La protéine Nestin

Nestin est une grosse protéine (>1600 acides aminés), possédant une structure similaire aux autres protéines de filaments intermédiaires. Un domaine central hautement conservé composé d’une hélice α de 300-330 acides aminés situé entre les domaines N et C- terminaux. Cette partie centrale est composée de plusieurs hélices α enroulées soit les hélices 1A et 1B

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séparées par un lien (L1), les hélices 1B et 2A séparées par L12, et les hélices 2A et 2B séparées par L2. [378] [365] (Voir Figure14. Structure de Nestin).

2 MultipleSclerosisInternational NF-L NF-M KSP1KSD KSP2 SP KErepeats Esegment KEsegment NF-H DEPPS KSPsegment Esegment Esegment Esegment KEPsegment α-internexin EEIIEE EEIIEE /KEpeptide 100amino acids A/Ckinase Akinase Peripherin Vimentin EEIIEE RDGpeptide RDGpeptide A/Ckinase A/C/cdc2kinase QErepeats

Head _— Rod _— Tail

Nestin

EEIIEE

Figur e1: Diagramof thesubunit proteinsof neurofilaments. NF-L, NF-M, NF-H, and α-internexin can beregarded asthemajor subunits of adult NFsthoughNFsmayalsocontain peripherin, vimentin, andnestin in certain locations, developmental stages, andpossiblydamage or diseasestates. Phosphorylationsitesfor proteinkinaseA(Akinase), proteinkinaseC(Ckinase), andcdc2kinases(cdc2kinase) havebeen characterized in theglobular “head” regionsof certain of thesemoleculesasindicated. Theregionsindicated by KSP, SP, KSD, and DEPPS areknown serinephosphorylation sitesinthe“tail” regions. EEIIEE, KErepeats, Tail a, Esegment, KEPsegment, KEsegment, RGD, andQE repeatseach refer tospecifickindsof sequencemotif. For further details, seeShaw1998[13].

2. Introduction toNeurofilament Proteins

Neurofilaments (NFs) are the major structural proteins of neurons. They aremost abundant in larger neuronsand are heavily concentrated in axons, in particular longprojection axons. Thesubunitsof NFsbelong to theintermediate(IF) family of proteins, which arecharacterized by astructurally conservedα-helical coiled-coil “rod” region whichformsthe backbone of the filament, with variable N and C terminal extensions (Figure 1). The IF subunits of vertebrates are divided into five classes based on protein characteristics, expressionpattern, andintronplacement. IFsubunitsClassI andII includetheepithelial keratins; theclassIII IFsinclude vimentin, desmin, and glial fibrillary acidicprotein (GFAP), while the major neurofilament subunits NF-Light (NF- L), NF-medium (NF-M), NF-Heavy (NF-H), α-internexin, and nestin form IF class IV. Class V IFs are the lamin proteins of the nuclear matrix. The expression profile of Class IV proteins is limited to the nervous system, with the exception of nestin, which may be found in stem cells throughout the body. All 5 Class IV genes share a distinct

intron pattern fromthat seen in other IFgenes, indicatinga closeevolutionary relationship. TheclassIV subunitsNF-H and NF-M have unusually long and complex C-terminal “tail” regions which are responsible for the wispy spacers seen by electron microscopy and thewider spacing of NFs comparedtoother IFs.NF-L,NF-M,NF-H,andα-internexin are all abundant proteins of the nervous systems of adult mammals, while nestin is expressed early in development and is normally downregulated in the adult. One class III IF protein, peripherin, is found copolymerized with NF-L, NF-M, NF-H, and α-internexin in theNFof someneurons insignificant amounts, particularlyintheperipheral nervous system.Finally,afewapparentlyunusual neuronsintheadult express another Class III protein, vimentin. Neuroblasts express this protein, but it is generally downregulated as development proceeds. However, in many damage and disease states, cells will re-express proteins which were downregulated developmentally, sothat expression of anyof the 7 subunits shown in Figure1 could be associated with specificdamageor diseasestates. Thevariousproteinswhich may be included in NFs are known to be phosphorylated,

2 MultipleSclerosisInternational NF-L NF-M KSP1KSD KSP2 SP KErepeats Esegment KEsegment NF-H DEPPS KSPsegment Esegment Esegment Esegment KEPsegment α-internexin EEIIEE EEIIEE /KEpeptide 100amino acids A/Ckinase Akinase Peripherin Vimentin EEIIEE RDGpeptide RDGpeptide A/Ckinase A/C/cdc2kinase QErepeats

