Approfondimento sulla specificità del legame peptidico al C-terminale del dominio PDZ dell’ossido nitrico sintasi neuronale

Studi precedenti hanno dimostrato che il c-jun e l’ossido nitrico sintasi neurale (nNOS) sono indotti nei motoneuroni danneggiati, ma il loro ruolo nella morte dei motoneuroni rimane poco chiaro.

  • Abbiamo ipotizzato che nNOS potrebbe essere un ulteriore effettore della chinasi N-terminale c-jun (JNK) / c-jun nella morte per distacco del motoneurone. Qui abbiamo scoperto che il distacco della radice brachiale ha indotto un aumento transitorio dell’attività di JNK e un aumento di tre e quattro volte rispettivamente di fosfo-c-jun e c-jun; tuttavia, il distacco brachiale ha ridotto l’espressione della proteina nNOS da 4 ore a 14 giorni dopo la lesione.
  • A 14 giorni dalla lesione, quasi tutti i motoneuroni nNOS-positivi erano localizzati insieme ai motoneuroni fosfo-c-jun positivi nelle corna addominali omolaterali.
  • L’inibitore JNK SP600125, applicato immediatamente dopo la lesione, ha aumentato il livello della proteina nNOS sia nel midollo spinale che nei motoneuroni feriti e ha causato una leggera riduzione della morte dei motoneuroni inibendo la fosforilazione di c-jun 14 giorni dopo la lesione.
  • I nostri risultati hanno mostrato che la via di trasduzione del segnale JNK / c-jun è coinvolta nel distacco della radice. L’inibizione della fosforilazione di c-jun impedisce che i livelli di nNOS scendano al di sotto della linea di base nel midollo spinale e mitiga parzialmente la morte del motoneurone dopo l’estrazione della radice.
  • Pertanto, l’inibizione della fosforilazione di c-jun o l’aumento del livello della proteina nNOS in un midollo spinale danneggiato in una fase iniziale potrebbero essere utilizzati in futuro come strategie di bersaglio molecolare per prevenire la degenerazione dei motoneuroni durante l’eliminazione delle radici.

 

La coda C-terminale (CT) dell’ossido nitrico sintasi neuronale (nNOS) è un elemento regolatorio che inibisce l’attività di nNOS in assenza di calmodulina legata (CaM).

 

Struttura cristallina del dominio nNOS reduttasi (nNOSr) (Garcin, ED, Bruns, CM, Lloyd, SJ, Hosfield, DJ, Tiso, M., Gachhui, R., Stuehr, DJ, Tainer, JA e Getzoff, ED (2004 ) J Biol. Chem. 279, 37918-37927) ha descritto come la prima metà di CT interagisce con nNOSr e ha quindi fornito un modello per uno studio dettagliato. Abbiamo generato mutanti troncati in nNOS e nNOSr per testare l’importanza di 3 diverse regioni CT.

  • L’eliminazione della metà terminale di CT (tutti i residui da Ile1413 a Ser1429), che è invisibile nella struttura cristallina, non ha avuto quasi alcun effetto sul rilascio di NADP +, riduzione della flavina, autoossidazione della flavina, riduzione dell’eme, attività reduttasi o NO attività di sintesi, ma ha impedito un aumento della schermatura FMN che normalmente si verifica in risposta al legame NADPH.
  • Un’ulteriore rimozione  dell’alfa-elica CT (residui da 1401 a 1412) ha aumentato significativamente il tasso di rilascio di NADP +, l’autoossidazione della flavina e l’attività della NADPH ossidasi e ha provocato un’esposizione eccessiva del cofattore FMN. Questi effetti sono stati associati con una maggiore attività della reduttasi e una riduzione leggermente ridotta dell’eme e della sintesi di NO.
  • L’ulteriore rimozione dei residui dopo Gly1396 (troncamento CT completo) ha migliorato gli effetti sopra menzionati e ha ulteriormente modificato l’interazione di NADP + con FAD, attenuando la soppressione cinetica della riduzione della flavina e ulteriormente ridotta la riduzione dell’eme e la sintesi di NO.
  •  I nostri risultati mostrano che la TC esercita effetti sia sfaccettati che regiospecifici sull’attività catalitica e sui comportamenti correlati, fornendo così nuove informazioni sui meccanismi che regolano la catalisi di nNOS.

