine (K) (thus designated "E200K")
  • a change from aspartic acid (D) at position 178 in the protein to asparagine (D178N) when it is accompanied by a polymorphism in both PRNP genes that encodes valine at position 129. When the polymorphism at codon 129 is Met on both genes, the D178N mutation produces Fatal Familial Insomnia instead.
  • a change from valine (V) at position at position 210 to isoleucine (V210I)

Extracts of autopsied brain tissue from these patients can transmit the disease to

  • apes (whose PRNP gene is probably almost identical to that of humans).
  • transgenic mice who have been given a Prnp gene that contains part of the human sequence.

These results lead to the important realization that prion diseases can only be transmitted to animals that already carry a PRNP gene with a sequence that is at least similar to the one that encoded the PrP Sc. In fact, knockout mice with no Prnp genes at all cannot be infected by PrP Sc .

Gerstmann-Sträussler-Scheinker disease (GSS)

This prion disease is caused by the inheritance of a PRNP gene with a mutations encoding most commonly

  • leucine instead of proline at position 102 (P102L) or
  • valine instead of alanine at position 117 (A117V)

Again, the disease is also strongly associated with homozygosity for a polymorphism at position 129 (both residues being methionine).

Brain extracts from patients with GSS can transmit the disease to

Transgenic mice expressing the P102L gene develop the disease spontaneously.

Fatal Familial Insomnia (FFI)

People with this rare disorder have inherited

  • a PRNP gene with asparagine instead of aspartic acid encoded at position 178 (D178N)
  • the susceptibility polymorphism of methionine at position 129 of the PRNP génov.

Extracts from autopsied brains of FFI victims can transmit the disease to transgenic mice.

Infectious Prion Diseases

Kuru was once found among the Fore tribe in Papua New Guinea whose rituals included eating the brain tissue of recently deceased members of the tribe. Since this practice was halted, the disease has disappeared. Before then, the disease was studied by transmitting it to chimpanzees using injections of autopsied brain tissue from human victims.

Scrapie

This disease of sheep (and goats) was the first TSE to be studied. It seems to be transmitted from animal to animal in feed contaminated with nerve tissue. It can also be transmitted by injection of brain tissue.

Bovine Spongiform Encephalopathy (BSE) or "Mad Cow Disease"

An epidemic of this disease began in Great Britain in 1985 and before it was controlled, some 800,000 cattle were sickened by it. Its origin appears to have been cattle feed that contained brain tissue from sheep infected with scrapie and had been treated in a new way that no longer destroyed the infectiousness of the scrapie prions.

The use of such food was banned in 1988 and after peaking in 1992, the epidemic declined quickly.

Creutzfeldt-Jakob Disease (CJD)

A number of humans have acquired CJD through accidental exposure to material contaminated with CJD prions.

  • Grafts of dura mater taken from patients with inherited CJD have transmitted the disease to 228 recipients.
  • Corneal transplants have also inadvertently transmitted CJD.
  • Instruments used in brain surgery on patients with CJD have transmitted the disease to other patients. Two years after their supposed sterilization, these instruments remained infectious.
  • 226 people have acquired CJD from injections of human growth hormone (HGH) or human gonadotropins prepared from pooled pituitary glands that inadvertently included glands taken from humans with CJD.

Now that both HGH and human gonadotropins are available through recombinant DNA technology, such disastrous accidents need never recur.

Variant Creutzfeldt-Jakob Disease (vCJD)

This human disorder appeared some years after the epidemic of BSE (Mad Cow Disease) swept through the cattle herds in Great Britain. Even though the cow and human PRNP genes differ at 30 codons, the sequence of their prions suggests that these patients (155 by 2005) acquired the disease from eating contaminated beef.

All the patients are homozygous for the susceptibility polymorphism of methionine at position 129. The BSE epidemic has waned, and slaughter techniques that allow cattle nervous tissue in beef for human consumption have been banned since 1989. Now we must wait to see whether more cases of vCJD are going to emerge or whether the danger is over.

