Name: subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment
Uploaded: 2017-09-25T15:00:00
Description: Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Dette arbejde blev støttet af en Ellison/AFAR postdoc stipendium i Aging Research Program til SJK, og en Glenn Foundation Award og NIH tilskud til PC (1P01AG034906, 1R01GM 090311, 1R01ES 020812). Alle forfattere synes i filmen.

1. peptid behandling til celler <ol><li>plade to millioner SH-SY5Y eller HEK293 celler (2 x 10 6) i et 10 cm parabol og dyrke dem i 2 dage</li> <li>(valgfrit) næste dag, cellerne vaskes med serumfrit Dulbecco &#39; s ændret Eagle Medium (DMEM) medier én gang, og der inkuberes med serum gratis DMEM natten hvis behandlingen skal gøres i serum frie medier.</li>
	<li>På dag 3, opløses S14G-humanin peptider i 0,2 µm filtreret, destilleret vand, og rekonstruere dem som 1mM stamopløsning. 
	Bemærk: P...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanin:+a+novel+central+regulator+of+peripheral+insulin+action.">Humanin: a novel central regulator of peripheral insulin action.</a> PloS one. 4 (7), 6334 (2009).</li><li>Lee, C., Yen, K., Cohen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanin:+a+harbinger+of+mitochondrial-derived+peptides.">Humanin: a harbinger of mitochondrial-derived peptides.</a> Trends Endocrinol. Metab. 24 (5), 222-228 (2013).</li><li>Kim, S. J., Guerrero, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial-derived+peptide+humanin+activates+the+ERK1/2,+AKT,+and+STAT3+signaling+pathways+and+has+age-dependent+signaling+differences+in+the+hippocampus.">The mitochondrial-derived peptide humanin activates the ERK1/2, AKT, and STAT3 signaling pathways and has age-dependent signaling differences in the hippocampus.</a> Oncotarget. 7 (30), 46899-46912 (2016).</li><li>Ying, G., Iribarren, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanin,+a+newly+identified+neuroprotective+factor,+uses+the+G+protein-coupled+formylpeptide+receptor-like-1+as+a+functional+receptor.">Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor.</a> J. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinase+signaling+cascades+in+the+mitochondrion:+a+matter+of+life+or+death.">Kinase signaling cascades in the mitochondrion: a matter of life or death.</a> Free Radic. Biol. Med. 38 (1), 2-11 (2005).</li><li>Aebersold, D. M., Shaul, Y. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+signal-regulated+kinase+1c+(ERK1c),+a+novel+42-kilodalton+ERK,+demonstrates+unique+modes+of+regulation,+localization.">Extracellular signal-regulated kinase 1c (ERK1c), a novel 42-kilodalton ERK, demonstrates unique modes of regulation, localization.</a> Mol Cell Biol. 24 (22), 10000-10015 (2004).</li><li>Zhu, J. -. H., Guo, F., Shelburne, J., Watkins, S., Chu, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+phosphorylated+ERK/MAP+kinases+to+mitochondria+and+autophagosomes+in+Lewy+body+diseases.">Localization of phosphorylated ERK/MAP kinases to mitochondria and autophagosomes in Lewy body diseases.</a> Brain Pathol. 13 (4), 473-481 (2003).</li><li>Cobb, L. J., Lee, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+mitochondrial-derived+peptides+are+age-dependent+regulators+of+apoptosis,+insulin+sensitivity,+and+inflammatory+markers.">Naturally occurring mitochondrial-derived peptides are age-dependent regulators of apoptosis, insulin sensitivity, and inflammatory markers.</a> Aging. 8 (4), 796-809 (2016).</li><li>Ihle, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stat+family+in+cytokine+signaling.">The Stat family in cytokine signaling.</a> Curr. Opin. Cell Biol. 13 (2), 211-217 (2001).</li><li>Xu, Y. S., Liang, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+Undergoes+Acetylation-dependent+Mitochondrial+Translocation+to+Regulate+Pyruvate+Metabolism.">STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism.</a> Sci. Rep. 6 (1), 39517 (2016).</li><li>You, L., Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+STAT3+in+autophagy.">The role of STAT3 in autophagy.</a> Autophagy. 11 (5), 729-739 (2015).</li><li>Hashimoto, Y., Niikura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+characterization+of+neuroprotection+by+a+rescue+factor+humanin+against+various+Alzheimer's+disease-relevant+insults.">Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults.</a> The J Neurosci. 21 (23), 9235-9245 (2001).</li><li>Ying, G., Iribarren, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanin,+a+newly+identified+neuroprotective+factor,+uses+the+G+protein-coupled+formylpeptide+receptor-like-1+as+a+functional+receptor.">Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor.</a> J. 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Her, viste vi at humanin peptid-medieret ERK1/2 Aktivering sker i to forskellige celletyper, og subcellulært lokaliseringen af aktiverede ERK1/2 kan være forskellige afhængig af betingelser (fx, dosis af peptid, tidspunkt og celle type). Det har vist sig at humanin signaler gennem to forskellige receptorer22,23, hvilket kan forklare forskelle i signalering mellem to cellelinier samt kravet om forskellige doser af humanin. De fysiologiske konsekvenser ...