I domini PDZ sono motivi di interazione proteica multifunzionale che spesso si legano al C-terminale delle proteine ​​​​bersaglio.

 L’ossido nitrico (NO), una molecola di segnalazione endogena, svolge un ruolo chiave nel funzionamento del sistema nervoso, immunitario e cardiovascolare. Sebbene ci siano molte funzioni fisiologiche per l’NO neuronale prodotto principalmente dalla NO sintasi neuronale (nNOS), l’eccessiva attività di nNOS media il danno cerebrale nell’ischemia cerebrale e nei modelli animali del morbo di Parkinson.

Pertanto, la localizzazione subcellulare dell’attività di nNOS deve essere strettamente regolata. Per determinare i ligandi del dominio nNOS PDZ, abbiamo cercato 13 miliardi di peptidi diversi e abbiamo scoperto che il dominio nNOS-PDZ si lega strettamente ai peptidi che terminano in Asp-X-ValQuesto differisce dall’unico consenso noto (Thr / Ser) -X-Val che interagisce con i domini PDZ di PSD-95. La preferenza per Asp in posizione -2 è mediata da Tyr-77 di nNOS. La sostituzione di Y77D78 con H77E78 cambia la specificità del legame da Asp-X-Val a Thr-X-Val. A seguito del consenso di Asp-X-Val, sono state identificate le proteine ​​candidate che interagiscono con nNOS, inclusi i recettori del glutammato e della melatonina. I nostri risultati mostrano che i domini PDZ hanno una chiara specificità di legame peptidico.

 

L’ossido nitrico sintasi neurale, a differenza delle sue controparti endoteliali e inducibili, mostra un dominio PDZ (PSD-95 / Dlg / ZO-1) situato al suo N-terminale coinvolto nel targeting subcellulare.

 Il C-terminale delle varie proteine ​​cellulari si inserisce nel solco di legame di questo dominio PDZ e definisce la distribuzione subcellulare della NOS neuronale (nNOS). I meccanismi molecolari alla base di queste interazioni sono poco conosciuti poiché il dominio nNOS PDZ può sembrare mostrare specificità di legame di classe I, classe II e classe III.

Inoltre  , è stato recentemente suggerito che il dominio nNOS PDZ si leghi con un’affinità molto bassa al C-terminale delle proteine ​​bersaglio e che la necessaria interazione laterale concomitante debba avvenire affinché avvenga il legame. Descriviamo qui che un dominio nNOS PDZ può comportarsi come un dominio PDZ di classe III in buona fede e legarsi a sequenze C-terminali con residui acidi nella posizione P-2 con costanti micromolari basse. Può verificarsi anche il legame alle sequenze C-terminali con un residuo idrofobo in P-2 e un residuo acido in P-3 (classe II), sebbene interazioni che coinvolgono residui che si estendono a P-7 mediano questo tipo di legame.

Questo comportamento promiscuo si estende anche alla sua associazione con sequenze di classe I, che devono mostrare un residuo di Glu in P-3 e un residuo di Thr in P-2. Mediante mutagenesi sito-diretta e spettroscopia NMR, siamo stati in grado di identificare i residui coinvolti in ogni tipo specifico di legame e razionalizzare i meccanismi utilizzati per riconoscere i partner vincolanti.

 

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Neuronal Nitric Oxide Synthase (Nnos) Antibody

20-abx114082 Abbexa
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Rat neuronal nitric oxide synthase(nNOS) Elisa Kit

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1-CSB-E13872h Cusabio
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Human Neuronal Nitric Oxide Synthase,nNOS ELISA KIT

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nNOS, aa1409-1429 (Neuronal Nitric Oxide Synthase, bNOS)

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Rat Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

EKC39476-5x96T Biomatik Corporation 5x96T 3216.7 EUR

Rat Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

EKC39476-96T Biomatik Corporation 96T 677.2 EUR

Rat Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

EK11198 SAB 96Т 799 EUR

Rat Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

AE58201RA-48Tests Abebio 48 Tests 360 EUR

Rat Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

AE58201RA-96Tests Abebio 96 Tests 680 EUR

Rat Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

CSB-E14034r-24T Cusabio 1 plate of 24 wells 198 EUR

Rat Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

1-CSB-E14034r Cusabio
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  • 1 plate of 96 wells
  • 10 plates of 96 wells each
  • 5 plates of 96 wells each

