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FLIPPING THE SWITCH ON PROTEIN ACTIVITY: ELASTIN-LIKE
POLYPEPTIDES ASSEMBLE INTO CELL SWITCHES AND
VESICLES
by
Martha K. Pastuszka
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERISTY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirement of the degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
May 2014
Object Description
| Title | Flipping the switch on protein activity activity: elastin-like polypeptides assemble into cell switches and vesicles |
| Author | Pastuszka, Martha K. |
| Author email | martha.past@gmail.com;martha.past@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Molecular Pharmacology and Toxicology |
| School | School of Pharmacy |
| Date defended/completed | 2014-05-22 |
| Date submitted | 2014-07-14 |
| Date approved | 2014-07-15 |
| Restricted until | 2014-07-15 |
| Date published | 2014-07-15 |
| Advisor (committee chair) | Mackay, John Andrew |
| Advisor (committee member) |
Hamm-Alvarez, Sarah F. Okamoto, Curtis Toshio |
| Abstract | This research project primarily focuses on the application of elastin‐like polypeptides (ELPs) as intracellular protein switches. ELPs are biologic, environmentally responsive polymers comprised of repeats of the Val‐Pro‐Gly‐Xaa‐Gly motif, a sequence derived from the gene for human tropoelastin. Modulating the molecular weight and amino acid composition allows for precise control over the temperature that triggers polymeric self assembly, allowing for the formulation of polymers that form structures at physiologically relevant temperatures. Intracellular protein switches mainly focus on the genetic deletion of the protein of interest using methods such as siRNA or tetracycline inducible promoters. Some protein switches targeting the fully formed protein exist, however ease of implementation as well as reversibility remain a hindrance. With this limitation in mind, a novel ELP based switch that sequesters a protein of interest into an insoluble cytosolic microdomain would permit for the rapid and reversible inhibition of that protein’s function, effectively acting as a functional knock‐out. Studies assaying the feasibility of this approach reveal that ELPs can knock out a protein’s activity by sequestering it with a half life of minutes and can reinstate protein function equally quickly. The genetic encodability of ELPs make it easy to transfer this paradigm to alternate systems, presenting a novel, easily adaptable tool to study protein behavior in real time. ❧ ELP’s environmental responsive switch from disorder to order has previously been used in thermally directed chemotherapeutic applications, protein purification, as well as the formulation of thermally responsive hydrogels for cell culture and tissue engineering. However, this reversible self assembly had only been characterized in PBS and outside the cell, with no extensive intercellular applications having previously been described. Chapter 2 characterizes, for the first time, the biophysics of ELP assembly in mammalian cytosol. It supports the hypothesis that cytoplasmic GFP‐ELP fusion proteins will phase separate rapidly and reversibly. ELPs comprised of varying molecular weights and compositions were assessed for their ability to self assemble into microdomains upon thermal stimulation. A correlation between ELP self assembly in solution versus the cytosol is found, leading to the identification of an optimal formulation for further ELP protein switch applications. ❧ Chapter 3 looks extensively at the conditional blockage of clathrin‐mediated endocytosis. It seeks to address the postulated hypothesis that temperature‐triggered aggregation of clathrin‐light chain ELP fusion proteins will halt clathrin‐mediated endocytosis. ELPs are genetically engineered onto the N‐terminus of the clathrin‐light chain molecule, a key effector in the clathrin‐mediated endocytosis (CME) pathway. Prior to ELP assembly, the ELP – CLC fusion functions normally and allows for normal receptor mediated internalization. Raising the temperature induces ELP microdomain assembly, which brings in sequesters the fused CLC protein. The absence of CLC to function as a scaffold protein for CME inhibits the internalization of G‐protein coupled receptors. Dropping the temperature back down causes the ELPs to re‐solublize and restores CME activity. ❧ Fusing a polymer to a small peptide is a common technique used to increase the molecular weight of a low molecular weight drug. Testing the hypothesis that increasing the molecular weight of a peptide by attachment of an ELP will increase that peptide’s hydrodynamic radius, Chapter 4 describes a surprising new formulation for the assembly of polymeric vesicles using L4F‐ELP fusion proteins. Below their phase transition, ELPs assume monomeric form. Attaching L4F, an alpha helical peptide derived from the lipid‐binding domain of the ApoA1 protein, drives ELP assembly into nanoparticles below the ELP assembly temperature, greatly increasing the hydrodynamic radius neither L4F nor the ELP would have alone. Cell assays show that despite this vesicle formation, the L4F peptide still retains anti‐immunogenic properties, holding promise for future systemic applications of this nanoparticle. |
| Keyword | polymers; cell biology; bioengineering; nanoparticles; elastin‐like polypeptide; clathrin; confocal microscopy; receptor mediated internalization; fusion protein |
| Language | English |
| Format (imt) | application/pdf |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m |
| Contributing entity | University of Southern California |
| Rights | Pastuszka, Martha K. |
| Physical access | The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given. |
| Repository name | University of Southern California Digital Library |
| Repository address | USC Digital Library, University of Southern California, University Park Campus MC 7002, 106 University Village, Los Angeles, California 90089-7002, USA |
| Repository email | cisadmin@lib.usc.edu |
| Filename | etd-PastuszkaM-2681.pdf |
| Archival file | uscthesesreloadpub_Volume14/etd-PastuszkaM-2681.pdf |
Description
| Title | Page 1 |
| Repository email | cisadmin@lib.usc.edu |
| Full text | i FLIPPING THE SWITCH ON PROTEIN ACTIVITY: ELASTIN-LIKE POLYPEPTIDES ASSEMBLE INTO CELL SWITCHES AND VESICLES by Martha K. Pastuszka A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERISTY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirement of the degree DOCTOR OF PHILOSOPHY (MOLECULAR PHARMACOLOGY AND TOXICOLOGY) May 2014 |
