4d-imaging-of-protein-aggregation-in-live-cells

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Hebrew University of Jerusalem 

The eukaryotic 20 S proteasome contains the following 6 active sites: 2 chymotrypsin-like, 2 trypsin-like, and 2 caspase-like. We previously showed that hydrophobic peptide substrates of the chymotrypsin-like sites allosterically stimulate peptide hydrolysis by the caspase-like sites and their own cleavage. More thorough analysis revealed that these peptides also stimulate peptide hydrolysis by the trypsin-like site. This general activation by hydrophobic peptides occurred even if the chymotrypsin-like sites were occupied by a covalent inhibitor and was highly cooperative, with an average Hill coefficient of 7. Therefore, this stimulation of peptide hydrolysis at all active sites occurs upon binding of hydrophobic peptides to several non-catalytic sites. The stimulation by hydrophobic peptides was not observed in the yeast Delta N alpha 3 mutant 20 S proteasomes, in 20 S-PA26 complexes, or SDS-activated proteasomes and was significantly lower in 26 S proteasomes, all of which appear to have the gated channel in the alpha-rings in an open conformation and hydrolyze peptides at much faster rates than 20 S proteasomes. Also the hydrophobic peptides altered K(m), V(max) of active sites in a similar fashion as PA26 and the Delta N alpha 3 mutation. The activation by hydrophobic peptides was decreased in K(+)-containing buffer, which favors the closed state of the channels. Therefore, hydrophobic peptides stimulate peptide hydrolysis most likely by promoting the opening of the channels in the alpha-rings. During protein breakdown, this peptide-induced channel opening may function to facilitate the release of products from the proteasome.

Binding of hydrophobic peptides to several non-catalytic sites promotes peptide hydrolysis by all active sites of 20 S proteasomes. Evidence for peptide-induced channel opening in the alpha-rings.

Les eucaryotes 20 S proteasome contient 6 sites actifs suivants: 2 2 chymotrypsine-like, trypsine et 2 caspase-like. Nous avons montr&eacute; pr&eacute;c&eacute;demment que les substrats hydrophobes de peptide des chymotrypsine-like sites stimulent allost&eacute;riquement hydrolyse peptidique des caspase-comme sites et leur clivage. Une analyse plus approfondie a r&eacute;v&eacute;l&eacute; que ces peptides stimulent aussi hydrolyse peptidique par le site de la trypsine. Cette activation g&eacute;n&eacute;rale de peptides hydrophobes s&#39;est produite m&ecirc;me si les sites de la chymotrypsine-like ont &eacute;t&eacute; occup&eacute;es par un inhibiteur covalent et a &eacute;t&eacute; tr&egrave;s coop&eacute;ratifs, avec un coefficient moyen de colline de 7. Par cons&eacute;quent, cette stimulation de l&#39;hydrolyse peptidique &agrave; tous les sites actifs se produit lors de la liaison des peptides hydrophobes vers plusieurs sites non catalytiques. La stimulation par les peptides hydrophobes n&#39;a pas &eacute;t&eacute; observ&eacute;e dans la levure Delta N alpha 3 mutant 20 S prot&eacute;asomes, dans les 20 S-PA26 complexes, ou activ&eacute;s par le SDS prot&eacute;asomes et &eacute;tait significativement plus faible en 26 S prot&eacute;asomes, qui semblent ont la cha&icirc;ne ferm&eacute;e en alpha-anneaux dans une conformation ouverte et l&#39;hydrolyse de peptides &agrave; des taux beaucoup plus rapides que 20 S prot&eacute;asomes. Aussi, les peptides hydrophobes modifi&eacute; K(m), V(max) de sites actifs de fa&ccedil;on semblable comme PA26 et la mutation d&#39;alpha 3 Delta N. L&#39;activation par les peptides hydrophobes est diminu&eacute;e dans K (+)-contenant un tampon, ce qui favorise l&#39;&eacute;tat ferm&eacute; des canaux. Par cons&eacute;quent, peptides hydrophobes stimulent hydrolyse peptidique probablement en favorisant l&#39;ouverture des canaux dans les cercles d&#39;alpha. Au cours de la d&eacute;gradation des prot&eacute;ines, cet ouverture d&#39;un canal induite par le peptide peut fonctionner pour faciliter la lib&eacute;ration de produits par le prot&eacute;asome.

Liaison des peptides hydrophobes vers plusieurs sites non catalytiques favorise l&#39;hydrolyse peptidique par tous les sites actifs de 20 S prot&eacute;asomes. Preuve pour canal induite par le peptide d&#39;ouverture dans les cercles d&#39;alpha.

20 Eucariotica S proteasoma contiene i seguenti 6 siti attivi: 2 2 come chimotripsina, tripsina-like e 2 caspase-like. Abbiamo dimostrato in precedenza che substrati peptidi idrofobi dei siti chimotripsina-come stimolano allosterically idrolisi del peptide di caspase-come siti e la propria scissione. Un&#39;analisi pi&ugrave; approfondita ha rivelato che questi peptidi stimolano anche idrolisi del peptide da tripsina-come sito. Questa attivazione generale di peptidi idrofobi si &egrave; verificato anche se il chimotripsina-come siti sono stati occupati da un inibitore covalente ed era altamente cooperativi, con un coefficiente di Hill medio di 7. Pertanto, questa stimolazione dell&#39;idrolisi del peptide a tutti i siti attivi si verifica dell&#39;associazione dei peptidi idrofobi a diversi siti non catalitiche. La stimolazione di peptidi idrofobi non &egrave; stata osservata nel lievito N Delta alfa 3 mutante 20 S proteasomi, nei 20 S-PA26 complessi, o SDS-attivato proteasomi ed era significativamente pi&ugrave; bassa in 26 S proteasomi, che sembrano avere il canale gated in alfa-anelli in una conformazione aperta e idrolizzare peptidi a tassi molto pi&ugrave; veloci rispetto a 20 S proteasomi. Anche i peptidi idrofobi alterato K(m), FV di siti attivi in un modo simile come PA26 e la mutazione di alfa 3 Delta N. L&#39;attivazione di peptidi idrofobi &egrave; stata diminuita in K (+)-contenente tampone, che favorisce lo stato chiuso dei canali. Pertanto, peptidi idrofobi stimolano idrolisi del peptide pi&ugrave; probabile promuovendo l&#39;apertura dei canali alfa-anelli. Durante il catabolismo proteico, questo canale del peptide-indotta di apertura potrebbe funzionare per facilitare il rilascio di prodotti dal proteasoma.