Miscellaneous Infectious Prion Diseases

A number of TSEs have been found in other animals. Cats are susceptible to Feline Spongiform Encephalopathy (FSE). Mink are also susceptible to a TSE. Even though mad cow disease has not been seen in North America, a similar disease is found in elk and mule deer in the Rocky Mountains of the U.S.

Sporadic Prion Diseases

CJD and FFI occasionally occur in people who have no family history of the disease and no known exposure to infectious prions. The cause of their disease is uncertain.

  • Perhaps a spontaneous somatic mutation has occurred in one of the PRNP genes in a cell.
  • Perhaps their normal PrP C protein has spontaneously converted into the PrP Sc formulár.
  • Or perhaps the victims were simply unknowingly exposed to infectious prions, and sporadic prion diseases do not exist!

Whatever the answer, all the cases are found in people with a susceptibility polymorphism in their PRNP génov.

Prions in Yeast

Two proteins in yeast (Saccharomyces cerevisiae)

are able to form prions that is, they can exist either

  • v PrP C-like form that is functional or
  • v PrP Sc-like form that is not.

The greater ease with which yeast can be studied has proved that only protein is involved in prion formation and provided insight into the need for PrP Sc to find PrP C molecules of a similar primary structure in order to be able to convert them into the PrP Sc formulár.

Evidence that prions are a "protein-only" phenomenon

  • A few molecules of a PrP Sc form of the Sup35 protein, when introduced into yeast cells, convert the yeast cell's own Sup35 protein into prion aggregates. The resulting "disease" phenotype is then passed on to the cell's daughters.

The introduced protein was synthesized in bacteria making it unlikely that it could be contaminated by any gene-containing infectious agent of yeast.

Possible basis of species specificity of prions

  • A particular PrP Sc can only convert PrP C molecules of the same or at least similar primary structure.
  • This requirement of "like-with-like" resides in a short sequence at the N-terminal of the protein (rather like an antibody epitope).
  • Yeasts engineered to form two types of prion form two types of "pure" aggregates within the cell.
  • Even in the test tube, each type of prion finds and aggregates with others of its own type.

So the picture that emerges is that a molecule of PrP Sc acts as a "seed" providing a template for converting PrP C to more PrP Sc. These interact with each other to form small soluble aggregates. These interact with each other to form large insoluble deposits. Although only a small portion of the prion protein is responsible for its specificity, other parts of the molecule are needed for flipping the molecule from the alpha-helical to the beta conformation. All prion proteins contain tracts of repeated Gln-Asn residues which appear to be essential for the conversion process.

Other Pathogenic Prion-like Proteins

The deposits of PrP Scin the brain are called amyloid. Amyloid deposits are also found in other diseases.

  • Alzheimer's disease is characterized by amyloid deposits of
    • the peptide amyloid-beta (A&beta)
    • the protein tau

    With all of these diseases there is evidence that their amyloid-forming proteins, like PrP Sc , can act as a "seed" converting a correctly-folded protein into an incorrectly-folded one and have this effect spread from cell to cell. However, they do not seem to be able to be spread from person to person (unlike the TSEs). Perhaps this is because they are not so incredibly resistant to degradation as PrP Sc je.

    Most cells, including neurons in the brain, contain proteasomes that are responsible for degrading misfolded or aggregated proteins. In the various brain diseases characterized by a build-up of amyloid deposits, it appears that as the small insoluble amyloid precursors accumulate, they bind to proteasomes but cannot be degraded by them. Furthermore, this binding blocks the ability of the proteasomes to process other proteins that are normal candidates for destruction. Because of the critical role of proteasomes in many cell functions, such as mitosis, it is easy to see why this action leads to death of the cell.

    Prion-like proteins not always harmful

    CPEB ("cytoplasmic polyadenylation element binding protein") is a protein that

    • is found in neurons of the central nervous system (as well as elsewhere)
    • stimulates messenger RNA (mRNA) translation
    • is needed for long-term facilitation (LTF)
    • accumulates at activated (by serotonin) synapses
    • has the ability to undergo a change in tertiary structure that
      • persists for long periods
      • induces the same conformational change in other molecules of CPEB forming prion-like aggregates

      Perhaps the accumulation of these aggregates at a stimulated synapse causes a long-term change in its activity (memory).


