Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Summary

This protocol describes how to stimulate cells with mitochondrial-derived peptides and assess the signaling cascade and localization of phospho-proteins.

Abstract

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the mitochondrial genome. MDPs have a wide variety of biological effects such as protecting neurons from apoptosis, improving metabolic markers, and protecting cells from chemotherapy. Humanin was the first MDP to be discovered and is the most studied peptide among the MDP family. The membrane receptors and downstream signaling pathways of humanin have been carefully characterized. Additional MDPs such as MOTS-c and SHLP1-6 have been more recently discovered and the signaling mechanisms have yet to be elucidated. Here we describe a cell culture based method to determine the function of these peptides. In particular, cell fractionation techniques in combination with western blotting allow for the quantitative determination of activation and translocation of important signaling molecule. While there are other methods of cell fractionation, the one described here is an easy and straightforward method. These methods can be used to further elucidate the mechanism of action of these peptides and other therapeutic agents.

Introduction

Emerging studies show that mitochondrial-derived peptides (MDPs) play important roles in cytoprotection and metabolism^1,2,3. Understanding the signal transduction pathway in the presence of MDPs gives us insight into the mechanism by which MDPs modulate various functions. The first identified MDP, humanin, has been shown to increase extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation through its receptor binding^4,5. However, the downstream effect of ERK1/2 activation is still underexplored.

The ERK1/2 cascade serves as an essential mediator in a variety of cellular processes including proliferation, cell migration, cellular metabolism, survival, and apoptosis^6,7,8. To orchestrate all these distinct cellular processes, the activity and subcellular localization of ERK1/2 are tightly regulated by phosphatases and scaffold proteins^9,10. In addition to post-translational modification, the dynamic shuttling of ERK1/2 also regulates its signaling function, activity, and specificity^11,12. ERK1/2 is primarily localized in the cytosol¹³. A set of anchoring and scaffold proteins help retain ERK1/2 in cytoskeletal elements, on the surface of organelles, or diffused in the cytoplasm¹³. Upon stimulation, ERK1/2 is phosphorylated and becomes dissociated from its anchoring proteins, allowing the translocation of ERK1/2 to other subcellular compartments, including the nucleus, mitochondria, Golgi, and lysosomes^14,15,16.

Although humanin is known to activate the ERK1/2 signaling pathway, the activation of ERK1/2 is only observed in the total cell lysates. As described previously, since ERK1/2 subcellular localization plays a crucial role in its downstream effect, the analysis of both the subcellular localization and the total levels of phosphorylated ERK1/2 is necessary to provide a comprehensive understanding of humanin-induced ERK1/2 activation and the activation of downstream targets.

To understand the target organelles of activated ERK1/2, subcellular fractionation followed by western blotting for phosphorylated ERK1/2 was performed. This method can be easily implemented as it utilizes standard laboratory equipment and reagents. The isolated subcellular compartments are of high purity, allowing the results to be straightforwardly interpreted. Immunostaining of ERK1/2 may produce similar results. However, certain subcellular compartments are relatively hard to visualize and require special fixation and permeabilization methods. ERK1/2 levels vary in subcellular compartments, and this variation could cause false positive and false negative signals when looking at whole cell lysates. Therefore, an immunoblot using isolated subcellular compartments gives us a better understanding of ERK1/2 localization.

The versatility of the method enables modifications of the protocol to investigate effects of other stimulants including other MDPs or translocation of other signaling molecules such as STAT3. Recently discovered small humanin-like peptides (SHLPs) are encoded from 16S rRNA region where humanin is encoded, and they have similar but distinct properties compared to HN¹⁷. For example, SHLP2 and SHLP3 activate ERK1/2 after 8 h although humanin activates ERK1/2 within 5 min. Examining subcellular localization of ERK1/2 in response to different peptides will give us a better understanding of the biology of these peptides. Emerging evidence showed that the subcellular localization of signaling molecules plays a crucial role in their downstream effects. For instance, STAT3 traditionally is known to be primarily localized in the cytosol in resting cells, and then it translocates into the nucleus to activate gene expression in response to cytokines¹⁸. STAT3 also translocates to mitochondria and regulates the TCA cycle and ATP production¹⁹. Regarding autophagy regulation, different subcellular localization of STAT3 modulates autophagy in various ways²⁰. For example, nuclear STAT3 transcriptionally regulates autophagy-related genes and acts as an autophagy modulator. Cytoplasmic STAT3 constitutively inhibits autophagy by interacting with autophagy signaling molecules. Mitochondrial STAT3 inhibits and prevents mitophagy by suppressing oxidative stress induced autophagy. Therefore, this subcellular compartment isolation method is crucial for understanding the role of other signaling molecules as well as ERK1/2.