Associazione dei peptidi idrofobi a diversi siti non catalitiche promuove l&#39;idrolisi del peptide da tutti i siti attivi di 20 S proteasomi. Prove per il canale del peptide-indotta in alfa-anelli di apertura.

Los eucariotas 20 S proteasoma contiene los siguientes sitios activos 6: 2 2 tipo quimotripsina, tripsina y 2 caspasa-como. Hemos demostrado previamente que sustratos de p&eacute;ptido hidrof&oacute;bico de los quimotripsina-como sitios allosterically estimularon la hidr&oacute;lisis del p&eacute;ptido de los caspasa-como sitios y su escote. An&aacute;lisis m&aacute;s minucioso revelaron que estos p&eacute;ptidos tambi&eacute;n estimularon la hidr&oacute;lisis del p&eacute;ptido por el sitio de la tripsina. Esta activaci&oacute;n general por p&eacute;ptidos hidr&oacute;fobos ocurri&oacute; aunque los quimotripsina-como sitios fueron ocupados por un inhibidor covalente y fue muy cooperativos, con un coeficiente de Hill promedio de 7. Por lo tanto, esta estimulaci&oacute;n de hidr&oacute;lisis del p&eacute;ptido en todos los sitios activos se produce sobre la Uni&oacute;n de p&eacute;ptidos hidr&oacute;fobos varios sitios no catal&iacute;tica. La estimulaci&oacute;n por p&eacute;ptidos hidrof&oacute;bicos no se observ&oacute; en la levadura N Delta alpha 3 mutante 20 S proteasomas, en 20 S PA26 complejos, o proteasomas SDS-activado y fue significativamente menor en 26 S proteasomas, todos los cuales parecen haber el canal cerrado en los anillos de alfa en una conformaci&oacute;n abierta e hidrolizan los p&eacute;ptidos a tasas mucho m&aacute;s r&aacute;pidas que 20 S proteasomas. Tambi&eacute;n los p&eacute;ptidos hidr&oacute;fobos alteraci&oacute;n k, V(max) de sitios activos de manera similar como PA26 y la mutaci&oacute;n de la alfa 3 de N de Delta. La activaci&oacute;n por p&eacute;ptidos hidr&oacute;fobos disminuy&oacute; en K (+)-con tamp&oacute;n, que favorece el estado cerrado de los canales. Por lo tanto, hidrof&oacute;bicos p&eacute;ptidos estimulan la hidr&oacute;lisis del p&eacute;ptido probablemente promoviendo la apertura de los canales en los anillos de alfa. Durante la descomposici&oacute;n de prote&iacute;nas, este canal inducida por el p&eacute;ptido apertura puede funcionar para facilitar la liberaci&oacute;n de productos de la proteasoma.

Uni&oacute;n de p&eacute;ptidos hidr&oacute;fobos varios sitios no catal&iacute;tica promueve la hidr&oacute;lisis del p&eacute;ptido por todos sitios activos de 20 S proteasomas. Evidencia de canal p&eacute;ptido-inducida en los anillos de alfa.

Achieving the correct balance between folding and degradation of misfolded proteins is critical for cell viability. The importance of defining the mechanisms and factors that mediate cytoplasmic quality control is underscored by the growing list of diseases associated with protein misfolding and aggregation. Molecular chaperones assist protein folding and also facilitate degradation of misfolded polypeptides by the ubiquitin-proteasome system. Here we discuss emerging links between folding and degradation machineries and highlight challenges for future research.

Protein quality control: chaperones culling corrupt conformations.

Atteindre le juste &eacute;quilibre entre le pliage et la d&eacute;gradation des prot&eacute;ines mal repli&eacute;es est critique pour la viabilit&eacute; des cellules. L&#39;importance de d&eacute;finir les m&eacute;canismes et les facteurs qui assurent la m&eacute;diation cytoplasmique contr&ocirc;le de la qualit&eacute; est soulign&eacute;e par la liste croissante des maladies associ&eacute;es au repliement et d&#39;agr&eacute;gation. Chaperons mol&eacute;culaires aider le repliement des prot&eacute;ines et &eacute;galement facilitent la d&eacute;gradation des polypeptides mal repli&eacute;es par le syst&egrave;me ubiquitine-prot&eacute;asome. Ici, nous discutons des liens &eacute;mergents entre machines de pliage et de la d&eacute;gradation et de mettre en &eacute;vidence les d&eacute;fis pour la recherche future.

Contr&ocirc;le de qualit&eacute; des prot&eacute;ines : accompagnateurs abattage conformations corrompues.

Raggiungere il corretto equilibrio tra folding e degradazione delle proteine malpiegate &egrave; fondamentale per la vitalit&agrave; delle cellule. L&#39;importanza di definire i meccanismi e i fattori che mediano citoplasmatica di controllo di qualit&agrave; &egrave; sottolineata dalla crescente elenco di malattie associate con la proteina misfolding e aggregazione. Chaperoni molecolari assistono il protein folding e anche facilitano la degradazione dei polipeptidi malpiegate dal sistema ubiquitina-proteasoma. Qui discutiamo emergenti collegamenti tra macchine folding e degradazione ed evidenziare le sfide per la ricerca futura.

