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

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

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

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

20-abx329607 Abbexa
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Rat neuronal Nitric Oxide synthase, NOS,nNOS GENLISA ELISA

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Rat Nitric Oxide Synthase 1, Neuronal / nNOS (NOS1) CLIA Kit

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Rat Nitric Oxide Synthase 1, Neuronal / nNOS (NOS1) CLIA Kit

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Rat NOS1/nNOS(Nitric Oxide Synthase 1, Neuronal) ELISA Kit

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Rat Nitric Oxide Synthase 1, Neuronal / nNOS (NOS1) ELISA Kit

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  • 10 × 96 tests
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Rat NOS1/nNOS(Nitric Oxide Synthase 1, Neuronal) ELISA Kit

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Mouse NOS1/nNOS(Nitric Oxide Synthase 1, Neuronal) ELISA Kit

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Human Nitric Oxide Synthase 1, Neuronal / nNOS (NOS1) ELISA Kit

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  • 10 × 96 tests
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Human NOS1/nNOS(Nitric Oxide Synthase 1, Neuronal) ELISA Kit

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Human NOS1/nNOS(Nitric Oxide Synthase 1, Neuronal) ELISA Kit

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CLIA kit for Rat NOS1/nNOS (Nitric Oxide Synthase 1, Neuronal)

E-CL-R0746 Elabscience Biotech 1 plate of 96 wells 700.8 EUR

Chicken Nitric Oxide Synthase 1, Neuronal / nNOS (NOS1) ELISA Kit

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ELISA kit for Rat NOS1/nNOS (Nitric Oxide Synthase 1, Neuronal)

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CLIA kit for Mouse NOS1/nNOS (Nitric Oxide Synthase 1, Neuronal)

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ELISA kit for Mouse NOS1/nNOS (Nitric Oxide Synthase 1, Neuronal)

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ELISA kit for Human NOS1/nNOS (Nitric Oxide Synthase 1, Neuronal)

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NOS1/nNOS ELISA Kit| Mouse Nitric Oxide Synthase 1, Neuronal ELI

EF013784 Lifescience Market 96 Tests 826.8 EUR

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EF017083 Lifescience Market 96 Tests 826.8 EUR

Guinea pig Nitric Oxide Synthase 1, Neuronal / nNOS (NOS1) ELISA Kit

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Rat Neuronal Nitric Oxide Synthase ELISA kit

