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.

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 c....

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 treatment.......

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

List of materials used in this article

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