Controllo di qualit&agrave; della proteina: chaperones abbattimento conformazioni corrotti.

Lograr el equilibrio correcto entre plegable y la degradaci&oacute;n de prote&iacute;nas mal plegadas es fundamental para la viabilidad celular. Se destac&oacute; la importancia de definir los mecanismos y factores que median control citopl&aacute;smico de calidad por la creciente lista de enfermedades asociadas con el mal plegamiento de la prote&iacute;na y agregaci&oacute;n. Chaperonas moleculares a plegamiento de la prote&iacute;na y tambi&eacute;n facilitan la degradaci&oacute;n de polip&eacute;ptidos mal plegadas por el sistema ubiquitina-proteasoma. Aqu&iacute; discutimos v&iacute;nculos emergentes entre mecanismos plegables y degradaci&oacute;n y destacar los retos para la investigaci&oacute;n futura.

Control de calidad de la prote&iacute;na: chaperonas sacrificio conformaciones corruptos.

The accumulation of misfolded proteins in intracellular amyloid inclusions, typical of many neurodegenerative disorders including Huntington's and prion disease, is thought to occur after failure of the cellular protein quality control mechanisms. Here we examine the formation of misfolded protein inclusions in the eukaryotic cytosol of yeast and mammalian cell culture models. We identify two intracellular compartments for the sequestration of misfolded cytosolic proteins. Partition of quality control substrates to either compartment seems to depend on their ubiquitination status and aggregation state. Soluble ubiquitinated misfolded proteins accumulate in a juxtanuclear compartment where proteasomes are concentrated. In contrast, terminally aggregated proteins are sequestered in a perivacuolar inclusion. Notably, disease-associated Huntingtin and prion proteins are preferentially directed to the perivacuolar compartment. Enhancing ubiquitination of a prion protein suffices to promote its delivery to the juxtanuclear inclusion. Our findings provide a framework for understanding the preferential accumulation of amyloidogenic proteins in inclusions linked to human disease.

Misfolded proteins partition between two distinct quality control compartments.

L&#39;accumulation de prot&eacute;ines mal repli&eacute;es dans des inclusions intracellulaires amylo&iuml;des, typiques de nombreuses maladies neurod&eacute;g&eacute;n&eacute;ratives, y compris la maladie de Huntington et la maladie &agrave; prion, est cens&eacute;e se produire apr&egrave;s l&#39;&eacute;chec des m&eacute;canismes de contr&ocirc;le de la qualit&eacute; des prot&eacute;ines cellulaires. Nous examinons ici la formation d&#39;inclusions de prot&eacute;ines mal repli&eacute;es dans le cytosol eucaryote des levures et des mod&egrave;les de culture de cellules de mammif&egrave;res. Nous identifions deux compartiments intracellulaires pour la s&eacute;questration des prot&eacute;ines cytosoliques. Partition de substrats de contr&ocirc;le de la qualit&eacute; de des compartiments semble d&eacute;pendre de leur &eacute;tat de statut et d&#39;agr&eacute;gation de l&#39;ubiquitination. Soluble ubiquitin&eacute;es prot&eacute;ines s&#39;accumulent dans un compartiment juxtanucl&eacute;aire o&ugrave; sont concentr&eacute;s les prot&eacute;asomes. En revanche, les prot&eacute;ines agr&eacute;g&eacute;es en phase terminale sont s&eacute;questr&eacute;s dans une inscription de type p&eacute;rivacuolaire. Notamment, la huntingtine associ&eacute;s &agrave; la maladie et la prot&eacute;ine prion est pr&eacute;f&eacute;rentiellement r&eacute;alis&eacute;e dans le compartiment de type p&eacute;rivacuolaire. Renforcer l&#39;ubiquitination d&#39;une prot&eacute;ine prion suffit de promouvoir sa livraison &agrave; l&#39;inclusion de la substance. Nos r&eacute;sultats fournissent un cadre pour comprendre l&#39;accumulation pr&eacute;f&eacute;rentielle de prot&eacute;ines amylo&iuml;dog&egrave;ne dans la maladie li&eacute;e &agrave; l&#39;humain des inclusions.

Les prot&eacute;ines mal repli&eacute;es partition entre deux compartiments distincts contr&ocirc;le de la qualit&eacute;.

L&#39;accumulo di proteine malpiegate inclusioni intracellulari amiloide, tipici di molte malattie neurodegenerative, tra cui la Corea di Huntington e la malattia da prioni, pensa che si verificano dopo il fallimento dei meccanismi di controllo di qualit&agrave; della proteina cellulare. Qui esaminiamo la formazione di inclusioni di proteina malpiegata nel citosol eucariotico del lievito e modelli di coltura di cellule di mammifero. Identifichiamo due compartimenti intracellulari per il sequestro delle proteine citosoliche malpiegate. Partizione dei substrati di controllo di qualit&agrave; per ogni vano sembra dipendono dal loro stato di ubiquitination status e aggregazione. Ubiquitarie solubile malpiegate proteine si accumulano in un vano di juxtanuclear dove sono concentrati i proteasomi. Al contrario, proteine terminalmente aggregate sono sequestrate in un&#39;inclusione di perivacuolar. In particolare, dell&#39;huntingtina associata a malattia e le proteine prioniche sono preferenzialmente diretto al vano perivacuolar. Miglioramento ubiquitination di una proteina prionica &egrave; sufficiente per promuovere la sua consegna all&#39;inclusione di juxtanuclear. I nostri risultati forniscono un quadro per comprendere l&#39;accumulo preferenziale di proteine amiloidogeniche nella malattia legata all&#39;essere umano di inclusioni.

Proteine malpiegate partizione tra due compartimenti distinti di controllo di qualit&agrave;.