      Transmembrane PrP

      Three topological forms of PrP

      Most PrP C molecules are attached to the outer leaflet of the plasma membrane through a C-terminal glycosyl-phosphatidylinositol anchor (this topology is designated Sec PrP see Fig. 3). However, when PrP is synthesised in vitro, in transfected cells or in mouse brain, some of the molecules assume a transmembrane orientation 59 –66 . These species, designated Ntm PrP and Ctm PrP, span the lipid bilayer once via a highly conserved hydrophobic region in the centre of the molecule (amino acids 111–134), with either the N-terminus or C-terminus, respectively, on the extracytoplasmic side of the membrane ( Fig. 3).

      Three topological forms of PrP.

      Three topological forms of PrP.

      Ntm PrP and Ctm PrP are generated in small amounts (< 10% of the total) as part of the normal biosynthesis of wild-type PrP in the endoplasmic reticulum. However, mutations within or near the transmembrane domain, including A117V and P105L mutations linked to GSS as well as several ‘artificial’ mutations not seen in human patients, increase the relative proportion of Ctm PrP to as much as 20–30% of the total 59 ,61 ,63 –65 . Current evidence 61 ,64 indicates that the membrane topology of PrP is determined by competition at the translocon between two conflicting topological determinants in the polypeptide chain: (i) the signal sequence (residues 1–22) that directs translocation of the N-terminus of the polypeptide chain across the membrane to produce Sec PrP or Ntm PrP and (ii) the central hydrophobic domain (residues 111–134) that acts as a type II signal-anchor sequence, directing translocation of the C-terminus across the membrane to produce Ctm PrP.

      A proposed pathogenic role of Ctm PrP

      It has been hypothesised that Ctm PrP is a key pathogenic intermediate in both familial and infectiously acquired prion diseases. One piece of evidence for a role in familial forms comes from transgenic mice that synthesise PrP molecules carrying the A117V mutation, or one of the other Ctm PrP-favouring mutations 59 ,63 . Animals expressing these mutant proteins above a threshold level synthesise Ctm PrP in their brains, and spontaneously develop a scrapie-like neurological illness but without PrP Sc detectable by Western blotting or infectivity assays. Evidence for a role of Ctm PrP in infectiously acquired prion diseases comes from mice in which a wild-type hamster PrP transgene serves as a reporter of Ctm PrP formation 63 . When these animals are inoculated with mouse prions, the amounts of Ctm PrP as well as PrP Sc in the brain are found to increase during the course of the infection. This result has been interpreted to indicate that PrP Sc induces formation of Ctm PrP, which is then the proximate cause of neurodegeneration. Thus, the amount of Ctm PrP can be increased either directly by mutations in the PrP molecule, or indirectly via formation of PrP Sc .

      The cell biology of Ctm PrP

      To investigate further the hypothesis that Ctm PrP plays an important role in the pathogenesis of prion diseases, it is necessary to characterise the cell biological properties of this form, since very little is known about its localisation, metabolism, or mode of synthesis and processing in cells. Part of the difficulty in addressing these issues has been that it was not possible to produce Ctm PrP in the absence of the other two topological variants ( Ntm PrP and Sec PrP). We have overcome this limitation by identifying mutations in PrP that cause the protein to be synthesised exclusively with the Ctm PrP topology.

      The starting point for these studies was our discovery of a novel structural feature of Ctm PrP that had not been previously appreciated – Ctm PrP has an uncleaved, N-terminal signal peptide 66 . This feature can be rationalised by the fact that the N-terminus of the polypeptide chain does not enter the ER lumen where signal peptidase is located. We reasoned that mutations in the signal peptide itself might influence the amount of Ctm PrP. Consistent with this idea, we found that the substitution of a charged residue for a hydrophobic residue within the signal sequence (L9R) markedly increased the proportion of Ctm PrP to ∼50% after in vitro translation. Combining this mutation with 3AV, a mutation within the transmembrane domain, to create L9R/3AV resulted in a protein that was synthesised exclusively as Ctm PrP, in both in vitro translation reactions and transfected cells 66 .

