Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Yale School of Medicine

Howard Hughes Medical Institute

Bacterial heat-shock proteins, ClpB and DnaK form a bichaperone system that efficiently reactivates aggregated proteins. ClpB undergoes nucleotide-dependent self-association and forms ring-shaped oligomers. The ClpB-assisted dissociation of protein aggregates is linked to translocation of substrates through the central channel in the oligomeric ClpB. Events preceding the translocation step, such as recognition of aggregates by ClpB, have not yet been explored, and the location of the aggregate-binding site in ClpB has been under discussion. We investigated the reactivation of aggregated glucose-6-phosphate dehydrogenase (G6PDH) by ClpB and its N-terminally truncated variant ClpBDeltaN in the presence of DnaK, DnaJ, and GrpE. We found that the chaperone activity of ClpBDeltaN becomes significantly lower than that of the full-length ClpB as the size of G6PDH aggregates increases. Using a "substrate trap" variant of ClpB with mutations of Walker B motifs in both ATP-binding modules (E279Q/E678Q), we demonstrated that ClpBDeltaN binds to G6PDH aggregates with a significantly lower affinity than the full-length ClpB. Moreover, we identified two conserved acidic residues at the surface of the N-terminal domain of ClpB that support binding to G6PDH aggregates. Those N-terminal residues (Asp-103, Glu-109) contribute as much substrate-binding capability to ClpB as the conserved Tyr located at the entrance to the ClpB channel. In summary, we provided evidence for an essential role of the N-terminal domain of ClpB in recognition and binding strongly aggregated proteins.

The amino-terminal domain of ClpB supports binding to strongly aggregated proteins.

ClpB is a heat-shock protein that reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the family of AAA+ ATPases and forms ring-shaped oligomers: heptamers in the absence of nucleotides and hexamers in the presence of nucleotides. We investigated the thermodynamic stability of ClpB in its monomeric and oligomeric forms. ClpB contains six distinct structural domains: the N-terminal domain involved in substrate binding, two AAA+ ATP-binding modules, each consisting of two domains, and a coiled-coil domain inserted between the AAA+ modules. We produced seven variants of ClpB, each containing a single Trp located in each of the ClpB domains and measured the changes in Trp fluorescence during the equilibrium urea-induced unfolding of ClpB. We found that two structural domains: the small domain of the C-terminal AAA+ module and the coiled-coil domain were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation and/or interactions during formation of the ClpB rings.

Domain stability in the AAA+ ATPase ClpB from Escherichia coli.

Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ structures contain the nucleotide-binding P-loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA+ sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA+ ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker-A Thr with Asn in ClpB, a bacterial AAA+ chaperone which reactivates aggregated proteins. We found that the T-to-N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP-bound conformation and the high-affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker-A Thr in sensing the nucleotide's gamma-phosphate and in maintaining an allosteric linkage between the P-loop and the aggregate binding site of ClpB. We postulate that AAA+ ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate-remodeling activity.

Walker-A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB.

Bacterial AAA+ ATPase ClpB cooperates with DnaK during reactivation of aggregated proteins. The ClpB-mediated disaggregation is linked to translocation of polypeptides through the channel in the oligomeric ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and ClpB80, which does not contain the substrate-interacting N-terminal domain. The biological role of the truncated isoform ClpB80 is unknown. We found that resolubilization of aggregated proteins in Escherichia coli after heat shock and reactivation of aggregated proteins in vitro and in vivo occurred at higher rates in the presence of ClpB95 with ClpB80 than with ClpB95 or ClpB80 alone. Combined amounts of ClpB95 and ClpB80 bound to aggregated substrates were similar to the amounts of either ClpB95 or ClpB80 bound to the substrates in the absence of another isoform. The ATP hydrolysis rate of ClpB95 with ClpB80, which is linked to the rate of substrate translocation, was not higher than the rates measured for the isolated ClpB95 or ClpB80. We postulate that a reaction step that takes place after substrate binding to ClpB and precedes substrate translocation is rate-limiting during aggregate reactivation, and its efficiency is enhanced in the presence of both ClpB isoforms. Moreover, we found that ClpB95 and ClpB80 form hetero-oligomers, which are similar in size to the homo-oligomers of ClpB95 or ClpB80. Thus, the mechanism of functional cooperation of the two isoforms of ClpB may be linked to their heteroassociation. Our results suggest that the functionality of other AAA+ ATPases may be also optimized by interaction and synergistic cooperation of their isoforms.

Synergistic cooperation between two ClpB isoforms in aggregate reactivation.

In research for promising antibacterial and antifungal compounds, a series of 2-aryl 3-[1,2,4]triazol-5-yl 4-thiazolidinones 1 were synthesized by a domino reaction of 5-amino-1H-[1,2,4]triazoles 3, aromatic aldehydes, and α-mercaptoacids in boiling toluene in the presence of molecular sieves 4 Å. Of the twenty novel 3-[1,2,4]triazol-5-yl 4-thiazolidinone derivatives, four compounds 2-benzo[d][1,3]dioxol-6-yl-3-[(3-morpholin-4-yl)-1H-1,2,4-triazol-5-yl)]-1,3-thiazolidin-4-one (1i), 2-(4-chlorophenyl)-5-methyl-3-[3-(4-methylpiperazin-1-yl)-1H-1,2,4-triazol-5-yl]-1,3-thiazolidin-4-one (1p), 2-benzo[d][1,3]dioxol-6-yl-3-[3-(4-methylpiperazin-1-yl)-1H-1,2,4-triazol-5-yl]-1,3-thiazolidin-4-one (1s), 2-benzo[d][1,3]dioxol-6-yl-5-methyl-3-[3-(4-methylpiperazin-1-yl)-1H-1,2,4-triazol-5-yl]-1,3-thiazolidin-4-one (1t) exhibited MICs of 4 µg/mL or less versus Mycobacterium tuberculosis. Moreover, these compounds were screened against Candida albicans. Compounds 1p, 1s gave MICs of 1 µg/mL or less, and were fungicidal. Finally, compound 1s was evaluated against an expanded fungal panel and showed good activity against Cryptococcus neoformans. In addition, compound 1s also appeared to be fungicidal against Aspergillus arrhizus, with MIC <1 µg/mL.

Efficient regioselective three-component domino synthesis of 3-(1,2,4-Triazol-5-yl)-1,3-thiazolidin-4-ones as potent antifungal and antituberculosis agents.