Nye undersøgelser viser, at mitokondrie-afledte peptider (MDP'er) spille en vigtig rolle i cytoprotection og stofskifte1,2,3. Forståelse signaltransduktion pathway i overværelse af MDP'er giver os indsigt i den mekanisme, hvormed MDP'er modulere forskellige funktioner. Den første identificerede MDP, humanin, har vist sig at øge ekstracellulær signal-reguleret kinase 1/2 (ERK1/2) fosforylering gennem sin receptor bindende4,5. ERK1/2 aktivering downstream effekten er dog stadig underexplored.
ERK1/2 cascade fungerer som en vigtig mægler i en række forskellige cellulære processer herunder spredning, celle migration, cellulære metabolisme, overlevelse og apoptose6,7,8. For at orkestrere alle reguleret disse forskellige cellulære processer, aktivitet og subcellulært lokalisering af ERK1/2 stramt af fosfataser og stillads proteiner9,10. Ud over posttranslationel modifikation regulerer den dynamiske shuttling af ERK1/2 også sin signaling funktion, aktivitet og specificitet11,12. ERK1/2 er primært lokaliseret i cytosol13. Et sæt af forankring og stillads proteiner bidrage til at bevare ERK1/2 i cytoskeletal elementer, på overfladen af organeller, eller diffust i cytoplasma13. Stimulering, ERK1/2 er fosforyleret og bliver adskilt fra sin forankring proteiner, så omplantning af ERK1/2 til andre subcellulært rum, herunder kerne, mitokondrier, Golgi og lysosomer14, 15 , 16.
Selv om humanin er kendt for at aktivere ERK1/2 signalering pathway, er aktivering af ERK1/2 kun observeret i den samlede cellelysater. Som beskrevet tidligere, da ERK1/2 subcellulært lokalisering spiller en afgørende rolle i sin downstream virkning, analyse af både den subcellulært lokalisering og de samlede niveauer af er fosforyleres ERK1/2 nødvendigt at fastsætte en omfattende forståelse humanin-induceret ERK1/2 aktivering og aktivering af downstream mål.
For at forstå målet organeller aktiveret ERK1/2, blev subcellulært fraktionering efterfulgt af western blotting for fosforyleres ERK1/2 udført. Denne metode kan gennemføres let, som det udnytter almindeligt laboratorieudstyr og reagenser. De isolerede subcellulært rum er af høj renhed, så resultaterne skal fortolkes ligefremt. Immunfarvning af ERK1/2 kan give lignende resultater. Visse subcellulært rum er dog relativt svært at visualisere og kræver særlige fiksering og permeabilization metoder. ERK1/2 niveauer variere i subcellulært rum, og denne variation kan forårsage falsk positive og falsk negative signaler, når man ser på hele cellelysater. Derfor giver en immunblot ved hjælp af isolerede subcellulært rum os en bedre forståelse af ERK1/2 lokalisering.
Alsidigheden af metoden gør det muligt for ændringer af protokollen til at undersøge virkningerne af andre stimulanser, herunder andre MDP'er eller omplantning af andre signaling molekyler såsom STAT3. For nylig er opdaget små humanin-lignende peptider (SHLPs) kodet fra 16S rRNA region, hvor humanin er kodet, og de har lignende men forskellige egenskaber i forhold til HN17. For eksempel, aktivere SHLP2 og SHLP3 ERK1/2 efter 8 h selv humanin aktiverer ERK1/2 inden for 5 min. undersøgelse subcellulært lokalisering af ERK1/2 som svar på forskellige peptider vil give os en bedre forståelse af biologi af disse peptider. Nye beviser viste at subcellulært lokaliseringen af signalering molekyler spiller en afgørende rolle i deres downstream effekter. For eksempel, STAT3 traditionelt er kendt for at være hovedsagelig lokaliseret i cytosol i resting celler, og derefter det translocates ind i kernen til at aktivere genekspression i svar til cytokiner18. STAT3 også translocates til mitokondrier og regulerer TCA cyklus og ATP produktion19. Vedrørende autophagy forskrift modulerer forskellige subcellulært lokalisering af STAT3 autophagy i forskellige måder20. For eksempel, nukleare STAT3 transcriptionally regulerer autophagy-relaterede gener og fungerer som en autophagy-modulator. Cytoplasmatisk STAT3 hæmmer constitutively autophagy ved at interagere med autophagy signalerer molekyler. Mitokondrie STAT3 hæmmer og forhindrer mitophagy ved at undertrykke oxidativt stress induceret autophagy. Derfor, denne subcellulært rum isolation metode er afgørende for at forstå rollen, andre signaling molekyler samt ERK1/2.