Protocol

1. Peptide Treatment to Cells

1. Plate two million SH-SY5Y or HEK293 cells (2 x 10⁶) into a 10 cm dish and grow them for 2 days
2. (Optional) The next day, wash the cells with serum-free Dulbecco's Modified Eagle Medium (DMEM) media once and incubate with serum free DMEM overnight if the treatment needs to be done in serum free media.
3. On day 3, dissolve S14G-humanin peptides in 0.2 µm filtered, distilled water, and reconstitute them as 1mM stock solution.
   NOTE: The peptides should be dissolved in the appropriate solvent, which can be determined by the sequence characteristics of the peptide (e.g., overall charge) and may be provided by the manufacturer if the peptides are commercially available. For example, S14G-humanin, SHLP2, and SHLP6, and MOTS-c dissolve in water. Aliquot the peptides in an appropriate volume (e.g., 50 μL) to avoid freeze and thaw cycles. Once thawed, do not use them again.
4. Aliquot the pre-warmed serum-containing or serum-free media in a 50-mL conical tube and add room temperature 1 mM stock solution to make it into a working concentration (e.g., 1 µM, 10 µM).
5. Replace the media with 6 mL peptide solution from step 1.4. and incubate the cells for the appropriate amount of time.
   NOTE: Choose the incubation time depending on your condition which activates ERK.

2. Subcellular Fractionation for Cytosol and Nucleus

1. Wash the cells twice with 10 mL of ice-cold PBS, scrape the cells off the plate in 5 mL of ice-cold PBS, and transfer the cell suspension into a 15 mL conical tube.
2. Centrifuge the cells at 500 x g for 5 min at 4 °C.
3. Aspirate the PBS and re-suspend the pellet in 200 µL of ice-cold, fractionation buffer (10 mM HEPES pH = 7.6, 3 mM MgCl₂, 10 mM KCl, 5% (v/v) glycerol, 1% non-ionic surfactant (C₁₃H₂₂O(C₂H₄O)_n), protease/phosphatase inhibitors).
   NOTE: To keep the proteins in their phosphorylation status, the phosphatase inhibitor as well as protease inhibitor should be freshly added and samples should be kept on ice at all times. Repeating freeze and thaw cycles of samples should be avoided. Aliquot samples into small amount (e.g. 50 μg) before freezing them at -80 °C.
4. Incubate the re-suspended pellet on ice for 15 min and then centrifuge at 250 x g for 5 min at 4 °C.
5. Collect the supernatant as the cytoplasmic fraction and keep the pellet for the nuclear fraction.
6. Centrifuge the supernatant to remove cellular debris and other contaminants at 18,000 x g for 10 min at 4 °C and then transfer the supernatant into a new microcentrifuge tube. This is the cytosol fraction.
7. Resuspend the pellet in 200 μL ice-cold wash buffer (10 mM HEPES pH = 7.6, 1.5 mM MgCl₂, 420 mM NaCl, 25% (v/v) glycerol, 0.2 mM EDTA, protease/phosphatase inhibitors) and centrifuge at 250 x g for 5 min at 4 °C
8. Remove the supernatant and resuspend the pellet in 100 μL ice-cold, nuclear extraction buffer (20 mM HEPES pH = 7.6, 1.5 mM MgCl₂, 420 mM NaCl, 25% (v/v) glycerol, 0.2 mM EDTA, protease/phosphatase inhibitors) and sonicate 10 times (5 s on, 10 s off, 30% amp.) on ice.
9. Centrifuge at 18,000 x g for 10 min at 4 °C.
10. Transfer the supernatant to a new microcentrifuge tube. This is the nuclear fraction.
11. Quantify the protein amount using a BCA assay