      The availability of L9R/3AV PrP provided us for the first time the ability to analyse the properties of Ctm PrP in a cellular context in the absence of the other two topological variants 66 . By labelling cells expressing L9R/3AV PrP with [ 3 H]-palmitate, we demonstrated that Ctm PrP contains a GPI anchor. This result implies that Ctm PrP has an unusual, dual mode of membrane attachment, including both a membrane-spanning domain and a C-terminal GPI anchor. We also found that L9R/3AV PrP (and hence Ctm PrP) is absent from the cell surface, and is completely retained in the ER when expressed in transfected cells. This observation suggests the hypothesis that Ctm PrP is toxic because it stimulates the activation of pro-apoptotic, ER stress-response pathways.

      Most pathogenic mutations do not alter the membrane topology of PrP

      Mutations associated with familial prion diseases are found throughout the length of the PrP sequence 32 . Although mutations in or around the central, hydrophobic region were known to increase the amount of Ctm PrP, the effect of mutations outside of this area had not been examined. Therefore, we carried out in vitro translations of PrP mRNA encoding disease-associated mutations that lie both N- and C-terminal to the central, hydrophobic segment 65 . We found that the proportion of Ctm PrP was not increased over wild-type levels by any of the mutations outside of the central, hydrophobic domain. These results argue against the idea that Ctm PrP is an obligate toxic intermediate in all forms of familial prion diseases.


      What does it mean to have a genetic prion disease?

      In the last two posts we introduced the concept of a prion and introduced the human prion diseases, better known as Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), a Gertsmann-Straussler-Scheinker syndrome (GSS). Altogether these diseases are pretty rare, with an incidence of about 1 in 1 million people falling ill each year [Holman 2010]. The genetic forms of prion disease are even more rare, accounting for probably just 15% of all cases [Appleby & Lyketsos 2011].

      To understand how these diseases arise, we’ll need to go back to some biology basics. The DNA in the nucleus of your cells is made up of 2 copies each of chromosomes 1-22, plus 2 copies of X (if you’re female) or 1 X and 1 Y (if you’re male). On chromosome 20 there’s a gene called the pr io n p rotein gene, or PRNP. Since you have two copies of chromosome 20 you have two copies of PRNP. You got one of those two copies of PRNP from your mother, and the other one from your father.

      DNA contains instructions that have to be converted into RNA, and then the message in the RNA is translated into protein. So the gene PRNP in your DNA spells out instructions for making, ultimately, the prion protein (PrP). Everyone has PrP on the surface of their cells, and most of the time this is a perfectly good and healthy thing.

      Genetické mutations – think of these as typos in DNA – change the instructions and end up producing a slightly different protein. Most of the time that’s fine, but some mutations are really bad.

      There are two main ways that mutations can be bad. First, they can make a protein that doesn’t do its job correctly. This is called a loss-of-function mutation. The other option is that they can make a protein that robí do something new that it shouldn’t be doing. This is called a gain-of-function mutation. Genetic prion diseases are caused by a gain-of-function mutation in PRNP. They produce a slightly altered version of prion protein (PrP). A person can be pretty much healthy and fine with this version of PrP for decades, but as people get older, this protein is likely to eventually misfold and form a prion which can then attack other PrP and spread across the brain.

      Prion protein mutations follow a dominantný inheritance pattern. That just means that, unfortunately, it only takes jeden bad copy of the gene to cause the disease. If you have a parent who had a genetic prion disease, they probably had one bad copy of PRNP, and there’s a 50% chance you inherited that one, and a 50% chance that you inherited the normal, good copy of PRNP.

      You’ll also hear genetic prion diseases described as being autozomálne. That just means that they’re not sex-linked – PRNP is on chromosome 20, not on a sex chromosome. Both sexes are affected equally by genetic prion disease.

