東華大學圖書館 |

語系: 繁體中文

說明(常見問題)

回圖書館首頁

手機版館藏查詢

登入

回首頁

切換: 標籤 | MARC模式 | ISBD

Using SAMDI-MS and Peptide Arrays to...

Huang, Che-Fan.

FindBook

Google Book

Amazon

博客來

Using SAMDI-MS and Peptide Arrays to Study Protein Tyrosine Phosphatases and Discover New Cellular Regulatory Mechanisms.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Using SAMDI-MS and Peptide Arrays to Study Protein Tyrosine Phosphatases and Discover New Cellular Regulatory Mechanisms./
作者:	Huang, Che-Fan.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2021,
面頁冊數:	162 p.
附註:	Source: Dissertations Abstracts International, Volume: 82-12, Section: B.
Contained By:	Dissertations Abstracts International82-12B.
標題:	Chemistry. -
電子資源:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28496444
ISBN:	9798516064487

Using SAMDI-MS and Peptide Arrays to Study Protein Tyrosine Phosphatases and Discover New Cellular Regulatory Mechanisms.
Huang, Che-Fan.

Using SAMDI-MS and Peptide Arrays to Study Protein Tyrosine Phosphatases and Discover New Cellular Regulatory Mechanisms. - Ann Arbor : ProQuest Dissertations & Theses, 2021 - 162 p.

Source: Dissertations Abstracts International, Volume: 82-12, Section: B.

Thesis (Ph.D.)--Northwestern University, 2021.

This item must not be sold to any third party vendors.

The opposing activities of phosphatases and kinases determine the phosphorylation status of proteins, yet kinases have received disproportionate attention in studies of cellular processes, with the roles of phosphatases remaining less understood. This dissertation describes the use of self-assembled monolayer laser desorption/ionization mass spectrometry (SAMDI-MS) together with peptide arrays to directly assay phosphatase activity, profile their substrate specificity and reveal novel cellar regulatory mechanisms protein tyrosine phosphatases (PTPs) are critically involved in. SHP2 is the first identified oncogenic phosphatase. In the first part of this work, we applied high-throughput SAMDI-MS assays for SHP2 drug discovery and reported an FDA-approved compound, adapalene, as a potent SHP2 inhibitor. We identified that the adamantyl functional group is crucial for its inhibition. SHP2 disease mutants were profiled with phosphotyrosine-containing peptide arrays to study the alteration of substrate specificity. We found that some mutants particularly favored aromatic residues at the -1 position adjacent to a phosphotyrosine.Extending the study to the whole PTP family in human proteome, twenty-two tyrosine phosphatases were characterized with the arrays to give a profile of their specificities. An analysis of the data revealed that certain residues in the substrates had a conserved effect on activity for all enzymes tested, including the general rule that inclusion of a basic lysine or arginine residue on either side of the phosphotyrosine decreased activity. This insight also provides a new perspective on the role of an R1152Q mutant in the insulin receptor, which is known to exhibit a lower phosphorylation level, and which our work suggests may be due to an increased activity towards phosphatase enzymes.We then identified more than 6,000 cancer mutations involving basic residues adjacent to known phosphotyrosine sites through a database search. Using two β-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K), we showed that T653-basic mutant β-catenins are less efficiently dephosphorylated by SHP1 phosphatase, leading to sustained Y654 phosphorylation and elevated downstream Wnt signal. This example rationalized how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6,000+ potentially relevant cancer mutations.Lastly, we showed that the same principle can be applied for a phosphorylation/chare-altering modification crosstalk via PTP. Using citrullination for example, we found the modification neutralized the basic arginine residue and increased the peptide dephosphorylation kcat/KM by 2.3-fold. This novel regulatory mechanism is generalizable because all PTPs lack activity towards substrates that have a basic residue proximal to the phosphotyrosine. This dissertation demonstrates the use of SAMDI-MS to provide a rapid and quantitative assay of phosphatase enzymes will be important to gaining a more complete understanding of the biochemistry and biology of this important enzyme class.

