in-vitro-aggregation-assays-using-hyperphosphorylated-tau-protein

<em>In Vitro</em> Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Michigan State University

We have modified the yeast two-hybrid system to enable the detection of protein-protein interactions that require a specific post-translational modification, using the acetylation of histones and the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II as test modifications. In this tethered catalysis assay, constitutive modification of the protein to be screened for interactions is achieved by fusing it to its cognate modifying enzyme, with the physical linkage resulting in efficient catalysis. This catalysis maintains substrate modification even in the presence of antagonizing enzyme activities. A catalytically inactive mutant of the enzyme is fused to the substrate as a control such that the modification does not occur; this construct enables the rapid identification of modification-independent interactions. We identified proteins with links to chromatin functions that interact with acetylated histones, and proteins that participate in RNA polymerase II functions and in CTD phosphorylation regulation that interact preferentially with the phosphorylated CTD.

A tethered catalysis, two-hybrid system to identify protein-protein interactions requiring post-translational modifications.

Post-translational modification of proteins is a dynamic way of generating new protein-protein interaction interfaces that are critical for signaling networks in diverse cellular functions. Purified recombinant proteins frequently lack these signature modifications. Using the tumor suppressor p53 as the model protein, we present here a tethered catalysis approach for the production of acetylated p53 in vivo. P53 is a major tumor suppressor protein that protects the cell from various oncogenic stresses. Upon DNA damage, p53 is stabilized and activated by a plethora of post-translational modifications, including acetylation. Here, we show that constitutively acetylated p53 can be expressed and purified from both yeast and Escherichia coli. This method is highly suitable for studying protein-protein interactions in the conventional yeast two-hybrid screen that requires a constitutively acetylated state of p53. Furthermore, effective production and purification of acetylated p53 from E. coli supports future biochemical and structural characterization. The method described in this work can be applied to other proteins and modifications, and thus has widespread use in the fields of signal transduction and proteomic research.

Production of constitutively acetylated recombinant p53 from yeast and Escherichia coli by tethered catalysis.

Gcn5 protein is a prototypical histone acetyltransferase that controls transcription of multiple yeast genes. To identify molecular functions that act downstream of or in parallel with Gcn5 protein, we screened for suppressors that rescue the transcriptional defects of HIS3 caused by a catalytically inactive mutant Gcn5, the E173H mutant. One bypass of Gcn5 requirement gene (BGR) suppressor was mapped to the REG1 locus that encodes a semidominant mutant truncated after amino acid 740. Reg1(1-740) protein does not rescue the complete knockout of GCN5, nor does it suppress other gcn5- defects, including the inability to utilize nonglucose carbon sources. Reg1(1-740) enhances HIS3 transcription while HIS3 promoter remains hypoacetylated, indicating that a noncatalytic function of Gcn5 is targeted by this suppressor protein. Reg1 protein is a major regulator of Snf1 kinase that phosphorylates Ser10 of histone H3. However, whereas Snf1 protein is important for HIS3 expression, replacing Ser10 of H3 with alanine or glutamate neither attenuates nor augments the BGR phenotypes. Overproduction of Snf1 protein also preferentially rescues the E173H allele. Biochemically, both Snf1 and Reg1(1-740) proteins copurify with Gcn5 protein. Snf1 can phosphorylate recombinant Gcn5 in vitro. Together, these data suggest that Reg1 and Snf1 proteins function in an H3 phosphorylation-independent pathway that also involves a noncatalytic role played by Gcn5 protein.

Histone H3 Ser10 phosphorylation-independent function of Snf1 and Reg1 proteins rescues a gcn5- mutant in HIS3 expression.

Methylation of Lys-9 of histone H3 has been associated with repression of transcription. G9a is a histone H3 Lys-9 methyltransferase localized in euchromatin and acts as a corepressor for specific transcription factors. Here we demonstrate that G9a also functions as a coactivator for nuclear receptors, cooperating synergistically with nuclear receptor coactivators glucocorticoid receptor interacting protein 1, coactivator-associated arginine methyltransferase 1 (CARM1), and p300 in transient transfection assays. This synergy depends strongly on the arginine-specific protein methyltransferase activity of CARM1 but does not absolutely require the enzymatic activity of G9a and is specific to CARM1 and G9a among various protein methyltransferases. Reduction of endogenous G9a diminished hormonal activation of an endogenous target gene by the androgen receptor, and G9a associated with regulatory regions of this same gene. G9a fused to Gal4 DNA binding domain can repress transcription in a lysine methyltransferase-dependent manner; however, the histone modifications associated with transcriptional activation can inhibit the methyltransferase activity of G9a. These findings suggest a link between histone arginine and lysine methylation and a mechanism for controlling whether G9a functions as a corepressor or coactivator.

Histone H3 lysine 9 methyltransferase G9a is a transcriptional coactivator for nuclear receptors.