      Another term you’ll hear tossed around is penetrance. Penetrance is the percentage of people with the bad copy of the gene who will get the disease. Most of the genetic prion diseases that we know of are completely penetrant in the sense that everyone who has the mutation will get the disease eventually, if they live long enough. Some of the genetic prion diseases tend to strike people in their 40s and 50s, and so (as far as we know) virtually everyone who has the mutation dies of it. Other genetic prion diseases tend to have an older age of onset, so that some percentage of people will end up dying of some other cause – heart attacks, cancer, and so on – before they ever have symptoms of prion disease.

      There are over 40 different mutations that can cause genetic prion disease [Beck 2010], and each mutation tends to have its own particular symptoms, disease course, age of onset, and so on [Kong 2003, Mastrianni 2010 (full text)]. In other words, the mutation defines the disease.

      Therefore it’s important to have a system for naming mutations. The system that we use involves a lot of seemingly random numbers and letters such as “E200K.” Let’s break down what this code means.

      Proteins are made of amino acids, and PrP in particular is made of 253 amino acids, which are referred to in order. So the 200 in E200K means there’s a mutation in amino acid #200. E is shorthand for the amino acid glutamate, which is the amino acid that should normally be at position #200. K is the shorthand for the amino acid lysine, which is the amino acid that appears at position #200 instead of glutamate. Putting it all together, “E200K” means lysine instead of glutamate at amino acid #200.

      Sometimes there are even more numbers and letters. For instance, fatal familial insomnia is caused by a mutation called D178N 129M. Here, D178N means asparagine (N) instead of aspartate (D) at amino acid #178, and 129M means … and then methionine (M) at amino acid #129. Fatal familial insomnia is unusual in that the D178N mutation príčin the disease, but the exact typu of disease is determined by which amino acid is found at position #129 [Goldfarb 1992].

      Still other cases of genetic prion disease are not caused by a change in just one amino acid. One part of PrP has a repeating stretch of the same 8 amino acids over and over again, and this is called the “octapeptide repeat”. Some genetic prion diseases are caused by having too many copies of this repeat, and these are named by the number of extra copies – for instance, 𔄞-OPRI” means 6 extra copies of the octapeptide repeat inserted.

      Genetic counselors give people with genetic mutations advice on what their mutation means. Often this might include some statement about the typical age of disease onset for a person’s mutation. Estimates like these can be useful, but it is worth taking them with a bit of caution. For most genetic prion disease mutations, the age of onset can vary by a few decades. The statistics are usually based on a review of patients’ cases that doctors have reported in medical journals [Kong 2003] and there are many possible biases here. Younger patients might be more likely to be reported, people who die of something else first are almost never reported, and the statistics often fail to take into account the people still alive with the mutation. Moreover, the predicted age of disease onset for any one person really depends on how old they are today – after all, a person who is 60 today can’t have an age of onset of 50, even if that is the average for their mutation. For all of these reasons, statistics like “average age of onset” should never be taken as a hard-and-fast prediction about one’s own life.

      Even more importantly, there are good reasons for optimism that things will change, and that the diseases that we consider fatal and untreatable today will eventually be things of the past. We at Prion Alliance are working hard for a future where prion diseases don’t kill people. And we’re not alone – there are lots of bright scientists working in the prion field, and potential treatments at a variety of stages in the research pipeline. We can’t say exactly when treatments will become available, but lots of excellent science is being done every day, and we think right now is a good time in history to be an optimist.


      Evolution without genes – prions can evolve and adapt too

      If you search for decent definitions of evolution, the chances are that you’ll see genes mentioned somewhere. The American Heritage Dictionary talks about natural selection acting on “genetic variation”, Wikipedia discusses “change in the genetic material of a population… through successive generations”, and TalkOrigins talks about changes that are inherited “via the genetic material”. But, as the Year of Darwin draws to a close, a new study suggests that all of these definitions are too narrow.

      Jiali Li from the Scripps Institute in Florida has found that prions – the infectious proteins behind mad cow disease, CJD and kuru – are capable of Darwinian evolution, all without a single strand of DNA or its sister molecule RNA.