Hsp100 family of molecular chaperones shows a unique capability to resolubilize and reactivate aggregated proteins. The Hsp100-mediated protein disaggregation is linked to the activity of other chaperones from the Hsp70 and Hsp40 families. The best-studied members of the Hsp100 family are the bacterial ClpB and Hsp104 from yeast. Hsp100 chaperones are members of a large super-family of energy-driven conformational "machines" known as AAA+ ATPases. This review describes the current mechanistic model of the chaperone-induced protein disaggregation and explains how the structural architecture of Hsp100 supports disaggregation and how the co-chaperones may participate in the Hsp100-mediated reactions.

Aggregate reactivation mediated by the Hsp100 chaperones.

The main aim of the European Water Framework Directive (WFD, 2000) is to commit European Union Member states to the achievement of good qualitative and quantitative status for all water bodies by 2015. To achieve this, a reference state has to be determined and appropriate monitoring has to be carried out. Based on the fact that the WFD classifies Lake Balaton, the largest shallow freshwater lake in Central Europe, as one water body, and due to the lack of funds, the number of sampling locations on the lake was decreased. The aim of this study was to determine how many sub-areas with different WFD-related attributes (in this case, parameters) can be delimited in the so-called one water body of Lake Balaton, so that a number of representative sampling locations might be retained. To determine Lake Balaton's different water quality areas (i.e. sub-areas of water body) 23 parameters (inorganic compounds) were examined from 10 sampling locations for the time interval 1985-2004 using cluster- and discriminant analysis, and Wilks' lambda distribution. With cluster analysis we were able to determine two time intervals (1985-1997 and 1998-2004) with three patterns of sub-areas, two from the first and one from the latter interval. These patterns pointed to the fact that for the whole investigated time interval (1985-2004) a total of five sub-areas were present, changing in number and alignment. Then the results were verified using discriminant analysis, and the parameters which influenced the sub-areas the most were determined using Wilks' lambda distribution. The conclusion was that to be able to follow the changes in alignment of the sub-areas and to get a comprehensive picture of Lake Balaton, a minimum of five sampling locations should be retained, one in each sub-area. Based on this study the Water Authorities chose to keep five out of ten sampling locations so that the sub-areas could be described. We consider this a great success and the methodology as an example for setting up sub-areas in a water body.

Delimiting sub-areas in water bodies using multivariate data analysis on the example of Lake Balaton (W Hungary).

ClpB &#x91CD;&#x65B0;&#x6FC0;&#x6D3B;&#x805A;&#x5408;&#x7684;&#x86CB;&#x767D; DnaK/J.&#x4E0E;&#x5408;&#x4F5C;ClpB &#x5355;&#x4F53;&#x5305;&#x542B;&#x4E24;&#x4E2A;&#x6838;&#x82F7;&#x9178;&#x7ED3;&#x5408;&#x57DF; &#xFF08;D1&#x3001; D2&#xFF09;&#x3001; &#x7EBF;&#x5708;&#x76D8;&#x7ED5;&#x57DF;&#xFF0C;&#x5E76;&#x4E14;&#x4E0E;&#x542B;&#x7518;&#x7518;&#x6BCD;&#x9898;&#x7684;&#x4E3A;&#x671F; 17 &#x4E2A;&#x6B8B;&#x7559;&#x7684;&#x975E;&#x7ED3;&#x6784;&#x5316;&#x94FE;&#x63A5;&#x5668;&#x9644;&#x52A0;&#x5230; D1 N &#x7EC8;&#x7AEF;&#x57DF;&#x3002;ClpB &#x4ECB;&#x5BFC;&#x86CB;&#x767D;&#x5206;&#x7C7B;&#x94FE;&#x63A5;&#x5230;&#x79FB;&#x4F4D;&#x7684;&#x57FA;&#x677F;&#x901A;&#x8FC7;&#x4E2D;&#x592E;&#x901A;&#x9053;&#x4E2D;&#x516D; ClpB&#xFF0C;&#x4F46;&#x4E4B;&#x524D;&#x6613;&#x4F4D;&#x7684;&#x4E8B;&#x4EF6;&#x5374;&#x4E0D;&#x751A;&#x4E86;&#x89E3;&#x3002;N &#x7EC8;&#x7AEF;&#x57DF;&#x5F62;&#x6210;&#x73AF;&#x5468;&#x56F4;&#x7684;&#x5165;&#x53E3;&#x901A;&#x9053;&#xFF0C;&#x5E76;&#x6709;&#x52A9;&#x4E8E;&#x805A;&#x5408;&#x5177;&#x6709;&#x7EA6;&#x675F;&#x529B;&#x3002;&#x6709;&#x4EBA;&#x5EFA;&#x8BAE;&#x7531;&#x975E;&#x7ED3;&#x6784;&#x5316;&#x94FE;&#x63A5;&#x5668;&#x7EF4;&#x62A4;&#x7684; N-&#x672B;&#x7AEF;&#x57DF;&#x6D41;&#x52A8;&#x6027;&#x53EF;&#x80FD;&#x63A7;&#x5236;&#x805A;&#x5408;&#x6062;&#x590D;&#x7684;&#x6548;&#x7387;&#x3002;&#x6211;&#x4EEC;&#x751F;&#x4EA7; ClpB &#x4E03;&#x4E2A;&#x7684;&#x53D8;&#x79CD;&#x4FEE;&#x6539; N &#x7EC8;&#x7AEF;&#x94FE;&#x63A5;&#x5668;&#x7684;&#x987A;&#x5E8F;&#x3002;&#x82E5;&#x8981;&#x589E;&#x52A0;&#x94FE;&#x63A5;&#x5668;&#x7684;&#x6784;&#x8C61;&#x7684;&#x7075;&#x6D3B;&#x6027;&#xFF0C;&#x6211;&#x4EEC;&#x63D2;&#x5165;&#x65C1;&#x8FB9;&#x7684; GG &#x6BCD;&#x9898;&#x7684;&#x6700;&#x591A;&#x56DB;&#x4E2A;&#x7518;&#x3002;&#x82E5;&#x8981;&#x51CF;&#x5C0F;&#x94FE;&#x63A5;&#x5668;&#x7684;&#x7075;&#x6D3B;&#x6027;&#xFF0C;&#x6211;&#x4EEC;&#x5220;&#x9664; GG &#x56FE;&#x6848;&#xFF0C;&#x5C06;&#x5176;&#x53D8;&#x6210; GP &#x548C; PP&#x3002;&#x6211;&#x4EEC;&#x53D1;&#x73B0;&#x4EFB;&#x4F55;&#x94FE;&#x63A5;&#x5668;&#x4FEE;&#x6539;&#x6291;&#x5236; ClpB ATPase &#x7684;&#x57FA;&#x5E95;&#x6D3B;&#x52A8;&#x6216;&#x5BF9;&#x7A97;&#x4F53;&#x9F50;&#x805A;&#x7269;&#x7684;&#x80FD;&#x529B;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x4FEE;&#x6539;&#x94FE;&#x63A5;&#x5668; ClpB &#x53D8;&#x79CD;&#x663E;&#x793A;&#x805A;&#x5408;&#x7684;&#x8461;&#x8404;&#x7CD6;-6-&#x78F7;&#x9178;&#x8131;&#x6C22;&#x9176;&#x548C;&#x8424;&#x706B;&#x866B;&#x8367;&#x5149;&#x7D20;&#x9176;&#x548C;&#x8F83;&#x4F4E;&#x7684;&#x805A;&#x5408;&#x7ED1;&#x5B9A;&#x6548;&#x7387;&#x6BD4; wt ClpB &#x91CD;&#x65B0;&#x6FC0;&#x6D3B;&#x7387;&#x8F83;&#x4F4E;&#x3002;&#x6211;&#x4EEC;&#x5F97;&#x51FA;&#x7ED3;&#x8BBA;&#x94FE;&#x63A5;&#x5668;&#x4E0D;&#x4F1A;&#x53EA;&#x662F;&#x8FDE;&#x63A5; N &#x7EC8;&#x7AEF;&#x57DF;&#xFF0C;&#x4F46;&#x5B83;&#x652F;&#x6301; ClpB &#x7684;&#x4F34;&#x4FA3;&#x6D3B;&#x52A8;&#x901A;&#x8FC7;&#x805A;&#x5408;&#x7ED1;&#x5B9A;&#x548C;&#x5206;&#x7C7B;&#x7684;&#x6548;&#x7387;&#x4F5C;&#x51FA;&#x8D21;&#x732E;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x94FE;&#x63A5;&#x5668;&#x5E8F;&#x5217;&#x7684;&#x9009;&#x62E9;&#x6027;&#x538B;&#x529B;&#x53EF;&#x80FD;&#x7EF4;&#x6301; ClpB.&#x86CB;&#x767D;&#x8D28; 2012 &#xFF1B; &#x4E8C;&#x6B21;&#x805A;&#x5408;&#x6D3B;&#x5316;&#x7684;&#x6700;&#x5927;&#x6548;&#x7387;&#x7684;&#x5173;&#x952E;&copy; 2012&#x5E74;&#x5A01;&#x5229;&#x671F;&#x520A;&#xFF0C;Inc.

