1. Andrei Bilog and Nikolas Nikolaidis
Characterization of the interaction between HspA1A, a major stress-inducible protein, and lipids in mammalian cells
Andrei Bilog and Nikolas Nikolaidis
Department of Biological Science, California State University Fullerton, Fullerton, CA
Background
Purpose Hypothesis
Predicted Results
During cellular stress, proteins denature and aggregate, resulting in cell death.
I predict that cell surface biotinylation and subcellular fractionation experiments will show that HspA1A is competed from membranes when a lipid-biosensor is present (Fig. 6), validating and confirming the imaging results.
The purpose of this project is to determine under which conditions HspA1A binds to phosphatidylserine and cardiolipin in human cells.
My hypothesis is that lipid-binding will increase under different types of stress and disease conditions that alter the native lipid content and membrane composition.
Experimental Design * *
Fig. 1 Cellular stress damages the ER, Golgi, mitochondria, lysosomes ,and the plasma membrane, and causes protein denaturation, leading to cell death. (Adopted from Ritcher et al The Heat Shock Response: Life on the Verge of Death.)
Cells overexpressing HspA1A
To combat the deleterious effects of stress, a highly conserved family of stress response proteins, known as the 70-kilodalton heat shock proteins (Hsp70s) has evolved . HSPA1A is the major stress-inducible cytosolic member of the human Hsp70 family, and is a key player in several signaling pathways that regulate protein homeostasis and cell survival (1).
Transfect fluorescent lipid biomarkers using lipid-based delivery
Heat
ROS
Cell Surface Biotinylation
Subcellular Fractionation
Hypoxia
Fig. 5 Predicted cell surface biotinylation and subcellular fractionation results. Membrane localization of over-expressed HspA1A increases after recovery both at the PM and mitochondria. Subcellular fractionation was done for the mitochondria and cell surface biotiniylation for PM. N=3; bars= S.D.; p-value <0.05; (-) = without lipid biomarker and serves as a control; (+) = with lipid biomarker.
Ethanol
Predicted Results
Significance
In addition to their indispensable functions in protein-chaperoning and cell signaling, HspA1A also functions at the plasma membrane (PM), organelle membranes, and the extracellular environment (2), where it associates and binds to various lipids, like phosphatidylserine (PS) and cardiolipin (CL) in vitro (3).
Lipid-binding is a newly described property of HspA1A that recently received a lot of attention because the protein is expressed at the membrane of cancer cells, is then actively secreted, and able to activate the immune system. However, with the exception of phosphatidylserine (PS) at the plasma membrane (5), nothing is known about the interaction of HspA1A with other lipids, such as cardiolipin (CL) at the mitochondria, found in human cells
Furthermore, with the exception of heat-shock and to a lesser extent hypoxia (2, 5), the stresses that promote the interaction of HspA1A with lipids remain unknown. The results of this project will bridge this major gap in the knowledge, and refine the understanding of the cellular stress response and its association with cellular lipids and membranes.
Lastly, the discovery of the conditions that favor or induce HspA1A-lipid association in multiple cell types will establish the required foundation to directly assess the biological implications of this newly described and largely uncharacterized function of Hsp70s.
Confocal microscopy will reveal that HspA1A plasma membrane and mitochondria localization will increase after stress (Fig. 3-4) in the absence of a lipid-biosensor. However, in accordance to my hypothesis, I predict that the HspA1A membrane localization will significantly decrease in the presence of high amounts of a lipid-biosensor.
Phosphatidylserine
Cardiolipin
HSPA1A-GFP
C2-RFP
Colocalization
HSPA1A-GFP
C11Orf83-RFP
Colocalization
Fig. 3 Predicted images of mammalian cells expressing GFP-HSPA1A fusion protein and lipid biomarkers (RFP). [+ve PDM is the number of pixels with a positive Product of the Differences of the Mean (PDM) value; the PDM = (red intensity- mean red intensity)×(green intensity – mean green intensity)]. (Images from NN) *
References
Fig. 2 Hsp70 binding to various lipids during times of stress. HspA1A binding to lipids regulates important biological phenomena because binding to extracellular phosphatidylserine (PS) induces apoptosis and suppress tumor growth and binding to bis-(monoacylglycero)-phosphate (BMP) prevents permeabilization of the lysosomal membrane. (Image from NN). *
1. Lindquist S, Craig EA the Heat-Shock Proteins. Campbell, A 631–678.
2. Kirkegaard T, Roth AG, Petersen NHT, Mahalka AK, Olsen OD, Moilanen I, Zylicz A, Knudsen J, Sandhoff K, Arenz C, Kinnunen PKJ, Nylandsted J, Jäättelä M Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology. Nature 463:549–53.
3. McCallister C, Siracusa MC, Shirazi F, et al (2015b) Functional diversification and specialization of cytosolic 70-kDa heat shock proteins. Sci Rep 5:9363. doi: /srep09363
4. Multhoff G Heat shock protein 70 (Hsp70): Membrane location, export and immunological relevance. Methods 43:229–237.
5. De Maio A Extracellular heat shock proteins, cellular export vesicles, and the Stress Observation System: A form of communication during injury, infection, and cell damage: It is never known how far a controversial finding will go! Dedicated to Ferruccio Ritossa. Cell Stress Chaperones 16:235–249. ** * ** ** **
Extracellular and membrane bound HspA1A, which does not contain known secretory signals or lipid-binding domains (LBDs), is now recognized to play critical biological roles, as it marks several types of cancer cells, activates the immune system, and regulates apoptosis (4-5). However, the conditions that favor the interaction between the chaperone and specific lipids within human cells remain unknown.
Acknowledgments
Fig. 4 Predicted CTCF (corrected total cell fluorescence) ratio between red fluorescence (PM or mitochondrial membrane) and green fluoresce (GFP-HspA1A) will determine HspA1A localization at the PM or mitochondrial membrane. [CTCF = integrated density – (area of selected cell x mean fluorescence of background readings)]. N=100; bars= S.D.; p-value <0.05; (-) = without lipid-biosensor and serves as a control; (+) = in the presence of the lipid-biosensor.
I would like to thank my graduate buddies in the NN lab! Shout out to Ryan, Peter, and Rogue!
I also want to thank my BIOL500A/B cohort for editing this poster and their constructive criticism.