La acumulaci&oacute;n de prote&iacute;nas mal plegadas en inclusiones de amiloides intracelulares, t&iacute;picas de muchas enfermedades neurodegenerativas incluyendo la enfermedad de Huntington y la enfermedad pri&oacute;nica, se piensa para ocurrir despu&eacute;s del fracaso de los mecanismos de control de calidad de la prote&iacute;na celular. Aqu&iacute; examinamos la formaci&oacute;n de inclusiones de prote&iacute;nas mal plegadas en el citosol eucariota de levadura y modelos de cultivo de c&eacute;lulas de mam&iacute;fero. Identificamos dos compartimientos intracelulares para el secuestro de prote&iacute;nas mal plegadas citos&oacute;licas. Divisi&oacute;n de control de calidad de sustratos a cualquiera de los compartimientos parece depender de su estado de agregaci&oacute;n y estado de ubiquitinaci&oacute;n. Ubiquitinated soluble prote&iacute;nas mal plegadas se acumulan en un compartimiento de juxtanuclear donde se concentran proteasomas. Por el contrario, prote&iacute;nas terminal agregadas son secuestradas en una inclusi&oacute;n de perivacuolar. En particular, asociada a la enfermedad de la huntingtina y prote&iacute;nas pri&oacute;nicas se dirigen preferentemente del compartimiento de perivacuolar. Mejorar la ubiquitinaci&oacute;n de una prote&iacute;na del pri&oacute;n es suficiente para promover su entrega a la inclusi&oacute;n de juxtanuclear. Nuestros resultados proporcionan un marco para entender la acumulaci&oacute;n preferencial de prote&iacute;nas amyloidogenic en enfermedad vinculada al ser humano de inclusiones.

Prote&iacute;nas mal plegadas partici&oacute;n entre dos compartimientos distintos de control de calidad.

The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of 'individuality' of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed.

Equivalent mutations in the eight subunits of the chaperonin CCT produce dramatically different cellular and gene expression phenotypes.

Eucaryotes cytoplasmique contenant chaperonin TCP-1 (CCT) est un complexe form&eacute; par deux anneaux dos &agrave; dos empil&eacute;es h&eacute;t&eacute;ro-et qui aide le pliage d&#39;actines tubulines et autres prot&eacute;ines d&#39;une mani&egrave;re d&eacute;pendante de l&#39;ATP. Ici, nous avons test&eacute; l&#39;importance de la nature h&eacute;t&eacute;ro-oligom&egrave;re de CCT dans sa fonction en introduisant &agrave; son tour, dans chacun des huit sous-unit&eacute;s identique d&#39;une mutation &agrave; un poste qui est conserv&eacute;e dans toutes les sous-unit&eacute;s et participe &agrave; l&#39;hydrolyse de l&#39;ATP, afin d&#39;&eacute;tablir l&#39;ampleur de le &laquo; individualit&eacute; &raquo; des sous-unit&eacute;s diff&eacute;rentes. Nos r&eacute;sultats montrent que ces mutations identiques m&egrave;nent &agrave; radicalement diff&eacute;rents ph&eacute;notypes. Pour exemple, les cellules de levure Saccharomyces cerevisiae avec la mutation dans la sous-unit&eacute; CCT2 affichage chaleur sensibilit&eacute; et la sensibilit&eacute; au froid pour la croissance, ont un exc&egrave;s de correctifs de l&#39;actine et sont la seule souche g&eacute;n&eacute;r&eacute;e ici c&#39;est pseudo-diploid. En revanche, les cellules avec la mutation dans la sous-unit&eacute; CCT7 sont les seuls &agrave; accumuler des agr&eacute;gats de prot&eacute;ine juxtanucl&eacute;aire qui peuvent refl&eacute;ter une r&eacute;action de stress alt&eacute;r&eacute;e chez cette souche. Au niveau du syst&egrave;me analyse des souches &agrave; l&#39;aide de puces &agrave; RNA r&eacute;v&egrave;le les connexions entre la CCT et plusieurs r&eacute;seaux cellulaires, y compris la biog&eacute;n&egrave;se des ribosomes et TOR2, qui permettent d&#39;expliquer la variabilit&eacute; ph&eacute;notypique observ&eacute;e.

Mutations &eacute;quivalentes dans les huit sous-unit&eacute;s du TDC chaperonine produisent consid&eacute;rablement diff&eacute;rents ph&eacute;notypes d&#39;expression cellulaire et g&eacute;nique.

L&#39;eucariotica citoplasmatici contenenti chaperonin TCP-1 (CCT) &egrave; un complesso formato da due anelli di back to back impilati hetero-octameric che assiste il ripiegamento dell&#39;actina, tubulins e altre proteine in modo ATP-dipendente. Qui, abbiamo testato il significato della natura etero-oligomerica della CCT in sua funzione introducendo, in ciascuna delle otto subunit&agrave; a sua volta, una mutazione identica a una posizione che &egrave; conservata in tutte le unit&agrave; secondarie ed &egrave; coinvolto nell&#39;idrolisi dell&#39;ATP, al fine di stabilire la misura del &#39;individualit&agrave;&#39; delle varie subunit&agrave;. I nostri risultati mostrano che queste mutazioni identiche portano a drammaticamente diversi fenotipi. Per esempio, le cellule di lievito Saccharomyces cerevisiae con la mutazione nella subunit&agrave; CCT2 display calore sensibilit&agrave; e sensibilit&agrave; fredda per la crescita, hanno un eccesso di patch di actina e sono l&#39;unico ceppo generato qui che &egrave; pseudo-diploid. Al contrario, le cellule con la mutazione nella subunit&agrave; CCT7 sono gli unici ad accumulare juxtanuclear aggregati di proteina che possono riflettere una risposta allo stress alterata in questo ceppo. Livello di sistema di analisi dei ceppi usando i microarrays RNA rivelano connessioni tra CCT e diverse reti cellulari, tra cui la biogenesi del ribosoma e TOR2, che aiutano a spiegare la variabilit&agrave; fenotipica osservata.