&#x67D4;&#x6027;&#x8FDE;&#x63A5;&#x7684; ClpB &#x4E2D;&#x7684; N-&#x672B;&#x7AEF;&#x57DF;&#x8C03;&#x8282;&#x57FA;&#x677F;&#x7ED1;&#x5B9A;&#x548C;&#x805A;&#x5408;&#x6062;&#x590D;&#x6548;&#x7387;&#x3002;

ClpB reactivates aggregated proteins in cooperation with DnaK/J. The ClpB monomer contains two nucleotide-binding domains (D1, D2), a coiled-coil domain, and an N-terminal domain attached to D1 with a 17-residue-long unstructured linker containing a Gly-Gly motif. The ClpB-mediated protein disaggregation is linked to translocation of substrates through the central channel in the hexameric ClpB, but the events preceding the translocation are poorly understood. The N-terminal domains form a ring surrounding the entrance to the channel and contribute to the aggregate binding. It was suggested that the N-terminal domain's mobility that is maintained by the unstructured linker might control the efficiency of aggregate reactivation. We produced seven variants of ClpB with modified sequence of the N-terminal linker. To increase the linker's conformational flexibility, we inserted up to four Gly next to the GG motif. To decrease the linker's flexibility, we deleted the GG motif and converted it into GP and PP. We found that none of the linker modifications inhibited the basal ClpB ATPase activity or its capability to form oligomers. However, the modified linker ClpB variants showed lower reactivation rates for aggregated glucose-6-phosphate dehydrogenase and firefly luciferase and a lower aggregate-binding efficiency than wt ClpB. We conclude that the linker does not merely connect the N-terminal domain, but it supports the chaperone activity of ClpB by contributing to the efficiency of aggregate binding and disaggregation. Moreover, our results suggest that selective pressure on the linker sequence may be crucial for maintaining the optimal efficiency of aggregate reactivation by ClpB. Proteins 2012; Â© 2012 Wiley Periodicals, Inc.

Flexible connection of the N-terminal domain in ClpB modulates substrate binding and the aggregate reactivation efficiency.

ClpB r&eacute;active les prot&eacute;ines agr&eacute;g&eacute;es en collaboration avec DnaK/J. Le monom&egrave;re ClpB contient deux domaines de liaison nucl&eacute;otidique (D1, D2), un domaine coiled-coil et un domaine N-terminal attach&eacute; &agrave; la D1 avec un linker non structur&eacute; 17-r&eacute;sidu de long contenant un motif de Gly-Gly. La d&eacute;sagr&eacute;gation de la prot&eacute;ine induite par le ClpB est li&eacute;e &agrave; la translocation des substrats par la voie centrale dans le hexam&eacute;rique ClpB, mais les &eacute;v&eacute;nements pr&eacute;c&eacute;dant la translocation sont mal connues. Les domaines N-terminaux forment un anneau entourant l&#39;entr&eacute;e du chenal et contribuent &agrave; la liaison d&#39;agr&eacute;gation. Il a &eacute;t&eacute; sugg&eacute;r&eacute; que la mobilit&eacute; du domaine N-terminal qui est maintenue par l&#39;&eacute;diteur de liens non structur&eacute; peut-&ecirc;tre limiter l&#39;efficacit&eacute; de la r&eacute;activation globale. Nous avons produit sept variantes du ClpB avec la s&eacute;quence modifi&eacute;e de l&#39;&eacute;diteur de liens N-terminale. Pour augmenter la flexibilit&eacute; conformationnelle de l&#39;&eacute;diteur de liens, nous avons ins&eacute;r&eacute; jusqu&#39;&agrave; quatre Gly &agrave; c&ocirc;t&eacute; du motif GG. Pour diminuer la flexibilit&eacute; de l&#39;&eacute;diteur de liens, nous avons supprim&eacute; le motif GG et converti en GP et PP. Nous avons constat&eacute; qu&#39;aucune des modifications de l&#39;&eacute;diteur de liens a inhib&eacute; l&#39;activit&eacute; ClpB ATPase basale ou sa capacit&eacute; d&#39;oligom&egrave;res de forme. Cependant, les variantes du ClpB mis &agrave; jour le linker a montr&eacute; des taux inf&eacute;rieurs de r&eacute;activation pour agr&eacute;g&eacute;es glucose-6-phosphate d&eacute;shydrog&eacute;nase et lucif&eacute;rase luciole et une efficacit&eacute; d&#39;agr&eacute;gat-liaison plus faible que wt ClpB. Nous concluons que l&#39;&eacute;diteur de liens ne connecte pas simplement le domaine N-terminal, mais il prend en charge l&#39;activit&eacute; chaperon du ClpB en contribuant &agrave; l&#39;efficacit&eacute; de la liaison globale et la ventilation. En outre, nos r&eacute;sultats sugg&egrave;rent que la pression s&eacute;lective sur la s&eacute;quence de l&#39;&eacute;diteur de liens peut &ecirc;tre cruciale pour le maintien de l&#39;efficacit&eacute; optimale de r&eacute;activation agr&eacute;gation de prot&eacute;ines ClpB. 2012 ; &copy; 2012 Wiley p&eacute;riodiques, Inc.