Rat neuronal nitric oxide synthase,NOS1;nNOS ELISA KIT

E0542Ra-1096T Jiaxing Korain Biotech Ltd (BT Labs) 10*96T 4122 EUR

Rat neuronal nitric oxide synthase,NOS1;nNOS ELISA KIT

E0542Ra-48wells Jiaxing Korain Biotech Ltd (BT Labs) 48 wells 300 EUR

Rat neuronal nitric oxide synthase,NOS1;nNOS ELISA KIT

E0542Ra-596T Jiaxing Korain Biotech Ltd (BT Labs) 5*96T 2061 EUR

Rat neuronal nitric oxide synthase,NOS1;nNOS ELISA KIT

E0542Ra-96wells Jiaxing Korain Biotech Ltd (BT Labs) 96 wells 458 EUR

Rat Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

AE58201RA-48T Abebio 48T 360 EUR

Rat Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

AE58201RA-96T Abebio 96T 680 EUR

Rat Neuronal Nitric Oxide Synthase, nNOS/NOS1 ELISA Kit

MBS704610-10x96StripWells MyBiosource 10x96-Strip-Wells 5325 EUR

Rat Neuronal Nitric Oxide Synthase, nNOS/NOS1 ELISA Kit

MBS704610-24StripWellsLIMIT1 MyBiosource 24-Strip-Wells(LIMIT1) 275 EUR

Rat Neuronal Nitric Oxide Synthase, nNOS/NOS1 ELISA Kit

MBS704610-48StripWells MyBiosource 48-Strip-Wells 480 EUR

Rat Neuronal Nitric Oxide Synthase, nNOS/NOS1 ELISA Kit

MBS704610-5x96StripWells MyBiosource 5x96-Strip-Wells 2795 EUR

Rat Neuronal Nitric Oxide Synthase, nNOS/NOS1 ELISA Kit

MBS704610-96StripWells MyBiosource 96-Strip-Wells 675 EUR

Mouse neuronal nitric oxide synthase(NOS,nNOS)ELISA Kit

QY-E21072 Qayee Biotechnology 96T 433.2 EUR

Mouse Neuronal Nitric Oxide Synthase (nNOS/NOS1) Elisa Kit

EK731356 AFG Bioscience LLC 96 Wells 0.77 EUR

Mouse Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

CSB-E13273m-24T Cusabio 1 plate of 24 wells 198 EUR

Mouse Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

1-CSB-E13273m Cusabio
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  • 1 plate of 96 wells
  • 10 plates of 96 wells each
  • 5 plates of 96 wells each