Histone acetylation and methylation are linked to a variety of nuclear activities, most notably transcriptional regulation. Both synergistic and antagonistic relationships between these two modifications have been reported in different systems. Here we show that the budding yeast histone H4 arginine 3 (R3) methyltransferase Hmt1p binds acetylated histones H3 and H4, and importantly, that acetylated H4 is a significantly better methylation substrate for Hmt1p. Kinetic studies show that acetylation at any of the four acetylatable lysine residues of histone H4 results in more efficient methylation. Among the four, K8 acetylation imposes the strongest effect on reducing K(M), consistent with the observed acetylation-stimulated interaction. In vivo, hmt1Delta cells rescue the transcriptional defect caused by GCN5 deletion, indicating that one of the functions of Gcn5p is to neutralize the negative effect of Hmt1p. Mutating either K8 or R3 to alanine causes similar growth defects in selective histone and gcn5 mutant background, suggesting that these two residues function in the same pathway for optimal vegetative growth. Together, these results reveal a functional connection between histone acetylation, methylation, and two of the responsible enzymes, Gcn5p and Hmt1p.

Functional connection between histone acetyltransferase Gcn5p and methyltransferase Hmt1p.

The budding yeast Gcn5p is a prototypic histone acetyltransferase controlling transcription of diverse genes. Here we show that Gcn5p is itself regulated by Snf1p and Spt3p. Snf1p likely controls Gcn5p via direct interaction. Mutating four residues in the Gcn5p catalytic domain, T203, S204, T211, and Y212 (TSTY), phenocopies snf1 null cells, including Gcn5p hypophosphorylation, hypoacetylation at the HIS3 promoter, and transcriptional defects of the HIS3 gene. However, overexpressing Snf1p suppresses the above phenotypes associated with the phosphodeficient TSTY mutant, suggesting that it is the interaction with Snf1p important for Gcn5p to activate HIS3. A likely mechanism by which Snf1p potentiates Gcn5p function is to antagonize Spt3p, because the HIS3 expression defects caused by snf1 knockout, or by the TSTY gcn5 mutations, can be suppressed by deleting SPT3. In vitro, Spt3p binds Gcn5p, but the interaction is drastically enhanced by the TSTY mutations, indicating that a stabilized Spt3p-Gcn5p interaction may be an underlying cause for the aforementioned HIS3 transcriptional defects. These results suggest that Gcn5p is a target regulated by the competing actions of Snf1p and Spt3p.

Snf1p regulates Gcn5p transcriptional activity by antagonizing Spt3p.

It has been firmly established that many interphase nuclear functions, including transcriptional regulation, are regulated by chromatin and histones. How mitotic progression and quality control might be influenced by histones is less well characterized. We show that histone H3 plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis. Prior to anaphase, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. Lack of tension due to erroneous attachment activates the spindle assembly checkpoint, which corrects the mistakes and ensures segregation fidelity. A histone H3 mutation impairs the ability of yeast cells to activate the checkpoint in a tensionless crisis, leading to missegregation and aneuploidy. The defects in tension sensing result directly from an attenuated H3-Sgo1p interaction essential for pericentric recruitment of Sgo1p. Reinstating the pericentric enrichment of Sgo1p alleviates the mitotic defects. Histone H3, and hence the chromatin, is thus a key factor transmitting the tension status to the spindle assembly checkpoint.

Histone h3 exerts a key function in mitotic checkpoint control.

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Like many microalgae, Chlamydomonas reinhardtii forms lipid droplets rich in triacylglycerols when nutrient deprived. To begin studying the mechanisms underlying this process, nitrogen (N) deprivation was used to induce triacylglycerol accumulation and changes in developmental programs such as gametogenesis. Comparative global analysis of transcripts under induced and noninduced conditions was applied as a first approach to studying molecular changes that promote or accompany triacylglycerol accumulation in cells encountering a new nutrient environment. Towards this goal, high-throughput sequencing technology was employed to generate large numbers of expressed sequence tags of eight biologically independent libraries, four for each condition, N replete and N deprived, allowing a statistically sound comparison of expression levels under the two tested conditions. As expected, N deprivation activated a subset of control genes involved in gametogenesis while down-regulating protein biosynthesis. Genes for components of photosynthesis were also down-regulated, with the exception of the PSBS gene. N deprivation led to a marked redirection of metabolism: the primary carbon source, acetate, was no longer converted to cell building blocks by the glyoxylate cycle and gluconeogenesis but funneled directly into fatty acid biosynthesis. Additional fatty acids may be produced by membrane remodeling, a process that is suggested by the changes observed in transcript abundance of putative lipase genes. Inferences on metabolism based on transcriptional analysis are indirect, but biochemical experiments supported some of these deductions. The data provided here represent a rich source for the exploration of the mechanism of oil accumulation in microalgae.

Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism.