Raccordement flexible du domaine N-terminal &agrave; ClpB module de liaison du substrat et l&#39;efficacit&eacute; globale de r&eacute;activation.

ClpB reaktiviert aggregierte Proteine in Zusammenarbeit mit DnaK/J. Die ClpB-Monomer enth&auml;lt zwei Nukleotid-Bindung-Dom&auml;nen (D1, D2), eine aufgerollte Spule, auch eine N-terminalen Dom&auml;ne befestigt auf D1 mit einem 17-R&uuml;ckstand lange unstrukturierte Linker mit einem Gly-Gly-Motiv. Die ClpB-vermittelte Protein Entzweiung mit Translokation der Substrate durch den zentralen Kanal in der Hexameric ClpB verkn&uuml;pft ist, aber die Vorgeschichte der Translokation sind oft missverstanden. Die N-terminalen Dom&auml;nen bilden einen Ring um den Eingang zum Kanal und der aggregierten Bindung beitragen. Es wurde vermutet, dass der N-terminalen Dom&auml;ne-Mobilit&auml;t, die durch den unstrukturierten Linker verwaltet wird die Effizienz der aggregierten Reaktivierung kontrollieren k&ouml;nnte. Wir produzierten sieben Varianten des ClpB mit ver&auml;nderten Sequenz von der N-terminalen Linker. Um den Linker Konformations&auml;nderungen Flexibilit&auml;t zu erh&ouml;hen, eingef&uuml;gt wir bis zu vier Gly neben dem GG-Motiv. Um den Linker Flexibilit&auml;t zu verringern, haben wir gel&ouml;scht das GG-Motiv und wandelte sie in GP und PP. Wir fanden, dass keiner der Linker Modifikationen der basalen ClpB ATPase-Aktivit&auml;t oder seine F&auml;higkeit, Form Oligomere gehemmt. Jedoch zeigte die modifizierte Linker ClpB Varianten niedriger Reaktivierung Preise f&uuml;r aggregierte Glukose-6-phosphat-Dehydrogenase und Gl&uuml;hw&uuml;rmchen Luciferase und eine niedrigere Aggregat-Bindung-T&uuml;chtigkeit als wt ClpB. Wir schlie&szlig;en, dass der Linker nicht nur die N-terminale Dom&auml;ne schlie&szlig;t, unterst&uuml;tzt aber die Anstandsdame-Aktivit&auml;t von ClpB durch einen Beitrag zur Effizienz der aggregierten Bindung und Entzweiung. Dar&uuml;ber hinaus schlagen unsere Ergebnisse, dass selektive Druck auf die Linker-Sequenz entscheidend f&uuml;r die Aufrechterhaltung der optimalen Effizienz der aggregierten Reaktivierung durch ClpB. Proteine 2012 sein kann; &copy; 2012 Wiley Periodika, Inc.

Flexible Anbindung der N-terminalen Dom&auml;ne in ClpB moduliert Substrat Bindung und die Effizienz der aggregierten Reaktivierung.

ClpB riattiva proteine aggregate in collaborazione con DnaK, J. Il monomero ClpB contiene due domini nucleotide-binding (D1, D2), un dominio coiled-coil e un dominio N-terminale collegato al D1 con un linker non strutturato lungo 17-residui contenenti un motivo Gly-Gly. La disaggregazione della proteina ClpB-mediata &egrave; legata alla traslocazione di substrati attraverso il canale centrale in esamerica ClpB, ma gli eventi che precedono la traslocazione sono capiti male. I domini N-terminali formano un anello che circonda l&#39;ingresso al canale e contribuiscono all&#39;associazione aggregata. &Egrave; stato suggerito che mobilit&agrave; del dominio N-terminale che &egrave; mantenuto dal linker non strutturati potrebbe controllare l&#39;efficienza della riattivazione di aggregazione. Abbiamo prodotto sette varianti di ClpB con sequenza modificata del linker N-terminale. Per aumentare la flessibilit&agrave; conformazionale del linker, abbiamo inserito fino a quattro Gly accanto il motivo GG. Per diminuire la flessibilit&agrave; del linker, abbiamo eliminato il motivo GG e lo trasform&ograve; in GP e PP. Abbiamo trovato che nessuna delle modifiche del linker inibito l&#39;attivit&agrave; dell&#39;ATPasi ClpB basale o la capacit&agrave; di oligomeri di forma. Tuttavia, le varianti di ClpB modificato del linker ha mostrato tassi inferiori di riattivazione per aggregati glucosio-6-fosfato deidrogenasi e luciferasi firefly e una bassa efficienza di aggregazione-associazione che wt ClpB. Concludiamo che il linker non semplicemente collegare il dominio N-terminale, ma supporta l&#39;attivit&agrave; di chaperone di ClpB contribuendo all&#39;efficienza dell&#39;associazione aggregazione e disaggregazione. Inoltre, i nostri risultati suggeriscono che la pressione selettiva sulla sequenza linker potrebbe essere cruciale per mantenere l&#39;efficienza ottimale di riattivazione aggregazione di proteine ClpB. 2012; &copy; 2012 Wiley periodici, Inc.

Collegamento flessibile del dominio N-terminale in ClpB modula il legame del substrato e l&#39;efficienza complessiva riattivazione.