<table>
	<tbody>
		<tr>
			<td>p44/42 MAPK (ERK1/2)</td>
			<td>Cell signaling</td>
			<td>9102</td>
			<td>Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)</td>
			<td>Cell signaling</td>
			<td>4370</td>
			<td>Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>Lamin B1</td>
			<td>Cell signaling</td>
			<td>12586</td>
			<td>Nuclear Marker, Dilution 1:1,000</td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>Cell signaling</td>
			<td>5174</td>
			<td>Cytoplasmic marker, Dilution 1:2,000</td>
		</tr>
		<tr>
			<td>Tom20</td>
			<td>Santa cruz</td>
			<td>SC-17764</td>
			<td>Mitochondria marker, Dilution 1:2,000</td>
		</tr>
		<tr>
			<td>anti-Rabbit-HRP conjugated</td>
			<td>Cell signaling</td>
			<td>7074</td>
			<td>Dilution 1:30,000</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Culture Dish</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>12556002</td>
			<td></td>
		</tr>
		<tr>
			<td>HNG peptide</td>
			<td>Genescript</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25mm sylinge filter</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>09-719A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>3609</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP152-1</td>
			<td></td>
		</tr>
		<tr>
			<td>HCL</td>
			<td>Sigma</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>FALCON</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt&#8482; Protease and Phosphatase Inhibitor Cocktail (100X)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>78440</td>
			<td></td>
		</tr>
		<tr>
			<td>Thomas Pestle Tissue Grinder Assemblies with Smooth Pestles</td>
			<td>Thomas Scientific</td>
			<td>3432S90</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>8-16% Mini-PROTEAN TGX Precast Protein Gels</td>
			<td>BIO RAD</td>
			<td>4561104</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Module</td>
			<td>BIO RAD</td>
			<td>1658030</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer System</td>
			<td>BIO RAD</td>
			<td>1704150</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo RTA Mini PVDF Transfer Kit</td>
			<td>BIO RAD</td>
			<td>1704272</td>
			<td></td>
		</tr>
		<tr>
			<td>Clarity Western ECL Blotting Substrates</td>
			<td>BIO RAD</td>
			<td>1705060</td>
			<td></td>
		</tr>
		<tr>
			<td>Restore Western blot stripping buffer</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>21059</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator, Medel: FB120</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>695320-07-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

subcellular fractionation for erk activation upon mitochondrial-derived peptide treatment

Mitokondrie-afledte peptider (MDP'er) er en ny klasse af peptider, der er kodet ved små åbne læserammer inden for andre kendte gener af mitokondrie genomet. MDP'er har en bred vifte af biologiske effekter såsom beskytte neuroner mod apoptose, forbedrer metaboliske markører og beskytte celler fra kemoterapi. Humanin var den første MDP at blive opdaget og er den mest studerede peptid blandt familien MDP. Membranreceptorer og downstream signaling veje af humanin er blevet nøje karakteriseret. Yderligere MDP'er MOTS-c og SHLP1-6 har været for nylig opdaget og signalering mekanismer har endnu at blive belyst. Her beskriver vi en celle kultur baseret metode til at bestemme funktionen af disse peptider. Især mulighed celle fraktionering teknikker i kombination med western blotting for kvantitativ bestemmelse af aktivering og omplantning af vigtig signalfunktion molekyle. Mens der er andre metoder til celle fraktionering, er beskrevet her en nem og ligetil metode. Disse metoder kan bruges til yderligere belyse virkningsmekanismen af disse peptider og andre terapeutiske agenter.

Ved hjælp af den procedure, der præsenteres her, vi behandlet HEK293 og SH-SY5Y celler med 1 μM og 100 μM S14G-humanin, en potent humanin analog21, henholdsvis i komplet media til de angivne perioder (figur 1A og figur 1 B). vi derefter undersøgt den samlede og fosforyleres form for ERK1/2 på Thr202/Tyr204 fra total protein ekstrakter. S14G-hum...

Watch this Scientific Journal Video about Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment at JoVE.com

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Denne protokol beskriver hvordan man stimulerer celler med mitokondrie-afledte peptider og vurdere signaling cascade og lokalisering af phospho-proteiner.

Subcellulært fraktionering for ERK aktivering på mitokondrie-afledte peptid behandling

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

Research

JoVE Journal

Biology

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

RhoC GTPase Activation Assay

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Metabolisk mærkning og membranfraktionering for Komparativ proteomiske analyse af<em&gt; Arabidopsis thaliana</em&gt; Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Intravital mikroskopi af Imaging Subcellulære Strukturer Live mus, der udtrykker fluorescerende proteiner

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Imaging Cell Membrane Skade og Subcellulære processer involveret i reparation

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Metoder til at vurdere subcellulære afsnit af muskler i<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...