Comme de nombreux micro-algues, Chlamydomonas reinhardtii forme de gouttelettes lipidiques riches en triglyc&#xE9;rides lorsque des &#xE9;l&#xE9;ments nutritifs priv&#xE9;s. Pour commencer &#xE0; &#xE9;tudier les m&#xE9;canismes sous-jacents de ce processus, de l&#x26;#39;azote (N) de d&#xE9;favorisation a &#xE9;t&#xE9; utilis&#xE9; pour induire l&#x26;#39;accumulation de triglyc&#xE9;rides et les changements dans les programmes de d&#xE9;veloppement tels que la gam&#xE9;togen&#xE8;se. L&#x26;#39;analyse comparative globale des transcriptions dans des conditions non induites induits et a &#xE9;t&#xE9; appliqu&#xE9; comme une premi&#xE8;re approche de l&#x26;#39;&#xE9;tude des changements mol&#xE9;culaires qui favorisent l&#x26;#39;accumulation de triglyc&#xE9;rides ou d&#x26;#39;accompagner dans les cellules qui rencontrent un environnement nouveau nutriment. Pour atteindre cet objectif, la technologie de s&#xE9;quen&#xE7;age &#xE0; haut d&#xE9;bit a &#xE9;t&#xE9; utilis&#xE9; pour g&#xE9;n&#xE9;rer un grand nombre de marqueurs de s&#xE9;quence exprim&#xE9;e de huit biblioth&#xE8;ques biologiquement ind&#xE9;pendants, quatre pour chaque condition, N et N regorge priv&#xE9;, ce qui permet une comparaison statistiquement valable des niveaux d&#x26;#39;expression dans les deux conditions test&#xE9;es. Comme pr&#xE9;vu, la privation N activ&#xE9; un sous-ensemble de g&#xE8;nes impliqu&#xE9;s dans la gam&#xE9;togen&#xE8;se de contr&#xF4;le tout en bas-r&#xE9;gulation de la biosynth&#xE8;se des prot&#xE9;ines. Les g&#xE8;nes pour les composants de la photosynth&#xE8;se ont &#xE9;galement &#xE9;t&#xE9; r&#xE9;gul&#xE9;e &#xE0; la baisse, &#xE0; l&#x26;#39;exception du g&#xE8;ne PSBS. La privation N conduit &#xE0; une r&#xE9;orientation marqu&#xE9;e du m&#xE9;tabolisme: la source primaire de carbone, l&#x26;#39;ac&#xE9;tate, a &#xE9;t&#xE9; plus convertis en blocs de construction des cellules par le cycle du glyoxylate et de la n&#xE9;oglucogen&#xE8;se, mais canalis&#xE9; directement dans la biosynth&#xE8;se des acides gras. D&#x26;#39;autres acides gras peuvent &#xEA;tre produites par remodelage des membranes, un processus qui est sugg&#xE9;r&#xE9; par les changements observ&#xE9;s dans l&#x26;#39;abondance transcription de g&#xE8;nes putatifs lipase. Inf&#xE9;rences sur le m&#xE9;tabolisme bas&#xE9;es sur l&#x26;#39;analyse de la transcription sont indirectes, mais des exp&#xE9;riences biochimiques pris en charge certaines de ces d&#xE9;ductions. Les donn&#xE9;es fournies ici repr&#xE9;sentent une source riche pour l&#x26;#39;exploration du m&#xE9;canisme de l&#x26;#39;accumulation d&#x26;#39;huile dans les microalgues.

Changements dans l&#x26;#39;abondance Transcription chez Chlamydomonas reinhardtii privation d&#x26;#39;azote apr&#xE8;s Pr&#xE9;dire Diversion du m&#xE9;tabolisme