DnaK/j. &#x3068;&#x5354;&#x529B;&#x3057;&#x3066;&#x96C6;&#x7D04;&#x3055;&#x308C;&#x305F;&#x86CB;&#x767D;&#x8CEA; ClpB &#x518D;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x5316;&#x3057;&#x307E;&#x3059;&#x3002;ClpB &#x30E2;&#x30CE;&#x30DE;&#x30FC;&#x306B;&#x306F;&#x3001;2 &#x3064;&#x306E;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x7D50;&#x5408;&#x30C9;&#x30E1;&#x30A4;&#x30F3; (D1, D2)&#x3001;&#x30B3;&#x30A4;&#x30EB;&#x30C9; &#x30B3;&#x30A4;&#x30EB; &#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3001;&#x304A;&#x3088;&#x3073; D1 &#x3092; Gly Gly &#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x3092;&#x542B;&#x3080; 17 &#x6B8B;&#x6E23;&#x9577;&#x3044;&#x69CB;&#x9020;&#x5316;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x30EA;&#x30F3;&#x30AB;&#x30FC;&#x3092;&#x6DFB;&#x4ED8; N &#x672B;&#x7AEF;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x304C;&#x542B;&#x307E;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x86CB;&#x767D;&#x8CEA; ClpB &#x3092;&#x4ECB;&#x3057;&#x305F;&#x30CA;&#x30CE;&#x30C0;&#x30A4;&#x30E4;&#x30E2;&#x30F3;&#x30C9; &#xFF0D;c ClpB &#x3067;&#x4E2D;&#x592E;&#x30C1;&#x30E3;&#x30F3;&#x30CD;&#x30EB;&#x3092;&#x4ECB;&#x3057;&#x3066;&#x57FA;&#x677F;&#x306E;&#x8EE2;&#x6D41;&#x306B;&#x30EA;&#x30F3;&#x30AF;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x304C;&#x3001;&#x8EE2;&#x6D41;&#x3092;&#x524D;&#x306B;&#x30A4;&#x30D9;&#x30F3;&#x30C8;&#x304C;&#x3088;&#x304F;&#x308F;&#x304B;&#x3063;&#x3066;&#x3044;&#x307E;&#x305B;&#x3093;&#x3002;N &#x672B;&#x7AEF;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306F;&#x3001;&#x30C1;&#x30E3;&#x30CD;&#x30EB;&#x3078;&#x306E;&#x5165;&#x308A;&#x53E3;&#x3092;&#x53D6;&#x308A;&#x5DFB;&#x304F;&#x74B0;&#x3092;&#x5F62;&#x6210;&#x3057;&#x3001;&#x96C6;&#x8A08;&#x30D0;&#x30A4;&#x30F3;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x306B;&#x8CA2;&#x732E;&#x3002;&#x69CB;&#x9020;&#x5316;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x30EA;&#x30F3;&#x30AB;&#x30FC;&#x306B;&#x3088;&#x3063;&#x3066;&#x7DAD;&#x6301;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3001;N &#x672B;&#x7AEF;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x30E2;&#x30D3;&#x30EA;&#x30C6;&#x30A3;&#x96C6;&#x7D04;&#x518D;&#x6D3B;&#x6027;&#x5316;&#x306E;&#x52B9;&#x7387;&#x5236;&#x5FA1;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005; &#x306F; N &#x672B;&#x7AEF;&#x306E;&#x30EA;&#x30F3;&#x30AB;&#x30FC;&#x306E;&#x5909;&#x66F4;&#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3092; ClpB &#x306E; 7 &#x3064;&#x306E;&#x4E9C;&#x7A2E;&#x3092;&#x751F;&#x7523;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30EA;&#x30F3;&#x30AB;&#x30FC;&#x306E;&#x69CB;&#x9020;&#x306E;&#x67D4;&#x8EDF;&#x6027;&#x3092;&#x9AD8;&#x3081;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x6700;&#x5927; 4 &#x3064;&#x306E; Gly GG &#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x6A2A;&#x3092;&#x633F;&#x5165;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30EA;&#x30F3;&#x30AB;&#x30FC;&#x306E;&#x67D4;&#x8EDF;&#x6027;&#x3092;&#x6E1B;&#x3089;&#x3059;&#x306B;&#x306F;&#x3001;GG &#x306E;&#x30E2;&#x30C1;&#x30FC;&#x30D5;&#x3092;&#x524A;&#x9664;&#x3057;&#x3001;GP PP &#x306B;&#x5909;&#x63DB;&#x3057;&#x307E;&#x3059;&#x3002;&#x6211;&#x3005; &#x306F;&#x3001;&#x30EA;&#x30F3;&#x30AB;&#x30FC;&#x306E;&#x4FEE;&#x6B63;&#x306E;&#x3069;&#x308C;&#x3082;&#x57FA;&#x5E95; ClpB ATPase &#x6D3B;&#x6027;&#x307E;&#x305F;&#x306F;&#x305D;&#x306E;&#x30D5;&#x30A9;&#x30FC;&#x30E0; &#x30AA;&#x30EA;&#x30B4;&#x30DE;&#x30FC;&#x80FD;&#x529B;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x767A;&#x898B;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x5909;&#x66F4;&#x30EA;&#x30F3;&#x30AB;&#x30FC; ClpB &#x4E9C;&#x7A2E;&#x96C6;&#x8A08;&#x30B0;&#x30EB;&#x30B3;&#x30FC;&#x30B9;-6-&#x30EA;&#x30F3;&#x9178;&#x30C7;&#x30D2;&#x30C9;&#x30ED;&#x30B2;&#x30CA;&#x30FC;&#x30BC;&#x3068;&#x30DB;&#x30BF;&#x30EB;&#x30EB;&#x30B7;&#x30D5;&#x30A7;&#x30E9;&#x30FC;&#x30BC;&#x3068;&#x9AA8;&#x6750;&#x30D0;&#x30A4;&#x30F3;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x306E; wt ClpB &#x3088;&#x308A;&#x4F4E;&#x52B9;&#x7387;&#x306E;&#x305F;&#x3081;&#x3088;&#x308A;&#x4F4E;&#x3044;&#x518D;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x5316;&#x7387;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005; &#x306F;&#x3001;&#x30EA;&#x30F3;&#x30AB;&#x30FC;&#x306F;&#x5358;&#x306A;&#x308B; N &#x672B;&#x7AEF;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x3092;&#x63A5;&#x7D9A;&#x3057;&#x306A;&#x3044;&#x304C;&#x305D;&#x308C; ClpB &#x306E;&#x30B7;&#x30E3;&#x30DA;&#x30ED;&#x30F3;&#x6D3B;&#x6027;&#x96C6;&#x8A08;&#x30D0;&#x30A4;&#x30F3;&#x30C7;&#x30A3;&#x30F3;&#x30B0;&#x3068;&#x5206;&#x89E3;&#x306E;&#x52B9;&#x7387;&#x306B;&#x8CA2;&#x732E;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3092;&#x7DE0;&#x7D50;&#x3057;&#x307E;&#x3059;&#x3002;&#x307E;&#x305F;&#x3001;&#x6211;&#x3005; &#x306E;&#x7D50;&#x679C;&#x9078;&#x629E;&#x5727;&#x30EA;&#x30F3;&#x30AB;&#x30FC; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x86CB;&#x767D;&#x8CEA; ClpB&#x3002; 2012&#x5E74;&#x306B;&#x3088;&#x308B;&#x96C6;&#x8A08;&#x306E;&#x518D;&#x6D3B;&#x6027;&#x5316;&#x306E;&#x6700;&#x9069;&#x306A;&#x52B9;&#x7387;&#x3092;&#x7DAD;&#x6301;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x91CD;&#x8981;&#x306A;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x305F;;&copy; 2012&#x5E74;&#x30EF;&#x30A4;&#x30EA;&#x30FC;&#x5B9A;&#x671F;&#x520A;&#x884C;&#x7269; &#xFF08;&#x682A;&#xFF09;