Equivalente mutazioni della otto subunit&agrave; del CCT chaperonin producono drammaticamente diversi fenotipi di espressione genica e cellulare.

El eucariotas citoplasm&aacute;tica que contiene la chaperonina TCP-1 (AAC) es un complejo formado por dos anillos de espalda con espalda apiladas hetero-oct&aacute;merico que el plegamiento de las actinias, tubulinas y otras prote&iacute;nas de una manera dependiente de ATP. Aqu&iacute;, hemos probado la importancia de la naturaleza hetero oligom&eacute;rica de CCT en su funci&oacute;n mediante la introducci&oacute;n, en cada una de las ocho subunidades a su vez, una mutaci&oacute;n id&eacute;ntica en una posici&oacute;n que se conserva en todas las subunidades y est&aacute; implicado en la hidr&oacute;lisis de ATP, con el fin de determinar el alcance de la &quot;individualidad&quot; de las distintas subunidades. Nuestros resultados muestran que estas mutaciones id&eacute;nticas permitan dram&aacute;ticamente diferentes fenotipos. Por ejemplo, c&eacute;lulas de levadura Saccharomyces cerevisiae con la mutaci&oacute;n en la subunidad CCT2 pantalla calor sensibilidad y sensibilidad fr&iacute;o para el crecimiento, tienen un exceso de parches de actina y son la &uacute;nica tensi&oacute;n generada aqu&iacute; es pseudo-diploid. Por el contrario, las c&eacute;lulas con la mutaci&oacute;n en la subunidad CCT7 son los &uacute;nicos que acumular agregados de prote&iacute;na juxtanuclear que pueden reflejar una respuesta de estr&eacute;s deteriora en esta variedad. Nivel del sistema an&aacute;lisis de las cepas mediante microarrays de RNA revelan las conexiones entre el CCT y varias redes celulares, incluyendo la biog&eacute;nesis de ribosomas y TOR2, que ayudan a explicar la variabilidad fenot&iacute;pica observada.

Equivalentes mutaciones en las subunidades de ocho de la chaperonina CCT producen dram&aacute;ticamente diferentes fenotipos de expresi&oacute;n celular y gen&eacute;tica.

Noting that the glutamine (Q) amino acid side-chain bears a striking resemblance to urea, the chemical denaturant, we argue on biophysical grounds that polyQ chains should possess a potent denaturant activity. Using live-cell confocal microscopy, we demonstrate that the surface of a polyQ inclusion denatures cytosolic proteins by binding and trapping them in an immobilized ring. We also show the reverse effect: that elevated local concentrations of unfolded protein in the cytosol can drive the co-localization and accumulation of short polyQ tracts that normally do not aggregate. Such a urea-like mechanism explains many past observations about polyQ-driven disruption of proteostasis and neurodegeneration.

Polyglutamine shows a urea-like affinity for unfolded cytosolic protein.

Notant que la cha&icirc;ne lat&eacute;rale de l&#39;acide amin&eacute; glutamine (Q) porte une ressemblance frappante avec ur&eacute;e, produit chimique d&eacute;naturant, nous plaidons pour des raisons biophysiques que polyQ cha&icirc;nes devraient poss&eacute;der une puissante activit&eacute; d&eacute;naturant. &Agrave; l&#39;aide de la microscopie confocale de cellules vivantes, nous d&eacute;montrons que la surface d&#39;une inclusion de polyQ d&eacute;nature les prot&eacute;ines cytosoliques en liant et en pi&eacute;geant dans un anneau immobilis&eacute;. Nous montrons aussi l&#39;effet inverse : que des concentrations locales &eacute;lev&eacute;es de prot&eacute;ine d&eacute;pli&eacute;e dans le cytosol peuvent conduire la co-localisation et l&#39;accumulation des tracts polyQ court qui normalement ne s&#39;agr&egrave;gent pas. Un tel m&eacute;canisme d&#39;ur&eacute;e comme explique les diff&eacute;rentes observations pass&eacute;es sur la perturbation ax&eacute;e sur la polyQ de prot&eacute;ostasie et de la neurod&eacute;g&eacute;n&eacute;rescence.

Polyglutamine montre une affinit&eacute; de type ur&eacute;e pour d&eacute;voil&eacute;e de prot&eacute;ine cytosolique.

Notando che l&#39;amminoacido glutammina (Q) side-chain porta una sorprendente somiglianza con urea, il prodotto chimico denaturante, litighiamo su basi biofisiche che catene polyQ dovrebbero possedere una potente attivit&agrave; denaturante. Mediante microscopia confocale live-cella, dimostriamo che la superficie di un&#39;inclusione polyQ denatura le proteine citosoliche associando e li intrappola in un anello immobilizzato. Mostriamo anche l&#39;effetto inverso: che elevate concentrazioni locali di spiegata della proteina nel citosol possono guidare la collocazione e l&#39;accumulo di polyQ brevi tratti che normalmente non si aggregano. Un tale meccanismo di urea-come spiega molte passate osservazioni sull&#39;interruzione polyQ-driven di proteostasis e di neurodegenerazione.

Delle poliglutamine dimostra un&#39;affinit&agrave; di urea-come per spiegata della proteina citosolica.