E02N0069-192T BlueGene 192 tests 1524 EUR

Rat Neuronal Nitric Oxide Synthase ELISA kit

E02N0069-48 BlueGene 1 plate of 48 wells 624 EUR

Rat Neuronal Nitric Oxide Synthase ELISA kit

E02N0069-96 BlueGene 1 plate of 96 wells 822 EUR

Dog Neuronal Nitric Oxide Synthase ELISA kit

E08N0069-192T BlueGene 192 tests 1524 EUR

Dog Neuronal Nitric Oxide Synthase ELISA kit

E08N0069-48 BlueGene 1 plate of 48 wells 624 EUR

Dog Neuronal Nitric Oxide Synthase ELISA kit

E08N0069-96 BlueGene 1 plate of 96 wells 822 EUR

Pig Neuronal Nitric Oxide Synthase ELISA kit

E07N0069-192T BlueGene 192 tests 1524 EUR

Pig Neuronal Nitric Oxide Synthase ELISA kit

E07N0069-48 BlueGene 1 plate of 48 wells 624 EUR

Pig Neuronal Nitric Oxide Synthase ELISA kit

E07N0069-96 BlueGene 1 plate of 96 wells 822 EUR

Rat Neuronal Nitric Oxide Synthase ELISA kit

E01A14954 BlueGene 96T 700 EUR

Goat Neuronal Nitric Oxide Synthase ELISA kit

E01A49860 BlueGene 96T 700 EUR

Goat Neuronal Nitric Oxide Synthase ELISA kit

E06N0069-192T BlueGene 192 tests 1524 EUR

Goat Neuronal Nitric Oxide Synthase ELISA kit

E06N0069-48 BlueGene 1 plate of 48 wells 624 EUR

Goat Neuronal Nitric Oxide Synthase ELISA kit

E06N0069-96 BlueGene 1 plate of 96 wells 822 EUR

Sheep Neuronal Nitric Oxide Synthase ELISA kit

E01A102154 BlueGene 96T 700 EUR

Human Neuronal Nitric Oxide Synthase ELISA kit

E01A6207 BlueGene 96T 700 EUR

Mouse Neuronal Nitric Oxide Synthase ELISA kit

E01A23701 BlueGene 96T 700 EUR

Mouse Neuronal Nitric Oxide Synthase ELISA kit

E03N0069-192T BlueGene 192 tests 1524 EUR

Mouse Neuronal Nitric Oxide Synthase ELISA kit

E03N0069-48 BlueGene 1 plate of 48 wells 624 EUR

Mouse Neuronal Nitric Oxide Synthase ELISA kit

E03N0069-96 BlueGene 1 plate of 96 wells 822 EUR

Human Neuronal Nitric Oxide Synthase ELISA kit

E01N0069-192T BlueGene 192 tests 1524 EUR

Human Neuronal Nitric Oxide Synthase ELISA kit

E01N0069-48 BlueGene 1 plate of 48 wells 624 EUR

Human Neuronal Nitric Oxide Synthase ELISA kit

E01N0069-96 BlueGene 1 plate of 96 wells 822 EUR

Rabbit Neuronal Nitric Oxide Synthase ELISA kit

E01A32425 BlueGene 96T 700 EUR

Monkey Neuronal Nitric Oxide Synthase ELISA kit

E01A76007 BlueGene 96T 700 EUR

Bovine Neuronal Nitric Oxide Synthase ELISA kit

E01A84727 BlueGene 96T 700 EUR

Canine Neuronal Nitric Oxide Synthase ELISA kit

E01A67290 BlueGene 96T 700 EUR

Monkey Neuronal Nitric Oxide Synthase ELISA kit

E09N0069-192T BlueGene 192 tests 1524 EUR

Monkey Neuronal Nitric Oxide Synthase ELISA kit

E09N0069-48 BlueGene 1 plate of 48 wells 624 EUR

Monkey Neuronal Nitric Oxide Synthase ELISA kit

E09N0069-96 BlueGene 1 plate of 96 wells 822 EUR

Rabbit Neuronal Nitric Oxide Synthase ELISA kit

E04N0069-192T BlueGene 192 tests 1524 EUR

Rabbit Neuronal Nitric Oxide Synthase ELISA kit

E04N0069-48 BlueGene 1 plate of 48 wells 624 EUR

Rabbit Neuronal Nitric Oxide Synthase ELISA kit

E04N0069-96 BlueGene 1 plate of 96 wells 822 EUR

Porcine Neuronal Nitric Oxide Synthase ELISA kit

E01A58578 BlueGene 96T 700 EUR

Chicken Neuronal Nitric Oxide Synthase ELISA kit

E01A93441 BlueGene 96T 700 EUR

Guinea Pig Neuronal Nitric Oxide Synthase ELISA kit

E01A41140 BlueGene 96T 700 EUR

Guinea pig Neuronal Nitric Oxide Synthase ELISA kit

E05N0069-192T BlueGene 192 tests 1524 EUR

Guinea pig Neuronal Nitric Oxide Synthase ELISA kit

E05N0069-48 BlueGene 1 plate of 48 wells 624 EUR

Guinea pig Neuronal Nitric Oxide Synthase ELISA kit

E05N0069-96 BlueGene 1 plate of 96 wells 822 EUR

Nitric Oxide Synthase 1, Neuronal (NOS1) Antibody

20-abx129333 Abbexa
  • Ask for price
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  • Ask for price
  • Ask for price
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Nitric Oxide Synthase 1, Neuronal (NOS1) Antibody

20-abx173833 Abbexa
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • 100 ug
  • 1 mg
  • 200 ug
  • 20 ug
  • 50 ug

Nitric Oxide Synthase 1, Neuronal (NOS1) Antibody

20-abx103472 Abbexa
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Nitric Oxide Synthase 1, Neuronal (NOS1) Antibody

20-abx103473 Abbexa
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Nitric Oxide Synthase 1, Neuronal (NOS1) Antibody

20-abx103474 Abbexa
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Nitric Oxide Synthase 1, Neuronal (NOS1) Antibody

20-abx103475 Abbexa
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Nitric Oxide Synthase 1, Neuronal (NOS1) Antibody

20-abx103476 Abbexa
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Nitric Oxide Synthase 1, Neuronal (NOS1) Antibody

20-abx103477 Abbexa
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Nitric Oxide Synthase 1, Neuronal (NOS1) Antibody

20-abx103478 Abbexa
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • Ask for price
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase protein

AP79849 SAB 1mg 2640 EUR

Carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase protein

AP81351 SAB 1mg 2640 EUR

Recombinant Nitric Oxide Synthase 1, Neuronal (NOS1)

RPU43055-100ug Biomatik Corporation 100ug 554.4 EUR

Recombinant Nitric Oxide Synthase 1, Neuronal (NOS1)

RPU43055-1mg Biomatik Corporation 1mg 2457 EUR

Recombinant Nitric Oxide Synthase 1, Neuronal (NOS1)

RPU43055-50ug Biomatik Corporation 50ug 445.5 EUR

Recombinant Nitric Oxide Synthase 1, Neuronal (NOS1)

RPU43056-100ug Biomatik Corporation 100ug 578.6 EUR

Recombinant Nitric Oxide Synthase 1, Neuronal (NOS1)

RPU43056-1mg Biomatik Corporation 1mg 2566.2 EUR

Recombinant Nitric Oxide Synthase 1, Neuronal (NOS1)

RPU43056-50ug Biomatik Corporation 50ug 465.3 EUR

Recombinant Nitric Oxide Synthase 1, Neuronal (NOS1)

RPU43057-100ug Biomatik Corporation 100ug 556.6 EUR

Recombinant Nitric Oxide Synthase 1, Neuronal (NOS1)

RPU43057-1mg Biomatik Corporation 1mg 2467.4 EUR

Recombinant Nitric Oxide Synthase 1, Neuronal (NOS1)

RPU43057-50ug Biomatik Corporation 50ug 447.7 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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