&#x67D4;&#x8EDF;&#x306A;&#x63A5;&#x7D9A; ClpB &#x306E; N &#x672B;&#x7AEF;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x57FA;&#x8CEA;&#x7D50;&#x5408;&#x3068;&#x96C6;&#x8A08;&#x306E;&#x518D;&#x6D3B;&#x6027;&#x5316;&#x52B9;&#x7387;&#x3092;&#x5909;&#x8ABF;&#x3057;&#x307E;&#x3059;&#x3002;

ClpB DnaK/&#xC81C;&#xC774; &#xD611;&#xB825;&#xC5D0; &#xC9D1;&#xACC4; &#xB41C; &#xB2E8;&#xBC31;&#xC9C8;&#xC744; &#xB2E4;&#xC2DC; &#xD65C;&#xC131;&#xD654; ClpB &#xBAA8;&#xB178; &#xBA38;&#xC5D0;&#xB294; &#xB450; &#xAC1C;&#xC758; &#xB274;&#xD074;&#xB808;&#xC624;&#xD2F0;&#xB4DC; &#xBC14;&#xC778;&#xB529; &#xB3C4;&#xBA54;&#xC778; (D1, D2), &#xCF54;&#xC77C; &#xCF54;&#xC77C; &#xB3C4;&#xBA54;&#xC778;&#xACFC; Gly Gly &#xBAA8;&#xD2F0;&#xBE0C;&#xB97C; &#xD3EC;&#xD568; 17 &#xCC0C; &#xAEBC; &#xAE30; &#xAE34; &#xAD6C;&#xC870;&#xD654; &#xB418;&#xC9C0; &#xC54A;&#xC740; &#xB9C1;&#xCEE4; &#xD568;&#xAED8; d 1&#xC5D0; &#xC5F0;&#xACB0; &#xB41C; N &#xB9E8;&#xB05D; &#xB3C4;&#xBA54;&#xC778; &#xD3EC;&#xD568; &#xB418;&#xC5B4; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. ClpB &#xC911;&#xC7AC; &#xB2E8;&#xBC31;&#xC9C8; &#xC704;&#xC870;&#xD488;&#xC740; ClpB, hexameric&#xC5D0;&#xC11C; &#xC911;&#xC559; &#xCC44;&#xB110;&#xC744; &#xD1B5;&#xD574; &#xAE30;&#xD310;&#xC758; &#xC804; &#xC88C;&#xC5D0; &#xC5F0;&#xACB0; &#xB418;&#xC5B4; &#xC788;&#xC9C0;&#xB9CC; &#xC55E;&#xC5D0; &#xC804; &#xC774;&#xBCA4;&#xD2B8;&#xB294; &#xC11C; &#xD22C;&#xB974; &#xAC8C; &#xC774;&#xD574;. N &#xB9E8;&#xB05D; &#xB3C4;&#xBA54;&#xC778; &#xC218;&#xB85C; &#xC785;&#xAD6C;&#xB97C; &#xB458;&#xB7EC;&#xC2FC; &#xB9C1;&#xC744; &#xD615;&#xC131; &#xD558; &#xACE0; &#xC9D1;&#xACC4; &#xBC14;&#xC778;&#xB529;&#xC5D0; &#xAE30;&#xC5EC;. &#xAD6C;&#xC870;&#xD654; &#xB418;&#xC9C0; &#xC54A;&#xC740; &#xB9C1;&#xCEE4; &#xC720;&#xC9C0; &#xAD00;&#xB9AC; &#xD558;&#xB294; N &#xB9E8;&#xB05D; &#xB3C4;&#xBA54;&#xC778; &#xC774;&#xB3D9;&#xC131; &#xC9D1;&#xACC4; &#xC7AC; &#xD65C;&#xC131;&#xD654;&#xC758; &#xD6A8;&#xC728;&#xC131;&#xC744; &#xC81C;&#xC5B4;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC81C;&#xC548; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC; N &#xD130;&#xBBF8;&#xB110; &#xB9C1;&#xCEE4;&#xC758; &#xC218;&#xC815;&#xB41C; &#xB41C; &#xC2DC;&#xD000;&#xC2A4; Clpb&#xC758; 7 &#xBCC0;&#xC885; &#xC0DD;&#xC0B0;. &#xB9C1;&#xCEE4;&#xC758; &#xAD6C;&#xC870;&#xC801; &#xC720;&#xC5F0;&#xC131; &#xC99D;&#xAC00;, &#xCD5C;&#xB300; 4 &#xAC1C;&#xC758; Gly GG &#xBAA8;&#xD2F0;&#xD504; &#xC606; &#xC0BD;&#xC785; &#xD558;&#xC2ED;&#xC2DC;&#xC624;. &#xB9C1;&#xCEE4;&#xC758; &#xC720;&#xC5F0;&#xC131; &#xAC10;&#xC18C;, GG &#xBAA8;&#xD2F0;&#xD504;&#xB97C; &#xC0AD;&#xC81C; &#xD558; &#xACE0; GP &#xBC0F; PP&#xB85C; &#xBCC0;&#xD658;. &#xB9C1;&#xCEE4; &#xC218;&#xC815; &#xC911; &#xAE30;&#xC800; ClpB ATPase &#xD65C;&#xB3D9; &#xB610;&#xB294; &#xC591;&#xC2DD; &#xC62C;&#xB9AC;&#xACE0; &#xD558;&#xB294; &#xADF8;&#xAC83;&#xC758; &#xAE30;&#xB2A5; &#xC800;&#xD574;&#xB97C; &#xCC3E;&#xC558;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC218;&#xC815;&#xB41C; &#xB9C1;&#xCEE4; ClpB &#xBCC0;&#xC885; &#xC9D1;&#xACC4; &#xB41C; &#xD3EC;&#xB3C4; &#xB2F9; 6 &#xC778;&#xC0B0; &#xC5FC; dehydrogenase&#xC640; &#xAC1C; &#xB625; &#xBC8C;&#xB808; &#xB8E8;&#xC2DC;&#xD398;&#xB77C;&#xC544;&#xC81C; wt ClpB &#xBCF4;&#xB2E4; &#xB0AE;&#xC740; &#xC9D1;&#xACC4; &#xBC14;&#xC778;&#xB529; &#xD6A8;&#xC728;&#xC131;&#xC5D0; &#xB300; &#xD55C; &#xB354; &#xB0AE;&#xC740; &#xB2E4;&#xC2DC; &#xD65C;&#xC131;&#xD654; &#xBE44;&#xC728;&#xC744; &#xBCF4;&#xC600;&#xB2E4;. &#xC6B0;&#xB9AC; &#xACB0;&#xB860; &#xB9C1;&#xCEE4;&#xB294; &#xB2E8;&#xC9C0; N &#xB9E8;&#xB05D; &#xB3C4;&#xBA54;&#xC778;&#xC744; &#xC5F0;&#xACB0; &#xD558;&#xC9C0; &#xC54A;&#xB294;&#xB2E4; &#xD558;&#xC9C0;&#xB9CC; &#xC9D1;&#xACC4; &#xBC14;&#xC778;&#xB529; &#xBC0F; &#xC704;&#xC870;&#xD488;&#xC758; &#xD6A8;&#xC728;&#xC131;&#xC5D0; &#xAE30;&#xC5EC; &#xD558; &#xC5EC; Clpb&#xC758; &#xBCF4;&#xD638;&#xC790; &#xD65C;&#xB3D9;&#xC744; &#xC9C0;&#xC6D0; &#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; &#xB9C1;&#xCEE4; &#xC2DC;&#xD000;&#xC2A4;&#xC5D0; &#xC120;&#xD0DD;&#xC801; &#xC555;&#xB825; ClpB. 2012; &#xB2E8;&#xBC31;&#xC9C8;&#xC5D0; &#xC758;&#xD574; &#xC9D1;&#xACC4; &#xC7AC; &#xD65C;&#xC131;&#xD654;&#xC758; &#xCD5C;&#xC801;&#xC758; &#xD6A8;&#xC728;&#xC744; &#xC720;&#xC9C0; &#xD558;&#xAE30; &#xC704;&#xD55C; &#xC911;&#xC694; &#xD55C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAD8C;&#xD574; &#xB4DC;&#xB9BD;&#xB2C8;&#xB2E4;. &copy; 2012&#xC640; &#xC77C;&#xB9AC; &#xC815;&#xAE30; &#xAC04;&#xD589;&#xBB3C; &#xC8FC;&#xC2DD; &#xD68C;&#xC0AC;