Mouse Neuronal Nitric Oxide Synthase,nNOS;NOS1 ELISA KIT

E1803Mo-1096T Jiaxing Korain Biotech Ltd (BT Labs) 10*96T 4122 EUR

Mouse Neuronal Nitric Oxide Synthase,nNOS;NOS1 ELISA KIT

E1803Mo-48wells Jiaxing Korain Biotech Ltd (BT Labs) 48 wells 300 EUR

Mouse Neuronal Nitric Oxide Synthase,nNOS;NOS1 ELISA KIT

E1803Mo-596T Jiaxing Korain Biotech Ltd (BT Labs) 5*96T 2061 EUR

Mouse Neuronal Nitric Oxide Synthase,nNOS;NOS1 ELISA KIT

E1803Mo-96wells Jiaxing Korain Biotech Ltd (BT Labs) 96 wells 458 EUR

Mouse Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

RK07085 Abclonal 96T 280 EUR

Mouse Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

MBS1603280-10x96StripWells MyBiosource 10x96-Strip-Wells 3955 EUR

Mouse Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

MBS1603280-48StripWells MyBiosource 48-Strip-Wells 305 EUR

Mouse Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

MBS1603280-5x96StripWells MyBiosource 5x96-Strip-Wells 2005 EUR

Mouse Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

MBS1603280-96StripWells MyBiosource 96-Strip-Wells 475 EUR

Canine Neuronal Nitric Oxide Synthase (NOS, NNOS) ELISA Kit

MBS1603356-10x96StripWells MyBiosource 10x96-Strip-Wells 4295 EUR

Canine Neuronal Nitric Oxide Synthase (NOS, NNOS) ELISA Kit

MBS1603356-48StripWells MyBiosource 48-Strip-Wells 410 EUR

Canine Neuronal Nitric Oxide Synthase (NOS, NNOS) ELISA Kit

MBS1603356-5x96StripWells MyBiosource 5x96-Strip-Wells 2185 EUR

Canine Neuronal Nitric Oxide Synthase (NOS, NNOS) ELISA Kit

MBS1603356-96StripWells MyBiosource 96-Strip-Wells 520 EUR

Nitric Oxide Synthase 1, Neuronal / nNOS (NOS1) Antibody

20-abx329607 Abbexa
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  • 100 ug
  • 50 ug

Rat neuronal Nitric Oxide synthase, NOS,nNOS GENLISA ELISA

KLR0542 Krishgen 1 x 96 wells 341 EUR

Bovine Neuronal Nitric Oxide Synthase, nNOS/NOS1 ELISA Kit

MBS9310041-10x96StripWells MyBiosource 10x96-Strip-Wells 6725 EUR

Bovine Neuronal Nitric Oxide Synthase, nNOS/NOS1 ELISA Kit

MBS9310041-48StripWells MyBiosource 48-Strip-Wells 550 EUR

Bovine Neuronal Nitric Oxide Synthase, nNOS/NOS1 ELISA Kit

MBS9310041-5x96StripWells MyBiosource 5x96-Strip-Wells 3420 EUR

Bovine Neuronal Nitric Oxide Synthase, nNOS/NOS1 ELISA Kit

MBS9310041-96StripWells MyBiosource 96-Strip-Wells 765 EUR

Chicken Neuronal Nitric Oxide Synthase(nNOS/NOS1)ELISA Kit

YLA0003CH-48T Shanghai YL Biotech 48T Ask for price

Chicken Neuronal Nitric Oxide Synthase(nNOS/NOS1)ELISA Kit

YLA0003CH-96T Shanghai YL Biotech 96T Ask for price

Chicken Neuronal Nitric Oxide Synthase,nNOS;NOS1 ELISA KIT

E0156Ch-1096T Jiaxing Korain Biotech Ltd (BT Labs) 10*96T 4482 EUR

Chicken Neuronal Nitric Oxide Synthase,nNOS;NOS1 ELISA KIT

E0156Ch-48wells Jiaxing Korain Biotech Ltd (BT Labs) 48 wells 360 EUR

Chicken Neuronal Nitric Oxide Synthase,nNOS;NOS1 ELISA KIT

E0156Ch-596T Jiaxing Korain Biotech Ltd (BT Labs) 5*96T 2241 EUR

Chicken Neuronal Nitric Oxide Synthase,nNOS;NOS1 ELISA KIT

E0156Ch-96wells Jiaxing Korain Biotech Ltd (BT Labs) 96 wells 498 EUR

Chicken Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

MBS1601905-10x96StripWells MyBiosource 10x96-Strip-Wells 4470 EUR

Chicken Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

MBS1601905-48StripWells MyBiosource 48-Strip-Wells 415 EUR

Chicken Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

MBS1601905-5x96StripWells MyBiosource 5x96-Strip-Wells 2270 EUR

Chicken Neuronal Nitric Oxide Synthase (nNOS/NOS1) ELISA Kit

MBS1601905-96StripWells MyBiosource 96-Strip-Wells 540 EUR

ELISA kit for Rat Neuronal Nitric Oxide Synthase (nNOS/NOS1)

KTE100825-48T Abbkine 48T 424.8 EUR

ELISA kit for Rat Neuronal Nitric Oxide Synthase (nNOS/NOS1)

KTE100825-5platesof96wells Abbkine 5 plates of 96 wells 2702.4 EUR

Infine, abbiamo analizzato l’associazione ad alta affinità del dominio nNOS PDZ con Claudin-3 e Claudin-14, due proteine ​​di membrana tetraspan strettamente collegate che sono componenti essenziali della barriera paracellulare.

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