Wie viele Mikroalgen bildet Chlamydomonas Reinhardtii Lipid-Tr&ouml;pfchen, die reich an Triacylglycerol, wenn N&auml;hrstoff entzogen. Zun&auml;chst lernen die Mechanismen dieses Prozesses wurde Stickstoff (N) Entbehrung nutzten, um Triacylglycerol Anh&auml;ufung und &Auml;nderungen in entwicklungspolitischen Programmen wie Gametogenese hervorzurufen. Vergleichende globale Analyse der Transkripte induzierten und noninduced Bedingungen wurde angewendet, als ein erster Ansatz zum Studium der molekularer Ver&auml;nderungen, die zu f&ouml;rdern oder Triacylglycerol Akkumulation in Zellen, die Begegnung mit einer neuen N&auml;hrstoffen-Umgebung zu begleiten. Zur Erreichung dieses Ziels Hochdurchsatz-Sequenzierungstechnologie wurde eingesetzt, um gro&szlig;e Anzahl von exprimierten Sequenz Datentr&auml;gern acht biologisch unabh&auml;ngige Bibliotheken, vier f&uuml;r jede Bedingung, N vollgestopft zu generieren und N beraubt, erm&ouml;glicht einen statistisch einwandfreier Vergleich der Ausdruck unter den beiden getesteten Bedingungen. Wie erwartet aktiviert N Entbehrungen eine Teilmenge der Kontrolle Gene Gametogenese beim Down-Regulierung der Proteinbiosynthese beteiligt. Genen f&uuml;r Komponenten der Photosynthese wurden auch unten-geregelt, mit Ausnahme der Ansiedlung-gen. N-Entzug f&uuml;hrte zu einer deutlichen Umleitung des Stoffwechsels: die prim&auml;re Kohlenstoffquelle, Acetat, war nicht mehr durch den Glyoxylat-Zyklus und Gluconeogenese in Zelle Bausteine umgewandelt, aber kanalisiert direkt in Fetts&auml;ure-Biosynthese. Zus&auml;tzliche Fetts&auml;uren kann hergestellt werden, mit Membran-Umbau, einen Prozess, der durch die Ver&auml;nderungen im Transkript F&uuml;lle von vermeintlichen Lipase Gene beobachtet suggeriert wird. R&uuml;ckschl&uuml;sse auf den Stoffwechsel, die transkriptionelle Analyse sind indirekte, doch biochemische Experimente unterst&uuml;tzt einige der diese Abz&uuml;ge. Die hier bereitgestellten Daten stellen eine reiche Quelle f&uuml;r die Erforschung des Mechanismus der &Ouml;l-Akkumulation in Mikroalgen.

&Auml;nderungen im Transkript &Uuml;berfluss in Chlamydomonas Reinhardtii nach Stickstoff Entbehrung Voraussagen Abzweigung des Stoffwechsels.

Come molte microalghe, Chlamydomonas reinhardtii forma goccioline di lipidi ricche in triacylglycerols quando privi di nutrienti. Per iniziare a studiare i meccanismi alla base di questo processo, privazione di azoto (N) &egrave; stata utilizzata per indurre l&#39;accumulo di triacilglicerolo e modifiche nei programmi di sviluppo come gametogenesis. Analisi comparativa globale delle trascrizioni in condizioni indotte e noninduced &egrave; stato applicato come un primo approccio per studiare i cambiamenti molecolari che promuovono o accompagnano triacilglicerolo accumulo nelle cellule incontrando un nuovo ambiente di nutrienti. Verso questo obiettivo, tecnologie high-throughput sequencing fu impiegata per generare un gran numero di tag expressed sequence di otto biblioteche biologicamente indipendenti, quattro per ogni condizione, zeppo di N e N privato, consentendo un confronto statisticamente suono dei livelli di espressione sotto le due condizioni testate. Come previsto, la privazione N attivato un sottoinsieme di controllo geni coinvolti in gametogenesis mentre gi&ugrave;-regolazione della biosintesi della proteina. Geni per componenti di fotosintesi sono stati anche down-regolato, ad eccezione del gene PSBS. Privazione N ha portato a una marcata redirezione del metabolismo: la fonte primaria del carbonio, acetato, non era pi&ugrave; convertita in blocchetti di costruzione delle cellule dalla gluconeogenesi e ciclo del gliossilato ma incanalata direttamente nella biosintesi degli acidi grassi. Altri acidi grassi pu&ograve; essere prodotta da membrana, un processo che &egrave; suggerito dai cambiamenti osservati in abbondanza di trascrizione dei geni putativi lipasi di rimodellamento. Inferenze sul metabolismo basato su analisi trascrizionale sono indirette, ma esperimenti biochimici sostegno alcune di queste detrazioni. I dati qui forniti rappresentano una ricca fonte per l&#39;esplorazione del meccanismo di accumulo di olio di microalghe.

Cambiamenti nell&#39;abbondanza di trascrizione in Chlamydomonas reinhardtii seguendo la privazione dell&#39;azoto predicono deviazione del metabolismo.

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4C;&#x3067;&#x304D;&#x308B;&#x3002;&#x8EE2;&#x5199;&#x89E3;&#x6790;&#x306B;&#x57FA;&#x3065;&#x304F;&#x4EE3;&#x8B1D;&#x3078;&#x306E;&#x63A8;&#x8AD6;&#x306F;&#x3001;&#x9593;&#x63A5;&#x7684;&#x3067;&#x3059;&#x304C;&#x3001;&#x751F;&#x5316;&#x5B66;&#x7684;&#x306A;&#x5B9F;&#x9A13;&#x3067;&#x306F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x63A7;&#x9664;&#x306E;&#x4E00;&#x90E8;&#x3092;&#x30B5;&#x30DD;&#x30FC;&#x30C8;&#x3057;&#x3066;&#x3044;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x3053;&#x3067;&#x63D0;&#x4F9B;&#x3055;&#x308C;&#x308B;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x5FAE;&#x7D30;&#x85FB;&#x985E;&#x306E;&#x6CB9;&#x306E;&#x84C4;&#x7A4D;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306E;&#x63A2;&#x6C42;&#x306E;&#x305F;&#x3081;&#x306E;&#x8C4A;&#x5BCC;&#x306A;&#x30BD;&#x30FC;&#x30B9;&#x3092;&#x8868;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30AF;&#x30E9;&#x30DF;&#x30C9;&#x30E2;&#x30CA;&#x30B9;&#x306B;&#x304A;&#x3051;&#x308B;&#x8EE2;&#x5199;&#x7523;&#x7269;&#x91CF;&#x306E;&#x5909;&#x5316;&#x306F;&#x4EE5;&#x4E0B;&#x306E;&#x7A92;&#x7D20;&#x6B20;&#x4E4F;&#x306F;&#x4EE3;&#x8B1D;&#x306E;&#x8EE2;&#x63DB;&#x3092;&#x4E88;&#x6E2C;&#x30AF;&#x30E9;&#x30DF;&#x30C9;&#x30E2;&#x30CA;&#x30B9;