Observando que la glutamina (Q) del amino&aacute;cido de cadena lateral tiene un sorprendente parecido con urea, la sustancia desnaturalizante, abogamos por motivos biof&iacute;sicos que poliQ cadenas deben poseer una potente actividad desnaturalizante. Usando microscopia confocal de c&eacute;lulas vivas, demostramos que la superficie de una inclusi&oacute;n de poliQ desnaturaliza prote&iacute;nas citos&oacute;licas vinculante y atraparlos en un anillo inmovilizado. Tambi&eacute;n mostramos el efecto inverso: que elevadas concentraciones locales de prote&iacute;nas desdobladas en el citosol pueden conducir la colocalizaci&oacute;n y acumulaci&oacute;n de tramos de poliQ corto que normalmente no se agregan. Este mecanismo de la urea como explica muchos m&aacute;s all&aacute; de observaciones basadas en poliQ interrupci&oacute;n de proteostasis y la neurodegeneraci&oacute;n.

Poliglutaminas muestran una afinidad de urea como prote&iacute;na citos&oacute;lica desplegado.

Despite the activity of cellular quality-control mechanisms, subsets of mature and newly synthesized polypeptides fail to fold properly and form insoluble aggregates. In some cases, protein aggregation leads to the development of human neurodegenerative maladies, including Alzheimer's and prion diseases. Aggregates of misfolded prion protein (PrP), which appear in cells after exposure to the drug cyclosporin A (CsA), and disease-linked PrP mutants have been found to accumulate in juxtanuclear deposition sites termed 'aggresomes'. Recently, it was shown that cells can contain at least two types of deposition sites for misfolded proteins: a dynamic quality-control compartment, which was termed 'JUNQ', and a site for terminally aggregated proteins called 'IPOD'. Here, we show that CsA-induced PrP aggresomes are dynamic structures that form despite intact proteasome activity, recruit chaperones and dynamically exchange PrP molecules with the cytosol. These findings define the CsA-PrP aggresome as a JUNQ-like dynamic quality-control compartment that mediates the refolding or degradation of misfolded proteins. Together, our data suggest that the formation of PrP aggresomes protects cells from proteotoxic stress.

Cyclosporin-A-induced prion protein aggresomes are dynamic quality-control cellular compartments.

Malgr&eacute; l&#39;activit&eacute; de contr&ocirc;le de la qualit&eacute; des m&eacute;canismes cellulaires, sous-ensembles de polypeptides matures et nouvellement synth&eacute;tis&eacute;s ne pas plier correctement et forment des agr&eacute;gats insolubles. Dans certains cas, agr&eacute;gation de la prot&eacute;ine entra&icirc;ne le d&eacute;veloppement de maladies neurod&eacute;g&eacute;n&eacute;ratives humaines, y compris les maladies d&#39;Alzheimer et prions. Agr&eacute;gats de prot&eacute;ine de forme anormale prion (PrP), qui apparaissent dans les cellules apr&egrave;s que exposition &agrave; la cyclosporine m&eacute;dicament un (CsA) et li&eacute; &agrave; la maladie des mutants de PrP ont &eacute;t&eacute; trouv&eacute;s &agrave; s&#39;accumuler dans les sites o&ugrave; les d&eacute;p&ocirc;ts juxtanucl&eacute;aire appel&eacute; &laquo; agr&eacute;somes &raquo;. R&eacute;cemment, il a &eacute;t&eacute; d&eacute;montr&eacute; que les cellules peuvent contenir au moins deux types de sites de d&eacute;p&ocirc;ts de prot&eacute;ines mal repli&eacute;es : un compartiment de contr&ocirc;le de la qualit&eacute; dynamique, qui a &eacute;t&eacute; appel&eacute; &laquo; JUNQ &raquo; et un site pour les prot&eacute;ines agr&eacute;g&eacute;es en phase terminale, appel&eacute; &laquo; IPOD &raquo;. Ici, nous montrons que les PrP induite par le CsA agr&eacute;somes sont des structures dynamiques qui forment malgr&eacute; l&#39;activit&eacute; du prot&eacute;asome intact, recrutent les chaperons et les &eacute;changent dynamiquement des mol&eacute;cules PrP avec le cytosol. Ces r&eacute;sultats d&eacute;finissent le CsA-PrP aggresome comme un JUNQ type dynamique compartiment de contr&ocirc;le de la qualit&eacute; qui intervient dans le repliement ou la d&eacute;gradation des prot&eacute;ines mal repli&eacute;es. Ensemble, nos r&eacute;sultats sugg&egrave;rent que la formation de PrP agr&eacute;somes prot&egrave;ge les cellules contre le stress proteotoxic.

Agr&eacute;somes prot&eacute;ine prion induite par la cyclosporine-A sont dynamiques compartiments cellulaires de contr&ocirc;le de la qualit&eacute;.

Nonostante l&#39;attivit&agrave; dei meccanismi di controllo di qualit&agrave; cellulari, sottoinsiemi di polipeptidi maturi e recentemente sintetizzati non riescono a piegare correttamente e formano aggregati insolubili. In alcuni casi, aggregazione proteica conduce allo sviluppo di malattie neurodegenerative umane, comprese le malattie di Alzheimer e da prioni. Aggregati di proteina malpiegata prionica (PrP), che compaiono nelle celle dopo l&#39;esposizione per la ciclosporina (CsA) di droga e malattia-collegati PrP mutanti sono stati trovati ad per accumulare in siti di deposizione juxtanuclear chiamati &#39;aggresomes&#39;. Recentemente, &egrave; stato dimostrato che le cellule possono contenere almeno due tipi di siti di deposizione di proteine malpiegate: un vano di qualit&agrave;-controllo dinamico, che &egrave; stato definito &#39;JUNQ&#39; e un sito per le proteine terminalmente aggregati chiamato &#39;IPOD&#39;. Mostriamo qui, che aggresomes PrP CsA-indotta sono strutture dinamiche che formano nonostante l&#39;attivit&agrave; del proteasoma intatto, reclutano chaperoni e scambiano dinamicamente PrP molecole con il citosol. Questi risultati definiscono il CsA-PrP aggresome come un JUNQ-come dinamica vano controllo qualit&agrave; che media il ripiegamento o la degradazione delle proteine malpiegate. Insieme, i nostri dati suggeriscono che la formazione del PrP aggresomes protegge le cellule dallo stress proteotoxic.