Clpb&#xC5D0;&#xC11C; N &#xB9E8;&#xB05D; &#xB3C4;&#xBA54;&#xC778;&#xC758; &#xC720;&#xC5F0;&#xD55C; &#xC5F0;&#xACB0; &#xAE30;&#xD310; &#xBC14;&#xC778;&#xB529; &#xBC0F; &#xC9D1;&#xACC4; &#xC7AC; &#xD6A8;&#xC728; &#xBCC0;&#xC870;.

ClpB reactiva prote&iacute;nas agregadas en cooperaci&oacute;n con DnaK/J. El mon&oacute;mero de ClpB contiene dos dominios de uni&oacute;n a nucle&oacute;tido (D1, D2), un dominio coiled-coil y un dominio N-terminal conectado a D1 con un enlazador no estructurado 17-residuo-largo que contiene un motivo de Gly-Gly. La desagregaci&oacute;n de la prote&iacute;na ClpB-mediada est&aacute; vinculada a la translocaci&oacute;n de los sustratos a trav&eacute;s del canal central en el hex&aacute;mero ClpB, pero los acontecimientos anteriores a la translocaci&oacute;n se entienden mal. Los dominios N-terminal forman un anillo que rodea la entrada al canal y contribuyan a la fijaci&oacute;n total. Se sugiri&oacute; que movilidad de dominio N-terminal que se mantiene por el enlazador no estructurado puede controlar la eficiencia de la reactivaci&oacute;n total. Produjimos siete variantes de ClpB con secuencia modificada del vinculador de N-terminal. Para aumentar la flexibilidad conformacional del vinculador, hemos insertado hasta cuatro Gly junto al motivo GG. Para disminuir la flexibilidad de la m&aacute;quina para hacer chorizos, hemos eliminado el motivo GG y hab&iacute;a convertido en GP y PP. Encontramos que ninguna de las modificaciones de vinculador inhibe la actividad de la ATPasa ClpB basal o su capacidad de olig&oacute;meros de forma. Sin embargo, las variantes de ClpB modificado vinculador mostraron tasas de reactivaci&oacute;n para agregados de glucosa-6-fosfato deshidrogenasa y luciferasa de la luci&eacute;rnaga y una menor eficacia de agregado vinculante que wt ClpB. Concluimos que el enlazador no simplemente conecta el dominio N-terminal, pero es compatible con la actividad de acompa&ntilde;ante de ClpB contribuyendo a la eficiencia del enlace agregado y desagregaci&oacute;n. Por otra parte, nuestros resultados sugieren que la presi&oacute;n selectiva en la secuencia de vinculador puede ser crucial para mantener la eficacia &oacute;ptima de reactivaci&oacute;n agregada por ClpB. prote&iacute;nas 2012; &copy; 2012 Wiley peri&oacute;dicos, Inc.

Conexi&oacute;n flexible del dominio N-terminal en ClpB modula el atascamiento del substrato y la eficacia de la reactivaci&oacute;n total.

Mechanisms involved with degeneration of motor neurons in amyotrophic lateral sclerosis (ALS; Lou Gehrig's Disease) are poorly understood, but genetically inherited forms, comprising ~10% of the cases, are potentially informative. Recent observations that several inherited forms of ALS involve the RNA binding proteins TDP43 and FUS raise the question as to whether RNA metabolism is generally disturbed in ALS. Here we conduct whole transcriptome profiling of motor neurons from a mouse strain, transgenic for a mutant human SOD1 (G85R SOD1-YFP), that develops symptoms of ALS and paralyzes at 5-6 months of age. Motor neuron cell bodies were laser microdissected from spinal cords at 3 months of age, a time when animals were presymptomatic but showed aggregation of the mutant protein in many lower motor neuron cell bodies and manifested extensive neuromuscular junction morphologic disturbance in their lower extremities. We observed only a small number of transcripts with altered expression levels or splicing in the G85R transgenic compared to age-matched animals of a wild-type SOD1 transgenic strain. Our results indicate that a major disturbance of polyadenylated RNA metabolism does not occur in motor neurons of mutant SOD1 mice, suggesting that the toxicity of the mutant protein lies at the level of translational or post-translational effects.