Head _— Rod _— Tail

Nestin

EEIIEE

Figur e1: Diagramof thesubunit proteinsof neurofilaments. NF-L, NF-M, NF-H, and α-internexin can beregarded asthemajor subunits of adult NFsthoughNFsmayalsocontain peripherin, vimentin, andnestin in certain locations, developmental stages, andpossiblydamage or diseasestates. Phosphorylationsitesfor proteinkinaseA(Akinase), proteinkinaseC(Ckinase), andcdc2kinases(cdc2kinase) havebeen characterized in theglobular “head” regionsof certain of thesemoleculesasindicated. Theregionsindicated by KSP, SP, KSD, and DEPPS areknown serinephosphorylation sitesinthe“tail” regions. EEIIEE, KErepeats, Tail a, Esegment, KEPsegment, KEsegment, RGD, andQE repeatseach refer tospecifickindsof sequencemotif. For further details, seeShaw1998[13].

2. Introduction toNeurofilament Proteins

Neurofilaments (NFs) are the major structural proteins of neurons. They aremost abundant in larger neuronsand are heavily concentrated in axons, in particular longprojection axons. Thesubunitsof NFsbelong to theintermediate(IF) family of proteins, which arecharacterized by astructurally conservedα-helical coiled-coil “rod” region whichformsthe backbone of the filament, with variable N and C terminal extensions (Figure 1). The IF subunits of vertebrates are divided into five classes based on protein characteristics, expressionpattern, andintronplacement. IFsubunitsClassI andII includetheepithelial keratins; theclassIII IFsinclude vimentin, desmin, and glial fibrillary acidicprotein (GFAP), while the major neurofilament subunits NF-Light (NF- L), NF-medium (NF-M), NF-Heavy (NF-H), α-internexin, and nestin form IF class IV. Class V IFs are the lamin proteins of the nuclear matrix. The expression profile of Class IV proteins is limited to the nervous system, with

intron pattern fromthat seen in other IFgenes, indicatinga closeevolutionary relationship. TheclassIV subunitsNF-H and NF-M have unusually long and complex C-terminal “tail” regions which are responsible for the wispy spacers seen by electron microscopy and thewider spacing of NFs comparedtoother IFs.NF-L,NF-M,NF-H,andα-internexin are all abundant proteins of the nervous systems of adult mammals, while nestin is expressed early in development and is normally downregulated in the adult. One class III IF protein, peripherin, is found copolymerized with NF-L, NF-M, NF-H, and α-internexin in theNFof someneurons insignificant amounts, particularlyintheperipheral nervous system.Finally,afewapparentlyunusual neuronsintheadult express another Class III protein, vimentin. Neuroblasts express this protein, but it is generally downregulated as development proceeds. However, in many damage and disease states, cells will re-express proteins which were downregulated developmentally, sothat expression of anyof the 7 subunits shown in Figure1 could be associated with

2 MultipleSclerosisInternational NF-L NF-M KSP1KSD KSP2 SP KErepeats Esegment KEsegment NF-H DEPPS KSPsegment Esegment Esegment Esegment KEPsegment α-internexin EEIIEE EEIIEE /KEpeptide 100amino acids A/Ckinase A kinase Peripherin Vimentin EEIIEE RDGpeptide RDGpeptide A/Ckinase A/C/cdc2kinase QErepeats

Head _— Rod _— Tail

Nestin

EEIIEE

Figur e1: Diagramof thesubunit proteinsof neurofilaments. NF-L, NF-M, NF-H, and α-internexin can beregarded asthemajor subunits of adult NFsthough NFsmayalsocontain peripherin, vimentin, andnestin in certain locations, developmental stages, andpossiblydamage or diseasestates. Phosphorylationsitesfor proteinkinaseA(Akinase), proteinkinaseC(Ckinase), andcdc2kinases(cdc2kinase) havebeen characterized in theglobular “head” regionsof certain of thesemoleculesasindicated. Theregionsindicated by KSP, SP, KSD, and DEPPS areknownserinephosphorylation sitesinthe“tail” regions. EEIIEE, KErepeats, Tail a, Esegment, KEPsegment, KEsegment, RGD, andQE repeatseach refer tospecifickindsof sequencemotif. For further details, seeShaw1998[13].