Aggresomes proteina prionica indotta da ciclosporina-A sono dinamici controllo qualit&agrave; compartimenti cellulari.

A pesar de la actividad de mecanismos de control de calidad de celulares, subconjuntos de polip&eacute;ptidos maduros y reci&eacute;n sintetizados no doblar correctamente y forman agregados insolubles. En algunos casos, la agregaci&oacute;n de la prote&iacute;na conduce al desarrollo de enfermedades neurodegenerativas humanas, incluidas las enfermedades de Alzheimer y del pri&oacute;n. Agregados de prote&iacute;na pri&oacute;nica (PrP), que aparecen en las c&eacute;lulas despu&eacute;s de la exposici&oacute;n a la ciclosporina A (CsA) de la droga y mutantes de PrP vinculados con la enfermedad se ha encontrado que se acumulan en sitios de deposici&oacute;n de juxtanuclear denominados &#39;aggresomes&#39;. Recientemente, se demostr&oacute; que las c&eacute;lulas pueden contener por lo menos dos tipos de sitios de deposici&oacute;n de prote&iacute;nas mal plegadas: un compartimiento din&aacute;mico de control de calidad, que se denomin&oacute; &#39;JUNQ&#39; y un sitio para el terminal agregados prote&iacute;nas llamadas &#39;IPOD&#39;. Aqu&iacute;, demostramos que la CsA-inducido PrP aggresomes son estructuras din&aacute;micas que forman a pesar de la actividad del proteasoma intacto, reclutan acompa&ntilde;antes y din&aacute;micamente intercambian mol&eacute;culas de PrP con el citosol. Estos resultados definen la aggresome CsA-PrP JUNQ como compartimiento de control de calidad din&aacute;mico que media la refolding como de degradaci&oacute;n de las prote&iacute;nas mal plegadas. Juntos, nuestros datos sugieren que la formaci&oacute;n de aggresomes de PrP protege las c&eacute;lulas contra el estr&eacute;s proteotoxic.

Aggresomes de la prote&iacute;na del pri&oacute;n inducida por ciclosporina-A son compartimentos celulares din&aacute;micos de control de calidad.

Neurodegenerative diseases constitute a class of illnesses marked by pathological protein aggregation in the brains of affected individuals. Although these disorders are invariably characterized by the degeneration of highly specific subpopulations of neurons, protein aggregation occurs in all cells, which indicates that toxicity arises only in particular cell biological contexts. Aggregation-associated disorders are unified by a common cell biological feature: the deposition of the culprit proteins in inclusion bodies. The precise function of these inclusions remains unclear. The starting point for uncovering the origins of disease pathology must therefore be a thorough understanding of the general cell biological function of inclusions and their potential role in modulating the consequences of aggregation. Here, we show that in human cells certain aggregate inclusions are active compartments. We find that toxic aggregates localize to one of these compartments, the juxtanuclear quality control compartment (JUNQ), and interfere with its quality control function. The accumulation of SOD1G93A aggregates sequesters Hsp70, preventing the delivery of misfolded proteins to the proteasome. Preventing the accumulation of SOD1G93A in the JUNQ by enhancing its sequestration in an insoluble inclusion reduces the harmful effects of aggregation on cell viability.

Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity.

Maladies neurod&eacute;g&eacute;n&eacute;ratives constituent une classe de maladies marqu&eacute;es par l&#39;agr&eacute;gation de la prot&eacute;ine pathologique dans le cerveau de personnes touch&eacute;es. Bien que ces troubles sont invariablement se caract&eacute;rise par la d&eacute;g&eacute;n&eacute;rescence des sous-populations hautement sp&eacute;cifiques de neurones, agr&eacute;gation des prot&eacute;ines se produit dans toutes les cellules, ce qui indique que la toxicit&eacute; se pose que dans les contextes biologique cellule particuli&egrave;re. Troubles associ&eacute;s &agrave; l&#39;agr&eacute;gation sont unifi&eacute;s par une caract&eacute;ristique biologique commune de cellule : le d&eacute;p&ocirc;t des prot&eacute;ines coupable dans des corps d&#39;inclusion. On ne sait pas la fonction pr&eacute;cise de ces inclusions. Le point de d&eacute;part pour d&eacute;couvrir les origines de la maladie doit donc &ecirc;tre une compr&eacute;hension approfondie de la fonction biologique cellulaire g&eacute;n&eacute;rale des inclusions et leur r&ocirc;le potentiel dans la modulation des cons&eacute;quences de l&#39;agr&eacute;gation. Ici, nous montrons que, dans les cellules humaines, certaines inclusions globales sont des compartiments actives. Nous trouvons que les agr&eacute;gats toxiques localiser &agrave; l&#39;un de ces compartiments, le compartiment de contr&ocirc;le de la qualit&eacute; juxtanucl&eacute;aire (JUNQ) et interf&egrave;rent avec sa fonction de contr&ocirc;le de la qualit&eacute;. L&#39;accumulation des agr&eacute;gats SOD1G93A s&eacute;questre Hsp70, emp&ecirc;chant l&#39;acheminement des prot&eacute;ines mal repli&eacute;es pour le prot&eacute;asome. Pr&eacute;venir l&#39;accumulation de SOD1G93A dans le JUNQ en am&eacute;liorant sa s&eacute;questration dans une inclusion insoluble r&eacute;duit les effets nocifs d&#39;agr&eacute;gation sur la viabilit&eacute; des cellules.

Compartimentation de la superoxyde dismutase 1 (SOD1G93A) les agr&eacute;gats d&eacute;termine leur toxicit&eacute;.