&#xB9CE;&#xC740; &#xBBF8;&#xC138; &#xCC98;&#xB7FC; Chlamydomonas reinhardtii &#xC601;&#xC591;&#xBD84;&#xC744; &#xBC15;&#xD0C8; &#xD558;&#xB294; &#xACBD;&#xC6B0; triacylglycerols&#xC774; &#xD48D;&#xBD80;&#xD55C; &#xC9C0;&#xC9C8; &#xBC29;&#xC6B8;&#xC744; &#xD615;&#xC131; &#xD55C;&#xB2E4;. &#xC774; &#xACFC;&#xC815;&#xC744; &#xAE30;&#xBCF8; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xACF5;&#xBD80; &#xD558;&#xB824;&#xBA74; &#xC9C8;&#xC18C; (N) &#xBD80;&#xC871; triacylglycerol &#xCD95;&#xC801; &#xBC0F; gametogenesis &#xB4F1;&#xC758; &#xAC1C;&#xBC1C; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC758; &#xBCC0;&#xD654;&#xB97C; &#xC720;&#xB3C4; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC0AC;&#xC6A9; &#xB418;&#xC5C8;&#xB2E4;. &#xB179;&#xCDE8; &#xB85D; &#xC720;&#xB3C4; &#xD558; &#xACE0; noninduced &#xC870;&#xAC74;&#xC5D0;&#xC11C;&#xC758; &#xBE44;&#xAD50; &#xAE00;&#xB85C;&#xBC8C; &#xBD84;&#xC11D;&#xC744; &#xD64D;&#xBCF4; &#xD558;&#xAC70;&#xB098; &#xC0C8;&#xB85C;&#xC6B4; &#xC601;&#xC591; &#xD658;&#xACBD; &#xBC1C;&#xC0DD; &#xD558;&#xB294; &#xC140;&#xC5D0; triacylglycerol &#xCD95;&#xC801;&#xC744; &#xB3D9;&#xBC18; &#xD558;&#xB294; &#xBD84;&#xC790; &#xBCC0;&#xD654;&#xB97C; &#xACF5;&#xBD80; &#xD558; &#xACE0; &#xCCAB; &#xBC88;&#xC9F8; &#xC811;&#xADFC; &#xBC29;&#xC2DD;&#xC73C;&#xB85C; &#xC801;&#xC6A9; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774; &#xBAA9;&#xD45C;&#xB97C; &#xD5A5;&#xD574; &#xB192;&#xC740; &#xCC98;&#xB9AC;&#xB7C9; &#xC2DC;&#xD000;&#xC2F1; &#xAE30;&#xC220; 4 N &#xCDA9;&#xB9CC; &#xAC01; &#xC870;&#xAC74;&#xC5D0; &#xB300; &#xD55C; 8 &#xAC1C;&#xC758; &#xC0DD;&#xBB3C;&#xD559;&#xC801;&#xC73C;&#xB85C; &#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xB77C;&#xC774;&#xBE0C;&#xB7EC;&#xB9AC;&#xC758; &#xD45C;&#xD604;&#xB41C; &#xC2DC;&#xD000;&#xC2A4; &#xD0DC;&#xADF8;&#xC758; &#xD070; &#xBC88;&#xD638;&#xB97C; &#xC0DD;&#xC131; &#xD558; &#xACE0;&#xC6A9; &#xD588;&#xB2E4; &#xD558; &#xACE0; N &#xBC15;&#xD0C8;, &#xD5C8;&#xC6A9; &#xB450; &#xC2DC;&#xD5D8;&#xB41C; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xC2DD; &#xB808;&#xBCA8;&#xC758; &#xD1B5;&#xACC4;&#xC801;&#xC73C;&#xB85C; &#xC0AC;&#xC6B4;&#xB4DC; &#xBE44;&#xAD50;. &#xC608;&#xC0C1; &#xB300;&#xB85C;, N &#xBD80;&#xC871; &#xC81C;&#xC5B4; &#xC720;&#xC804;&#xC790; &#xAD00;&#xC5EC; &#xB2E8;&#xBC31;&#xC9C8; &#xC0DD; &#xD569;&#xC131; &#xB2E4;&#xC6B4;&#xC744; &#xADDC;&#xC81C; &#xD558;&#xB294; &#xB3D9;&#xC548; gametogenesis&#xC758; &#xD558;&#xC704; &#xC9D1;&#xD569;&#xC744; &#xD65C;&#xC131;&#xD654;. &#xAD11;&#xD569;&#xC131;&#xC758; &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xC5D0; &#xB300; &#xD55C; &#xC720;&#xC804;&#xC790; PSBS &#xC720;&#xC804;&#xC790; &#xC81C;&#xC678;&#xD55C; &#xB2E4;&#xC6B4; &#xADDC;&#xC81C; &#xD588;&#xB2E4;. N &#xBD80;&#xC871; &#xC8FC;&#xB3C4; &#xB300;&#xC0AC;&#xC758; &#xD45C;&#xC2DC; &#xB41C; &#xB9AC;&#xB514;&#xB809;&#xC158;: &#xAE30;&#xBCF8; &#xD0C4;&#xC18C; &#xC18C;&#xC2A4;, &#xCD08; &#xC0B0;, &#xB354; &#xC774;&#xC0C1; glyoxylate &#xC0AC;&#xC774;&#xD074; gluconeogenesis&#xC5D0; &#xC758;&#xD574; &#xC140; &#xBE14;&#xB85D;&#xC73C;&#xB85C; &#xBCC0;&#xD658; &#xD558;&#xC9C0;&#xB9CC; &#xC9C0;&#xBC29;&#xC0B0; &#xC0DD; &#xD569;&#xC131;&#xC5D0; &#xC9C1;&#xC811; &#xC55E;. &#xCD94;&#xAC00; &#xC9C0;&#xBC29;&#xC0B0; putative lipase &#xC720;&#xC804;&#xC790;&#xC758; &#xC0AC;&#xBCF8; &#xD48D;&#xC694;&#xC5D0;&#xC11C; &#xAD00;&#xCC30; &#xB41C; &#xBCC0;&#xD654;&#xC5D0; &#xC758;&#xD574; &#xC81C;&#xC2DC; &#xB41C; &#xACFC;&#xC815;&#xC744; &#xAC1C;&#xC7A5; &#xD558;&#xB294; &#xB9C9;&#xC5D0; &#xC758;&#xD574; &#xC0DD;&#xC0B0; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xCD94;&#xB860; transcriptional &#xBD84;&#xC11D;&#xC744; &#xAE30;&#xBC18;&#xC73C;&#xB85C; &#xD558;&#xB294; &#xC2E0;&#xC9C4; &#xB300;&#xC0AC;&#xC5D0; &#xC9C1;&#xC811;, &#xD558;&#xC9C0;&#xB9CC; &#xC0DD;&#xD654;&#xD559; &#xC2E4;&#xD5D8;&#xC774; &#xACF5;&#xC81C;&#xC758; &#xC77C;&#xBD80; &#xC9C0;&#xC6D0;. &#xC5EC;&#xAE30;&#xC5D0; &#xC81C;&#xACF5; &#xB41C; &#xB370;&#xC774;&#xD130; &#xBBF8;&#xC138;&#xC5D0; &#xB204;&#xC801; &#xB41C; &#xC624;&#xC77C;&#xC758; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC758; &#xD0D0;&#xD5D8;&#xC5D0; &#xB300; &#xD55C; &#xD48D;&#xBD80;&#xD55C; &#xC18C;&#xC2A4;&#xB97C; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;.