ISBN: 9798516064487Subjects--Topical Terms:

516420
Chemistry.
Subjects--Index Terms:

Enzyme specificity

Using SAMDI-MS and Peptide Arrays to Study Protein Tyrosine Phosphatases and Discover New Cellular Regulatory Mechanisms.
LDR:04466nmm a2200409 4500 001 2282956
005 20211022115808.5
008 220723s2021 ||||||||||||||||| ||eng d
020 $a 9798516064487
035 $a (MiAaPQ)AAI28496444
035 $a AAI28496444
040 $a MiAaPQ $c MiAaPQ
100 1 $a Huang, Che-Fan. $0 (orcid)0000-0002-5799-1533 $3 3561842
245 1 0 $a Using SAMDI-MS and Peptide Arrays to Study Protein Tyrosine Phosphatases and Discover New Cellular Regulatory Mechanisms.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 2021
300 $a 162 p.
500 $a Source: Dissertations Abstracts International, Volume: 82-12, Section: B.
500 $a Advisor: Mrksich, Milan.
502 $a Thesis (Ph.D.)--Northwestern University, 2021.
506 $a This item must not be sold to any third party vendors.
520 $a The opposing activities of phosphatases and kinases determine the phosphorylation status of proteins, yet kinases have received disproportionate attention in studies of cellular processes, with the roles of phosphatases remaining less understood. This dissertation describes the use of self-assembled monolayer laser desorption/ionization mass spectrometry (SAMDI-MS) together with peptide arrays to directly assay phosphatase activity, profile their substrate specificity and reveal novel cellar regulatory mechanisms protein tyrosine phosphatases (PTPs) are critically involved in. SHP2 is the first identified oncogenic phosphatase. In the first part of this work, we applied high-throughput SAMDI-MS assays for SHP2 drug discovery and reported an FDA-approved compound, adapalene, as a potent SHP2 inhibitor. We identified that the adamantyl functional group is crucial for its inhibition. SHP2 disease mutants were profiled with phosphotyrosine-containing peptide arrays to study the alteration of substrate specificity. We found that some mutants particularly favored aromatic residues at the -1 position adjacent to a phosphotyrosine.Extending the study to the whole PTP family in human proteome, twenty-two tyrosine phosphatases were characterized with the arrays to give a profile of their specificities. An analysis of the data revealed that certain residues in the substrates had a conserved effect on activity for all enzymes tested, including the general rule that inclusion of a basic lysine or arginine residue on either side of the phosphotyrosine decreased activity. This insight also provides a new perspective on the role of an R1152Q mutant in the insulin receptor, which is known to exhibit a lower phosphorylation level, and which our work suggests may be due to an increased activity towards phosphatase enzymes.We then identified more than 6,000 cancer mutations involving basic residues adjacent to known phosphotyrosine sites through a database search. Using two β-catenin mutants associated with cancer (T653R/K) and a mouse model for intellectual disability (T653K), we showed that T653-basic mutant β-catenins are less efficiently dephosphorylated by SHP1 phosphatase, leading to sustained Y654 phosphorylation and elevated downstream Wnt signal. This example rationalized how basic mutations proximal to phosphotyrosines can restrict counter-regulation by phosphatases, providing new mechanismistic and treatment insights for 6,000+ potentially relevant cancer mutations.Lastly, we showed that the same principle can be applied for a phosphorylation/chare-altering modification crosstalk via PTP. Using citrullination for example, we found the modification neutralized the basic arginine residue and increased the peptide dephosphorylation kcat/KM by 2.3-fold. This novel regulatory mechanism is generalizable because all PTPs lack activity towards substrates that have a basic residue proximal to the phosphotyrosine. This dissertation demonstrates the use of SAMDI-MS to provide a rapid and quantitative assay of phosphatase enzymes will be important to gaining a more complete understanding of the biochemistry and biology of this important enzyme class.
590 $a School code: 0163.
650 4 $a Chemistry. $3 516420
650 4 $a Biochemistry. $3 518028
650 4 $a Cellular biology. $3 3172791
650 4 $a Biomedical engineering. $3 535387
650 4 $a Molecular biology. $3 517296
653 $a Enzyme specificity
653 $a High-throughput experiments
653 $a Peptide arrays
653 $a Phosphatases
653 $a SAMDI-MS
653 $a β-Catenin
690 $a 0485
690 $a 0487
690 $a 0379
690 $a 0541
690 $a 0307
710 2 $a Northwestern University. $b Chemistry. $3 1030729
773 0 $t Dissertations Abstracts International $g 82-12B.
790 $a 0163
791 $a Ph.D.
792 $a 2021
793 $a English
856 4 0 $u https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28496444