Malattie neurodegenerative costituiscono una classe di malattie caratterizzato da aggregazione proteica patologica nel cervello delle persone affette. Anche se questi disturbi sono invariabilmente caratterizzati da degenerazione di altamente specifiche sottopopolazioni di neuroni, aggregazione proteica che si verifica in tutte le cellule, che indica tale tossicit&agrave; si pone solo in contesti biologici cella particolare. Disturbi associati aggregazione sono unificate da una caratteristica biologica cella comune: la deposizione di proteine colpevole nei corpi di inclusione. La funzione precisa di queste inclusioni rimane poco chiara. Il punto di partenza per scoprire le origini della patologia di malattia deve essere pertanto una conoscenza approfondita della funzione biologica generale delle cellule di inclusioni e il loro potenziale ruolo nel modulare le conseguenze dell&#39;aggregazione. Mostriamo qui, che nelle cellule umane determinate inclusioni di aggregazione sono compartimenti attivi. Troviamo che aggregati tossici localizzare a uno di questi comparti, il vano del controllo di qualit&agrave; di juxtanuclear (JUNQ) e interferiscano con la sua funzione di controllo della qualit&agrave;. L&#39;accumulo di aggregati SOD1G93A sequestra Hsp70, impedendo la consegna delle proteine malpiegate del proteasoma. Prevenire l&#39;accumulo di SOD1G93A nella JUNQ aumentando il suo sequestro in un&#39;inclusione insolubile riduce gli effetti nocivi di aggregazione sull&#39;attuabilit&agrave; delle cellule.

Compartimentazione della superossido dismutasi 1 (SOD1G93A) aggregati determina la loro tossicit&agrave;.

The division of the S.Â cerevisiae budding yeast, which produces one mother cell and one daughter cell, is asymmetric with respect to aging. Remarkably, the asymmetry of yeast aging coincides with asymmetric inheritance of damaged and aggregated proteins by the mother cell. Here, we show that misfolded proteins are retained in the mother cell by being sequestered in juxtanuclear quality control compartment (JUNQ) and insoluble protein deposit (IPOD) inclusions, which are attached to organelles. Upon exposure to stress, misfolded proteins accumulate in stress foci that must be disaggregated by Hsp104 in order to be degraded or processed to JUNQ and IPOD. Cells that fail to deliver aggregates to an inclusion pass on aggregates to subsequent generations.

Confinement to organelle-associated inclusion structures mediates asymmetric inheritance of aggregated protein in budding yeast.

La division de la levure S. cerevisiae, qui produit une cellule-m&egrave;re et une seule cellule fille, est asym&eacute;trique en ce qui concerne le vieillissement. Fait remarquable, l&#39;asym&eacute;trie du vieillissement de la levure co&iuml;ncide avec h&eacute;ritage asym&eacute;trique des prot&eacute;ines endommag&eacute;es et agr&eacute;g&eacute;es par la cellule m&egrave;re. Ici, nous montrons que les prot&eacute;ines mal repli&eacute;es sont conserv&eacute;es dans la cellule-m&egrave;re par &ecirc;tre s&eacute;questr&eacute; dans juxtanucl&eacute;aire compartiment de contr&ocirc;le de la qualit&eacute; (JUNQ) et des inclusions de d&eacute;p&ocirc;t (IPOD) prot&eacute;ines insolubles, qui sont attach&eacute;s aux organelles. Lors de l&#39;exposition au stress, les prot&eacute;ines mal repli&eacute;es s&#39;accumulent dans des foyers de tension qui doivent &ecirc;tre ventil&eacute;es par Hsp104 pour &ecirc;tre d&eacute;grad&eacute;s ou trait&eacute;es &agrave; JUNQ et IPOD. Les cellules qui ne parviennent pas &agrave; fournir des agr&eacute;gats &agrave; une inclusion passent sur les agr&eacute;gats pour les g&eacute;n&eacute;rations suivantes.

Confinement dans des structures associ&eacute;es aux organites inclusion n&eacute;gocie l&#39;h&eacute;ritage asym&eacute;trique des prot&eacute;ines agr&eacute;g&eacute;es dans la levure de bi&egrave;re.

La divisione del lievito S. cerevisiae in erba, che produce una cellula madre e cellula figlia uno, &egrave; asimmetrica rispetto a invecchiamento. Notevolmente, l&#39;asimmetria dell&#39;invecchiamento lievito coincide con ereditariet&agrave; asimmetrica delle proteine danneggiate e aggregate dalla cellula madre. Qui, ci mostra che le proteine malpiegate sono mantenute nella cellula madre di essere sequestrato in vano di controllo di qualit&agrave; juxtanuclear (JUNQ) e proteine insolubili deposito (IPOD) inclusioni, che sono collegati a organelli. Dopo l&#39;esposizione allo stress, proteine malpiegate si accumulano in foci dello stress che devono essere disaggregati per Hsp104 al fine di essere degradati o trasformati JUNQ e IPOD. Le cellule che non riescono a consegnare gli aggregati di un&#39;inclusione passano sugli aggregati alle generazioni successive.

Confinamento alle strutture associate all&#39;organello inclusione media asimmetrica ereditariet&agrave; degli aggregati della proteina nel lievito gemmante.

Daniel Kaganovich

Department of Cell and Developmental Biology,
Alexander Silberman Institute of Life Sciences,
Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences

Hebrew University of Jerusalem

4D Imaging of Protein Aggregation in Live Cells

Daniel Kaganovich

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell and Developmental Biology,Alexander Silberman Institute of Life Sciences,Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences

Hebrew University of Jerusalem

4D Imaging of Protein Aggregation in Live Cells

Department of Cell and Developmental Biology,
Alexander Silberman Institute of Life Sciences,
Department of Cell and Developmental Biology, Alexander Silberman Institute of Life Sciences