関連する実験動画

Updated: Jun 5, 2025

Oligopeptide Competition Assay for Phosphorylation Site Determination

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

超分子主体:ゲスト配列サイト選択的にペプチドリン酸化とキナーゼ活動を認識する

Junyi Chen¹, Parisa Fasihianifard¹, Ria Lian¹

¹Department of Chemistry, University of California─Riverside, Riverside, California 92521, United States.

Journal of the American Chemical Society

|December 16, 2024

まとめ

この研究は,高精度でリン酸化ペプチドを検出するために染料と宿主を使用する新しい方法を導入しています. このシステムは,キナーゼベースの薬物スクリーニングに不可欠な,リン酸化部位の選択的なモニタリングを可能にします.

科学分野:

超分子化学
分析化学
生物化学

背景:

リン酸化ペプチドは細胞の信号伝達において重要な役割を果たします.
リン酸化部位と状態の正確な検出は,生物学的プロセスと疾患メカニズムを理解するために不可欠です.
リン酸化ペプチドを検出する現在の方法は複雑であり,修正が必要である.

研究の目的:

リン酸化ペプチドを識別するための選択的で敏感な方法を開発する.
ペプチドの様々なリン酸化状態と部位を区別する.
キナーゼ活動などの複雑な生物環境で適用可能なシステムを作成する.

主な方法:

カチオンのスタイリルピリジニウム染料と水溶性深い洞穴と宿主の相乗効果を活用する.
主要な2つの相互作用メカニズムの調査:直接の染料-ペプチド相互作用と宿主:染料-ペプチド三重複合体の形成.
データ分析とリン酸化パターンの差別化のための機械学習アルゴリズムを使用します.

主要な成果:

酸化ペプチドの場所と状態の選択的認識を達成した.
ペプチドの修正なしに,酸化状態と位置に基づいて,異なる光反応を示した.
最低3つの成分配列を使用して,異なる位置のセリンリン酸化を成功裏に区別しました.
タンパク質キナーゼA (PKA) とその共因子の存在における機能性を示した.

結論:

開発されたシステムは,リン酸化ペプチドの差分感知のための汎用的なプラットフォームを提供します.
PKAモチーフのような特定の部位で選択的にリン酸化をモニタリングする能力は,生物学的研究にとって有望です.
このアプローチは,キナーゼベースの薬物スクリーニングと診断において,重要な可能性を持っています.

さらに関連する動画

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Published on: November 29, 2014

Identification of Kinase-substrate Pairs Using High Throughput Screening

Identification of Kinase-substrate Pairs Using High Throughput Screening

Published on: August 29, 2015

関連する概念動画

Protein Kinases and Phosphatases

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...

Assembly of Signaling Complexes

Assembly of Signaling Complexes

Multiprotein signaling complexes are formed in a dynamic process involving protein-protein interactions at the cytoplasmic domain of transmembrane receptors or enzymatic and non-enzymatic proteins associated with the receptor. These complexes ensure the activation and propagation of intracellular signals that regulate cell functions.
Interaction domains in cell signaling
Interaction domains recognize exposed features of their binding partners containing post-translationally modified sequences,...

Ligand Binding and Linkage

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...

Phosphorylation

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...

Receptor Tyrosine Kinases

Receptor Tyrosine Kinases

Receptor tyrosine kinases or RTKs are membrane-bound receptors that phosphorylate specific tyrosine on protein substrates. RTKs regulate cellular growth, differentiation, survival, and migration. They contain an extracellular ligand binding domain, a transmembrane domain, and a cytosolic tail with intrinsic kinase activity. Several extracellular signaling molecules activate RTKs in one or more ways and relay the signal downstream. Ligands such as platelet-derived growth factor (PDGF) or...

Allosteric Proteins-ATCase

Allosteric Proteins-ATCase

Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...