RNA-Seq profiling of spinal cord motor neurons from a presymptomatic SOD1 ALS mouse.

Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.

Molecular chaperone Hsp110 rescues a vesicle transport defect produced by an ALS-associated mutant SOD1 protein in squid axoplasm.

Mutant humaine Cu/Zn superoxyde dismutase 1 (SOD1) est associ&eacute;e &agrave; la toxicit&eacute; du neurone moteur et de la mort dans une forme h&eacute;r&eacute;ditaire de la scl&eacute;rose lat&eacute;rale amyotrophique (SLA ; Maladie de Lou Gehrig). Un des aspects de la toxicit&eacute; dans les neurones moteurs implique diminution transport axonal rapide, observ&eacute; aussi bien chez les souris transg&eacute;niques et, plus r&eacute;cemment, dans l&#39;axoplasme isol&eacute; des axones g&eacute;ants de calmar. Ce dernier effet semble &ecirc;tre directement m&eacute;di&eacute;e par la SOD1 mal repli&eacute;es, dont l&#39;ajout active la phosphorylation de p38 MAPK et la phosphorylation de la kin&eacute;sine. Ici, nous observons que plusieurs &Eacute;tats oligom&egrave;res d&#39;une prot&eacute;ine de fusion, comprenant les associ&eacute;s &agrave; ALS humaine G85R SOD1 rejoint avec la prot&eacute;ine fluorescente jaune (G85R SOD1YFP), qui produit des ALS chez des souris transg&eacute;niques, inhibent le transport ant&eacute;rograde lorsque ajout&eacute; &agrave; axoplasme de calmar. L&#39;inhibition a &eacute;t&eacute; bloqu&eacute;e par une kinase de signal-r&eacute;gulation de l&#39;apoptose 1 (ASK1 ; Inhibiteur de la MAPKKK) et par un inhibiteur de la p38, indiquant le d&eacute;faut de transport est m&eacute;di&eacute;e par la cascade MAPK. En plus des incubations, nous avons observ&eacute; que l&#39;addition du chaperon mol&eacute;culaire chez les mammif&egrave;res Hsc70, abondamment associ&eacute; &agrave; G85R SOD1YFP dans la moelle &eacute;pini&egrave;re de souris transg&eacute;niques, exerc&eacute;e correction partielle de la d&eacute;fectuosit&eacute; de transport, associ&eacute;e &agrave; une phosphorylation r&eacute;duite de p38. Plus frappant, l&#39;ajout du chaperon mol&eacute;culaire Hsp110, dans une concentration sous-stoechiom&eacute;triques de prot&eacute;ine SOD1 mutant sauv&eacute; compl&egrave;tement tant le d&eacute;faut de transport et de la phosphorylation de p38. Hsp110 a &eacute;t&eacute; d&eacute;montr&eacute;e &agrave; agir comme un facteur d&#39;&eacute;change de nucl&eacute;otide pour Hsc70 et, plus r&eacute;cemment, pour &ecirc;tre en mesure de coop&eacute;rer avec elle &agrave; la m&eacute;diation de d&eacute;sagr&eacute;gation de prot&eacute;ines. Nous croyons qu&#39;il peut coop&eacute;rer avec calmars endog&egrave;ne Hsp (c) 70 &agrave; la m&eacute;diation obligatoire et/ou de d&eacute;sagr&eacute;gation des prot&eacute;ine SOD1 mutante, abrogeant la toxicit&eacute;.

Chaperon mol&eacute;culaire Hsp110 sauve un d&eacute;faut de transport v&eacute;siculaire produit par une prot&eacute;ine de SOD1 mutante ALS-associ&eacute;s dans l&#39;axoplasme de calmar.

Mutante umano Cu/Zn superossido dismutasi 1 (SOD1) sono associato a tossicit&agrave; del neurone del motore e la morte in una forma ereditaria di sclerosi laterale amiotrofica (SLA; Malattia di Lou Gehrig). Uno degli aspetti della tossicit&agrave; nei motoneuroni comporta diminuita trasporto assonale rapido, osservato sia in topi transgenici e, pi&ugrave; recentemente, nell&#39;assoplasma isolato da assoni giganti di calamari. L&#39;effetto di quest&#39;ultimo sembra essere mediata direttamente da malpiegata SOD1, cui inoltre attiva la fosforilazione di p38 MAPK e fosforilazione di chinesina. Qui, osserviamo che diversi stati oligomerici differenti di una proteina di fusione, comprendente ALS-collegato umano G85R SOD1 si un&igrave; con la proteina fluorescente gialla (G85R SOD1YFP), che produce ALS in topi transgenici, inibiscono trasporto anterograda quando aggiunto al assoplasma calamari. Inibizione &egrave; stato bloccato da un&#39;apoptosi segnale di regolazione della chinasi 1 (ASK1; Inibitore MAPKKK) e di un inibitore della p38, indicando il difetto di trasporto &egrave; mediata attraverso la cascata MAPK. In ulteriore incubazione, abbiamo osservato che aggiunta del chaperone molecolare dei mammiferi Hsc70, abbondantemente associato G85R SOD1YFP nel midollo spinale di topi transgenici, esercitata parziale correzione del difetto di trasporto, associato a diminuita fosforilazione di p38. Pi&ugrave; colpisce, l&#39;aggiunta del chaperone molecolare Hsp110, in una concentrazione substechiometrica il mutante SOD1 proteina, completamente salvato la fosforilazione di p38 e il difetto di trasporto. Hsp110 ha dimostrato di agire come un fattore di scambio di nucleotide per Hsc70 e, pi&ugrave; recentemente, per essere in grado di collaborare con essa per mediare la disgregazione proteica. Si ipotizzano che essa pu&ograve; collaborare con calamari endogeni Hsp (c) 70 a mediare l&#39;associazione e/o disgregazione della proteina mutante SOD1, abrogare la tossicit&agrave;.

Chaperone molecolare Hsp110 salv&ograve; un difetto di trasporto vescicola prodotto da una proteina SOD1 mutante ALS-associati nell&#39;assoplasma calamari.

Maria Nagy

Department of Genetics

Yale School of Medicine

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Maria Nagy

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Genetics

Yale School of Medicine

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Department of Genetics