Dissection of signaling networks using monobodies /

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Bibliographic Details
Author / Creator:Sha, Fern, author.
Ann Arbor : ProQuest Dissertations & Theses, 2015
Description:1 electronic resource (147 pages)
Format: E-Resource Dissertations
Local Note:School code: 0330
URL for this record:http://pi.lib.uchicago.edu/1001/cat/bib/10773275
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Other authors / contributors:University of Chicago. degree granting institution.
Notes:Advisors: Shohei Koide; Sean Crosson Committee members: Lucy Godley; Anthony Kossiakoff.
This item is not available from ProQuest Dissertations & Theses.
Dissertation Abstracts International, Volume: 77-02(E), Section: B.
Summary:Signaling networks are regulated by a complex array of protein functions and dysregulation of these functions often leads to aberrant signaling behavior and disease. Protein domains are the minimally functional unit of a protein and of a signaling network. These domains can be enzymatic domains, such as kinase and phosphatase domains, or interaction domains, such as Src-homology 2 (SH2). Determining how individual domains contribute to the overall signaling behavior helps us understand how signaling networks operate and how aberrant signaling arises, which may ultimately inform therapeutic strategies. However, selective perturbation of protein domains is challenging due to the high homology within protein domain families. I took advantage of a small and robust engineered protein scaffold based on the tenth human fibronectin type III (FN3) domain called a monobody in order to engineer potent and specific perturbants of protein domains for dissecting signaling networks.
I started by developing inhibitors for SH2 domain-containing proteins involved in interactions with GAB2, a critical scaffold protein in the BCR-ABL signaling network. I was successful in isolating exquisitely potent monobodies directed towards the SH2 domains of phospholipase C gamma 1 (PLCG1) and SH2 domain-containing phosphatase 2 (SHP2). Interactome analysis of these monobodies revealed they were essentially monospecific in cells, approaching the highest specificity achievable and these monobodies are the first selective reagents reported for PLCG1 and SHP2. The monobodies directed towards the C-SH2 of PLCG1 and both the N-SH2 and C-SH2 of SHP2 are inhibitors of phosphopeptides. Crystal structures of SHP2 SH2/monobody complexes reveal how inhibition and specificity is achieved. Interestingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity.
In order to demonstrate the utility of monobodies for dissecting signaling networks, the SHP2 SH2-binding monobodies were expressed in BCR-ABL-expressing cancer cells. Monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. These results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific tools for dissecting signaling networks in cells.
I next sought to develop monobodies targeting the protein tyrosine phosphatase (PTP) domain of SHP2, a critical regulator of ERK signaling. I successfully isolated monobodies that were potent inhibitors of SHP2 catalytic activity and the monobodies were specific for the PTP domain of SHP2 versus the close homologs SHP1 and PTP1B. These monobodies are the first selective inhibitors of this protein domain. A crystal structure of the SHP2 PTP/monobody complex reveals how inhibition and specificity is achieved. Along with the SHP2 SH2 monobodies, these reagents are among the first and most selective reagents to target a phosphatase, and will be of significant use for SHP2 studies. Together, this work demonstrates that monobodies are excellent perturbants of protein domains and are fantastic tools for dissecting the functions of protein domains in signaling networks.