Identification of The Pericyte Specific Secretome in an In Vitro Model of the Neurovascular Unit
(2020) MOBN03 20192Degree Projects in Molecular Biology
- Abstract
- Pericytes play an important role in the integrity of blood brain barrier. They have intricate communication with several other cell types in the brain, both through paracrine and endocrine signaling. We have previously shown that in vitro-cultured pericytes secrete various cytokines, chemokines and microparticles in disease model-systems in vitro. However, the temporal dynamics and specificity of the pericyte secretome in vitro in the neurovascular unit is not yet fully understood. The emergence of fast biotin labeling, such as TurboID, offers a solution to overcome this problem. We used a cytosol facing endoplasmic reticulum (ER) anchor to dock TurboID to the ER allowing it to label any newly synthesized proteins in the close vicinity of... (More)
- Pericytes play an important role in the integrity of blood brain barrier. They have intricate communication with several other cell types in the brain, both through paracrine and endocrine signaling. We have previously shown that in vitro-cultured pericytes secrete various cytokines, chemokines and microparticles in disease model-systems in vitro. However, the temporal dynamics and specificity of the pericyte secretome in vitro in the neurovascular unit is not yet fully understood. The emergence of fast biotin labeling, such as TurboID, offers a solution to overcome this problem. We used a cytosol facing endoplasmic reticulum (ER) anchor to dock TurboID to the ER allowing it to label any newly synthesized proteins in the close vicinity of the ER. The biotin label on the secreted protein can be utilized to track down the cell origin of the protein. Here we show the optimized avidin-biotin affinity purification method that is able to successfully isolate the secreted biotinylated protein from a mixture of secreted proteins. We demonstrate the difference in biotinylated protein level under hypoxic condition versus the control and conclude that the purification method still needs further optimization. (Less)
- Popular Abstract
- A method to screen proteins based on their cell origin
How can we identify secreted proteins coming from a certain cell in a mix cell culture? A cell-specific protein labeling method, such as biotin labeling method, can be utilized in this kind of scenario. Biotin ligase catalyzes the attachment of biotin molecule on the exposed side-chain lysine residue of any protein in the vicinity of the enzyme, allowing the proteins to be purified using biotin-avidin purification method. As the labeling is cell-specific, the purified biotinylated proteins are mostly coming from the cell of interest although any naturally biotinylated protein will also be isolated.
TurboID is a new generation of the biotin ligase. This enzyme offers shorter... (More) - A method to screen proteins based on their cell origin
How can we identify secreted proteins coming from a certain cell in a mix cell culture? A cell-specific protein labeling method, such as biotin labeling method, can be utilized in this kind of scenario. Biotin ligase catalyzes the attachment of biotin molecule on the exposed side-chain lysine residue of any protein in the vicinity of the enzyme, allowing the proteins to be purified using biotin-avidin purification method. As the labeling is cell-specific, the purified biotinylated proteins are mostly coming from the cell of interest although any naturally biotinylated protein will also be isolated.
TurboID is a new generation of the biotin ligase. This enzyme offers shorter labeling duration and higher number of biotin utilization allowing faster reaction time and higher chemi-luminescence signal during western blot analysis compared to its predecessors. We utilized TurboID in pericytes that were grown together with brain endothelial cells and astrocytes as a spheroid, a clump of cells that works as a blood-brain barrier in-vitro model. Before that, we also confirmed that TurboID worked in the pericytes and significant biotinylation only occurred when extra biotin was added into the media. We also confirmed that the enzyme was not released outside the cell minimizing the chances of any non-cell-specific protein labeling. Furthermore, the amount of biotinylated proteins was increased proportionally to the concentration and the duration of biotin added in the cell culture media.
Next, we designed and optimized the purification method of the biotinylated proteins. We were able to purify biotinylated proteins using monomeric avidin beads coupled with the washing protocol from chromatin immuneprecipitation purification as well as the SDS protein denaturation for releasing the proteins from the beads. Despite of the success, we also introduced a high degree of monomeric avidin contamination in the sample. To circumvent this, we decided to excise only the interesting bands from electrophoresis gels for any further protein analysis. We then tried this method on pericytes in a spheroid under a hypoxic condition and compared it to a normoxic condition. We showed that the secreted protein band profile under hypoxia was visually different to the one under normoxia when visualized using western blot. This result confirmed the prospect of TurboID utilization to isolate secreted proteins from a specific cell in a mixed cell culture system in vitro and possibly in vivo.
Master’s Degree Project in Molecular Biology 60 credits 2020
Department of Biology, Lund University
Advisor: Robert Carlsson
Translational Neurology, BMC (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9028460
- author
- Lewi Santoso, Marvel
- supervisor
- organization
- course
- MOBN03 20192
- year
- 2020
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 9028460
- date added to LUP
- 2020-09-07 15:14:17
- date last changed
- 2020-09-07 15:14:17
@misc{9028460,
abstract = {{Pericytes play an important role in the integrity of blood brain barrier. They have intricate communication with several other cell types in the brain, both through paracrine and endocrine signaling. We have previously shown that in vitro-cultured pericytes secrete various cytokines, chemokines and microparticles in disease model-systems in vitro. However, the temporal dynamics and specificity of the pericyte secretome in vitro in the neurovascular unit is not yet fully understood. The emergence of fast biotin labeling, such as TurboID, offers a solution to overcome this problem. We used a cytosol facing endoplasmic reticulum (ER) anchor to dock TurboID to the ER allowing it to label any newly synthesized proteins in the close vicinity of the ER. The biotin label on the secreted protein can be utilized to track down the cell origin of the protein. Here we show the optimized avidin-biotin affinity purification method that is able to successfully isolate the secreted biotinylated protein from a mixture of secreted proteins. We demonstrate the difference in biotinylated protein level under hypoxic condition versus the control and conclude that the purification method still needs further optimization.}},
author = {{Lewi Santoso, Marvel}},
language = {{eng}},
note = {{Student Paper}},
title = {{Identification of The Pericyte Specific Secretome in an In Vitro Model of the Neurovascular Unit}},
year = {{2020}},
}