&#xB300; &#xBCF8; &#xD48D;&#xBD80; &#xD558; &#xAC8C; Chlamydomonas reinhardtii &#xC9C8;&#xC18C; &#xBD80;&#xC871;&#xC5D0; &#xB530;&#xB77C; &#xBCC0;&#xD654; &#xC608;&#xCE21; &#xBB3C;&#xC9C8; &#xB300;&#xC0AC;&#xC758; &#xC591; &#xB3D9;.

Al igual que muchas de microalgas, Chlamydomonas reinhardtii forma gotas de l&#xED;pidos ricos en triglic&#xE9;ridos, cuando los nutrientes necesitados. Para empezar a estudiar los mecanismos que subyacen a este proceso, el nitr&#xF3;geno (N) la privaci&#xF3;n fue utilizado para inducir la acumulaci&#xF3;n de triglic&#xE9;ridos y cambios en los programas de desarrollo tales como la gametog&#xE9;nesis. El an&#xE1;lisis comparativo global de las transcripciones en condiciones inducidas y noninduced se aplic&#xF3; como una primera aproximaci&#xF3;n al estudio de los cambios moleculares que promueven o acompa&#xF1;ar a la acumulaci&#xF3;n de triglic&#xE9;ridos en las c&#xE9;lulas que encuentran un ambiente m&#xE1;s nuevos nutrientes. Para lograr este objetivo, de alto rendimiento de la tecnolog&#xED;a de secuenciaci&#xF3;n se utiliz&#xF3; para generar un gran n&#xFA;mero de etiquetas de secuencias expresadas de ocho bibliotecas biol&#xF3;gicamente independientes, cuatro para cada condici&#xF3;n, N y N repleta privados, lo que permite una comparaci&#xF3;n estad&#xED;sticamente sonido de los niveles de expresi&#xF3;n en las dos condiciones ensayadas. Como era de esperar, la privaci&#xF3;n N activado un subconjunto de los genes implicados en la gametog&#xE9;nesis de control, mientras que abajo de la regulaci&#xF3;n la bios&#xED;ntesis de prote&#xED;nas. Los genes para los componentes de la fotos&#xED;ntesis fueron tambi&#xE9;n las reguladas, con la excepci&#xF3;n del gen RSP. Privaci&#xF3;n N conducido a una marcada redirecci&#xF3;n del metabolismo: la fuente de carbono primario, acetato, ya no se convierte en bloques de construcci&#xF3;n de c&#xE9;lulas por el ciclo de glioxilato y gluconeog&#xE9;nesis pero canalizado directamente en la bios&#xED;ntesis de &#xE1;cidos grasos. Adicional &#xE1;cidos grasos pueden ser producidos por la remodelaci&#xF3;n de la membrana, un proceso que es sugerido por los cambios observados en la abundancia de transcripci&#xF3;n de los genes de lipasa putativo. Las inferencias sobre el metabolismo basado en el an&#xE1;lisis de la transcripci&#xF3;n son indirectos, pero los experimentos bioqu&#xED;micos apoyado algunas de estas deducciones. Los datos proporcionados aqu&#xED; representan una rica fuente para la exploraci&#xF3;n de los mecanismos de acumulaci&#xF3;n de aceite en las microalgas.

Los cambios en la abundancia de transcripci&#xF3;n en Chlamydomonas reinhardtii privaci&#xF3;n de nitr&#xF3;geno continuaci&#xF3;n Predecir desv&#xED;o del metabolismo

Signalling through chromatin modifications and protein-protein interactions.

When deprived of nitrogen (N), the photosynthetic microalga Chlamydomonas reinhardtii accumulates large quantities of triacylglycerols (TAGs), making it a promising source of biofuel. Prominent transcriptional changes associated with the conditions leading to TAG accumulation have been found, suggesting that the key enzymes for TAG metabolism might be among those that fluctuate in their expression during TAG synthesis and breakdown. Using a Saccharomyces cerevisiae lipase null mutant strain for functional complementation, we identified the CrLIP1 gene from Chlamydomonas based on its ability to suppress the lipase deficiency-related phenotypes of the yeast mutant. In Chlamydomonas, an inverse correlation was found between the CrLIP1 transcript level and TAG abundance when Chlamydomonas cultures were reversibly deprived of N. The CrLIP1 protein expressed and purified from Escherichia coli exhibited lipolytic activity against diacylglycerol (DAG) and polar lipids. The lipase domain of CrLIP1 is most similar to two human DAG lipases, DAGLα and DAGLβ. The involvement of CrLIP1 in Chlamydomonas TAG hydrolysis was corroborated by reducing the abundance of the CrLIP1 transcript with an artificial micro-RNA, which resulted in an apparent delay in TAG lipolysis when N was resupplied. Together, these data suggest that CrLIP1 facilitates TAG turnover in Chlamydomonas primarily by degrading the DAG presumably generated from TAG hydrolysis.

Rapid triacylglycerol turnover in Chlamydomonas reinhardtii requires a lipase with broad substrate specificity.

Following N deprivation, microalgae accumulate triacylglycerols (TAGs). To gain mechanistic insights into this phenomenon, we identified mutants with reduced TAG content following N deprivation in the model alga Chlamydomonas reinhardtii. In one of the mutants, the disruption of a galactoglycerolipid lipase-encoding gene, designated PLASTID GALACTOGLYCEROLIPID DEGRADATION1 (PGD1), was responsible for the primary phenotype: reduced TAG content, altered TAG composition, and reduced galactoglycerolipid turnover. The recombinant PGD1 protein, which was purified from Escherichia coli extracts, hydrolyzed monogalactosyldiacylglycerol into its lyso-lipid derivative. In vivo pulse-chase labeling identified galactoglycerolipid pools as a major source of fatty acids esterified in TAGs following N deprivation. Moreover, the fatty acid flux from plastid lipids to TAG was decreased in the pgd1 mutant. Apparently, de novo-synthesized fatty acids in Chlamydomonas reinhardtii are, at least partially, first incorporated into plastid lipids before they enter TAG synthesis. As a secondary effect, the pgd1 mutant exhibited a loss of viability following N deprivation, which could be avoided by blocking photosynthetic electron transport. Thus, the pgd1 mutant provides evidence for an important biological function of TAG synthesis following N deprivation, namely, relieving a detrimental overreduction of the photosynthetic electron transport chain.