2. Introduction toNeurofilament Proteins

Neurofilaments (NFs) are the major structural proteins of neurons. They aremost abundant in larger neuronsand are heavily concentrated in axons, in particular longprojection axons. Thesubunitsof NFsbelong to theintermediate(IF) family of proteins, which arecharacterized by astructurally conservedα-helical coiled-coil “rod” region whichformsthe backbone of the filament, with variable N and C terminal extensions (Figure 1). The IF subunits of vertebrates are divided into five classes based on protein characteristics, expressionpattern, andintronplacement. IFsubunitsClassI andII includetheepithelial keratins; theclassIII IFsinclude vimentin, desmin, and glial fibrillaryacidicprotein (GFAP), while the major neurofilament subunits NF-Light (NF- L), NF-medium (NF-M), NF-Heavy (NF-H), α-internexin, and nestin form IF class IV. Class V IFs are the lamin

intron pattern fromthat seen in other IFgenes, indicatinga closeevolutionary relationship. TheclassIV subunitsNF-H and NF-M have unusually long and complex C-terminal “tail” regions which are responsible for the wispy spacers seen by electron microscopy and the wider spacing of NFs comparedtoother IFs.NF-L,NF-M,NF-H,andα-internexin are all abundant proteins of the nervous systems of adult mammals, while nestin is expressed early in development and is normally downregulated in the adult. One class III IF protein, peripherin, is found copolymerized with NF-L, NF-M, NF-H, and α-internexin in theNFof someneurons insignificant amounts, particularlyintheperipheral nervous system.Finally,afewapparentlyunusual neuronsintheadult express another Class III protein, vimentin. Neuroblasts express this protein, but it is generally downregulated as development proceeds. However, in many damage and disease states, cells will re-express proteins which were Partie centrale

Partie

N-terminale C-terminale Partie

Partie N-terminale Partie C-terminale Partie centrale Répétition KE Segment E Segment KE Répétition QE Peptide RDG

A

B

Figure 14. Structure de la protéine Nestin (A) Un domaine central hautement conservé composé d’une hélice α de 300-330 acides aminés entre les domaines N et C- terminaux. La partie centrale de la protéine est composée de plusieurs hélices α enroulé soit les hélices 1A et 1B séparées par un lien (L1), les hélices 1B et 2A séparées par L12, et les hélices 2A et 2B séparées par L2. (B) Comparaison de la structure de Nestin avec le Neurofilament M et la protéine Vimentine. Les sites KSP, SP et KSD sont des sites de phosphorylation dans la partie C-terminal du Neurofilament M. Les segments E, KE et QE font référence à des séquences répétées. (modifié de [379] [369])

La région C-terminale de Nestin compte 1306 acides aminés et une séquence répétée conservée contrairement à la région N-terminale qui est plus courte que les autres protéines de filaments intermédiaires. [380] La variation de l’extrémité C-terminale permet à la protéine de former des complexes avec d’autres protéines de structure. La protéine humaine Nestin est

plus courte que celle du rat et de la souris par 87 et 203 acides aminés respectivement [380] [381] La différence dans la longueur est due à une variation dans le nombre de séquences répétées. (Voir Figure 14. Structure de Nestin). La région C-terminale est conserve à 55% et la région hélicoïdale est conservée à 82% entre l’humain et les rongeurs. [380, 381]

La phosphorylation de Nestin régule son assemblage et sa désorganisation. [362] Ainsi, il a été démontré qu’un faible taux de phosphorylation est associé avec l’assemblage de Nestin durant la mitose. La protéine Nestin et sa partenaire, Vimentine, se retrouvent 3 et 6 fois plus phosphorylées respectivement durant la division cellulaire. De plus, ces états de phosphorylation coïncident avec la réorganisation des filaments intermédiaires. [362] Un site de phosphorylation important pour l’assemblage de la protéine Nestin est le résidu thréonine 316, localisé dans l’extrémité C-terminal de la protéine. D’ailleurs, il a été démontré que la phosphorylation régule l’organisation spatiale des protéines de filaments intermédiaires comme Vimentine et Nestin. [382, 383] En fait, durant la mitose, la désorganisation de Vimentine requiert sa phosphorylation et celle de Nestin. [384] Le rôle de Nestin dans la désorganisation de Vimentine a été confirmé par la réduction spécifique de l’expression de la protéine Nestin par une approche d’ARN interférant in vitro. Ceci a bloqué la désorganisation de Vimentine dans les cellules mitotiques et a démontré que Nestin devait être présente. [384]