3. Crude Mitochondrial Fraction

1. Wash the cells in each 10 cm dish with 10 mL of ice-cold PBS, add 5 mL ice-cold PBS, and detach the cells using a cell scraper.
   NOTE: Use three 10 cm dishes of cells to get a good yield of mitochondria for Western Blotting.
2. Transfer the cell suspension to a 15 mL conical tube, and combine all from three 10 cm dishes into one 15 ml conical tube.
3. Centrifuge the cells at 600 x g for 10 min at 4 °C.
4. Aspirate the PBS and re-suspend the pellet in 1 mL of ice-cold mitochondria isolation buffer (10 mM Tris-MOPS, 1 mM EGTA/Tris, 200 mM Sucrose, adjust to pH = 7.4).
5. Homogenize the cells with 25 strokes of a 2 mL homogenizer with polytetrafluoroethylene coated pestle on ice.
   NOTE: This step is critical to maintain mitochondrial integrity and maximize the yield of the mitochondrial fraction. The number of strokes should be optimized for each cell type. Precool the homogenizer before starting the procedure.
6. Transfer the homogenate to a microcentrifuge tube and centrifuge it at 600 x g for 10 min at 4 °C to remove nuclei and unbroken cells.
7. Collect the supernatant, transfer it to a new microcentrifuge tube, and centrifuge it at 7,000 x g for 10 min at 4 °C.
   NOTE: The pellet is loose, collect the supernatant with care and try not to disturb the pellet.
8. Remove the supernatant, re-suspend the pellet with 200 μL of ice-cold mitochondria isolation buffer, and transfer the solution to a new microcentrifuge tube. Centrifuge the tube at 7,000 x g for 10 min at 4 °C.
9. Repeat step 3.8 to wash the pellet. It is not necessary to transfer the supernatant to a new microcentrifuge tube in this step.
10. Remove the supernatant from the washed pellet and re-suspend the pellet containing mitochondria with 50 μL of RIPA buffer.
11. Incubate the suspension on ice for 10 min.
12. Centrifuge the suspension at 16,000 x g for 15 min at 4 °C.
13. Transfer the supernatant to a new microcentrifuge tube. This is the mitochondrial fraction.
14. Quantify the protein amount using a BCA assay.

4. Western Blotting for Phospho-Specific Proteins

1. Perform an SDS-polyacrylamide gel electrophoresis (8-16% premade gel) and transfer the protein to a PVDF membrane⁴.
2. Incubate the membrane with 5% BSA in TBST (0.1% Polysorbate 20) for 30 min at room temperature to block background non-specific binding sites.
   NOTE: For phosphorylated protein detection, block the membrane with BSA not Milk. Milk contains casein, an abundant phosphoprotein, which results in high nonspecific signal.
3. Incubate the membrane with the primary antibody (anti-phospho-ERK1/2) at 4 °C overnight.
4. The next day, wash the membrane with TBST (0.1% polysorbate 20) three times for 5 min at room temperature.
5. Incubate the membrane with secondary antibody (anti-rabbit HRP) for 1 h at room temperature.
6. Wash the membrane with TBST (0.1% polysorbate 20) three times for 5 min at room temperature.
7. Incubate the membrane with ECL solution and image the membrane in the image analyzer.
8. Strip the membrane with stripping buffer for 15 min at room temperature and wash the membrane with TBST three times for 5 min.
9. Repeat steps 4.2 to 4.7 using the anti-total ERK1/2 antibody.
10. To check the purity of each fraction, run SDS-PAGE and perform immunoblots using antibodies recognizing markers for each compartment (e.g., Lamin B1 for nucleus, GAPDH for cytosol, and TOM20 for mitochondria).

Results

Using the procedure presented here, we treated HEK293 and SH-SY5Y cells with 1 μM and 100 μM S14G-humanin, a potent humanin analog²¹, respectively, in complete media for the indicated time periods (Figure 1A and Figure 1B). We then examined the total and phosphorylated form of ERK1/2 at Thr²⁰²/Tyr²⁰⁴ from total protein extracts. S14G-humanin...

Discussion

Here, we demonstrated that humanin peptide-mediated ERK1/2 activation occurs in two different cell types, and the subcellular localization of activated ERK1/2 can be different depending on the conditions (e.g., dose of peptide, time point, and cell type). It has been shown that humanin signals through two different receptors^22,23, which may explain the differences in signaling between the two cell lines as well as the requirement for different doses of h...

Materials

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