A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii.

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.

Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779.

Selective enrichment of phosphopeptides prior to their analysis by mass spectrometry (MS) is vital for identifying protein phosphorylation sites involved in cellular regulation. This study describes modification of porous nylon substrates with TiO2 nanoparticles to create membranes that rapidly enrich phosphopeptides. Membranes with a 22-mm diameter bind 540 nmol of phosphoangiotensin and recover 70% of the phosphopeptides in mixtures with a 15-fold excess of nonphosphorylated proteins. Recovery is 90% for a pure phosphopeptide. Insertion of small membrane disks into HPLC fittings allows rapid enrichment from 5 mL of 1 fmol/μL phosphoprotein digests and concentration into small-volume (tens of microliters) eluates. The combination of membrane enrichment with tandem mass spectrometry reveals seven phosphorylation sites from in vivo phosphorylated tau (p-tau) protein, which is associated with Alzheimer's disease.

Phosphopeptide enrichment with TiO2-modified membranes and investigation of tau protein phosphorylation.

Protein acetylation and phosphorylation are key modifications that regulate both normal and pathological protein functions. The gel systems currently used for analyzing modified proteins require either expensive reagents or time-consuming second dimension electrophoresis. Here we present a neutral pH gel system that allows the analysis of acetylated and phosphorylated proteins. The neutral pH urea Triton-polyacrylamide gel electrophoresis (NUT-PAGE) system separates proteins based on their charge at pH 7.0 and generates discrete bands from each acetylated and/or phosphorylated species. In addition, the gel is composed of common and inexpensive laboratory reagents and requires only a single dimension of electrophoresis. We demonstrate the effectiveness of this system by analyzing the phosphorylated species of an acidic protein, α-synuclein, and both acetylated and phosphorylated species of a basic protein, histone H3. NUT-PAGE thus provides a cost-effective alternative for resolving acetylated and phosphorylated proteins, and potentially proteins with other post-translational modifications that alter net charge.

Resolving acetylated and phosphorylated proteins by neutral urea Triton-polyacrylamide gel electrophoresis: NUT-PAGE.

Many biomedically critical proteins are underrepresented in proteomics and biochemical studies due to difficulties of their production in E. coli. These proteins may possess a posttranslational modification vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator", such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and applications of a PIMAX system (protein interaction module-assisted function X) that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of hyperphosphorylated tau protein intimately linked to Alzheimer's disease, and an enzymatically active cyclin-dependent kinase complex. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3β and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer's disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad impacts on proteomics and biomedical research.

Article	Year
A tethered catalysis, two-hybrid system to identify protein-protein interactions requiring post-translational modifications. Nature biotechnology\| PubMed ID: 15208639	2004
Production of constitutively acetylated recombinant p53 from yeast and Escherichia coli by tethered catalysis. Protein expression and purification\| PubMed ID: 15866730	2005
Histone H3 Ser10 phosphorylation-independent function of Snf1 and Reg1 proteins rescues a gcn5- mutant in HIS3 expression. Molecular and cellular biology\| PubMed ID: 16287868	2005
Histone H3 lysine 9 methyltransferase G9a is a transcriptional coactivator for nuclear receptors. The Journal of biological chemistry\| PubMed ID: 16461774	2006
Functional connection between histone acetyltransferase Gcn5p and methyltransferase Hmt1p. Biochimica et biophysica acta\| PubMed ID: 19358899	2009
Snf1p regulates Gcn5p transcriptional activity by antagonizing Spt3p. Genetics\| PubMed ID: 19841091	2010
Histone h3 exerts a key function in mitotic checkpoint control. Molecular and cellular biology\| PubMed ID: 19917722	2010
Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism. Plant physiology\| PubMed ID: 20935180	2010
Signalling through chromatin modifications and protein-protein interactions. Biotechnology & genetic engineering reviews\| PubMed ID: 22530505	2006
Rapid triacylglycerol turnover in Chlamydomonas reinhardtii requires a lipase with broad substrate specificity. Eukaryotic cell\| PubMed ID: 23042128	2012
A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii. The Plant cell\| PubMed ID: 23161887	2012
Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779. PLoS genetics\| PubMed ID: 23166516	2012
Phosphopeptide enrichment with TiO2-modified membranes and investigation of tau protein phosphorylation. Analytical chemistry\| PubMed ID: 23638980	2013
Resolving acetylated and phosphorylated proteins by neutral urea Triton-polyacrylamide gel electrophoresis: NUT-PAGE. BioTechniques\| PubMed ID: 25109292	2014
PIMAX approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex. Molecular & cellular proteomics : MCP\| PubMed ID: 25385071	2014

Min-Hao Kuo

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