      Prions are rogue version of a protein called PrP. Like all proteins, they are made up of chains of amino acids that fold into a complex three-dimensional structure. Prions are versions of PrP that have folded incorrectly and this misfolded form, called PrPSc, is social, evangelical and murderous. It converts normal prion proteins into a likeness of its abnormal self, and it rapidly gathers together in large clumps that damage and kill surrounding tissues.

      Li has found that variation can creep into populations of initially identical prions. Their amino acid sequence stays the same but their already abnormal structures become increasingly twisted. These “mutant” forms have varying degrees of success in different environments. Some do well in brain tissue others thrive in other types of cell. In each case, natural selection culls the least successful ones. The survivors pass on their structure to the “next generation”, by altering the folds of normal prion proteins.

      This process follows the principles of Darwinian evolution, the same principles that shape the genetic material of viruses, bacteria and other living things. In DNA, mutations manifest as changes in the bases that line the famous double helix. In prions, mutations are essentially different styles of molecular origami. In both cases, they are selectively inherited and they can lead to adaptations such as drug resistance. In prions, it happens in the absence of any genetic material.

      If prions can evolve, and if they can show the same sort of adaptive resistance as bacteria or fungi, does this mean that they are alive? Charles Weissman, who heads up Li’s lab, doesn’t think so on the grounds that prions are completely dependent on their hosts for reproduction. They need normal proteins that are encoded within the genome of their host to make more copies of themselves.. He says, “The remarkable finding that prions can mutate and adapt to their environment imbues them with a further attribute of living things, without however elevating them to the status of being ‘alive’.”

      There are many distinct strains of prion. Each is a version of PrPSc folded in a subtly different way, and new strains can arise out of the blue. Working out their exact structure has been difficult and they’re usually characterised by the symptoms and disease they cause, and how long it takes for these to become apparent.

      Li found that prions taken from brain tissue are different to those grown in cells cultured in a laboratory. The brain-adapted prions are capable of infected nerve tissue and they’re resistant to a drug called swainsonine (swa) that completely blocks the growth of other strains. The cell-adapted prions lack both these abilities but they’re better at growing in cell cultures.

      When Li transferred brain prions into cell cultures, she found that they gradually adapted to their new environment. By the 12th ‘generation’, they were indistinguishable from cell-adapted prions. They had lost their ability to infect nerve tissue in favour of the ability to grow faster in cultured cells. When Li returned these prions back to brain tissue, the brain-adapted forms once again rose to dominance.

      Li also found that prions are capable to evolving resistance to drugs. She treated the cell-prions with swa. At first, the drug blitzed the prion population, slashing the proportion of infected cells by five times from 35% to 7%. But the rogue proteins staged a resurgence, bouncing back to infect around 25% of the cells. After just two rounds of growth, prions from cells that were exposed to swa completely resisted the drug. If the drug was removed, they faded into the background once more as the non-resistant forms took over again.

      Further experiments showed that the resistant strains were already there in the population. But their slower growth rates mean that they’re typically in the minority, accounting for just 1 in 200 prions. When swa blasted the population, these resistant few rose to dominance. Li says that prion populations consist of a multitude of strains and substrains, all of which are different ways of folding the same sequence of amino acids. Evolutionary pressures from the environment determine which of these strains is in power.

      But mutants can arise out of the blue too. Even if a population consists entirely of the same strain (which you can set up through cloning), resistant or sensitive mutants develop spontaneously in a very short span of time. Prions, it seems, are very quick to adapt.

      The fact that prions can evolve drug resistance so quickly is important news for scientists trying to find new treatments for prion diseases, such as Creutzfeld-Jacob Disease (CJD) and bovine spongiform encephalitis (BSE). Rather than trying to target the abnormal proteins themselves, it might be better to reduce the levels of the production of the normal PrP in the first place. The former tactic could be easily thwarted by the rise of resistant strains, while the latter tactic denies natural selection of raw materials to work with.

      Referencia: Li et al. 2009. Darwinian Evolution of Prions in Cell Culture. Science DOI: 10.1126/science.1183218