プロテインの脂質改変はソルターゼ触媒によるトランスペプチデーションによって行われる
John M Antos1, Gwenn M Miller, Gijsbert M Grotenbreg
1Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Journal of the American Chemical Society
|November 8, 2008
まとめ
研究者たちは,ソルターゼAを用いて脂質をタンパク質に結合させるための化学酵素法を開発した. この技術は,哺乳類の細胞と結合し,膜やエンドソームに局所するリポタンパク質を効率的に生成します.
科学分野:
- バイオケミストリー バイオケミストリー
- 化学生物学 化学生物学とは
- プロテイン工学は,タンパク質の
背景:
- 脂質化は,タンパク質の機能と局所化にとって極めて重要です.
- サイト固有の脂化のための既存の方法は,しばしば複雑または非効率である.
研究 の 目的:
- サイト固有のタンパク質脂化のための一般的で効率的な化学酵素法を開発する.
- 細胞研究のための機能性リポタンパク質を作成する.
主な方法:
- 利用されたソルターゼA酵素は,部位特異なリガーションを行う.
- 脂質改変オリゴグリシンペプチドをタンパク質C-terminiに添加した.
- 結合後のソルターゼA酵素除去のための方法を開発しました.
主要な成果:
- さまざまな水性改変で優れた収穫率 (60-90%) を達成しました.
- 記述された化学酵素学的アプローチを用いて,リポタンパク質を成功裏に製造した.
- リポプロテインと哺乳類の細胞との脂質尾に依存する関連が実証されています.
結論:
- 開発された化学酵素法により,タンパク質の脂質化のための多用途のプラットフォームが提供されています.
- その結果生成されるリポタンパク質は,哺乳類の細胞と効果的に結合し,その細胞内に局所する.
- このテクニックは,リポプロテイン-タンパク質の相互作用と細胞の局所化の研究を容易にする.
関連する概念動画
01:26Protein Modifications in the RER
Modification of secretory and transmembrane proteins entering the rough ER begins in the ER lumen. These modifications aid in protein folding and stabilize the acquired tertiary structure. Protein modifications in the rough ER co-occur at different stages of protein folding.
Broadly, these modifications can be categorized into four main categories — glycosylation, formation of disulfide bonds, assembly of protein subunits, and specific proteolytic cleavages like removal of signal sequences.
Broadly, these modifications can be categorized into four main categories — glycosylation, formation of disulfide bonds, assembly of protein subunits, and specific proteolytic cleavages like removal of signal sequences.
01:27Post-translational Translocation of Proteins to the RER
A sizable fraction of proteins destined for ER are first synthesized in the cell cytosol and then transported across the ER membrane–a process called post-translational translocation. Similar to cotranslationally translocated proteins, these proteins also use the Sec translocon complex to enter the ER lumen.
Targeting proteins to the ER
Hsp40 and Hsp70 chaperone molecules bind the translated proteins in the cytosol to prevent their folding. The chaperone binding helps to keep the signal...
Targeting proteins to the ER
Hsp40 and Hsp70 chaperone molecules bind the translated proteins in the cytosol to prevent their folding. The chaperone binding helps to keep the signal...
01:45Overview of Protein Sorting and Transport
Eukaryotic cells have different membrane-bound organelles with distinct protein requirements. The process by which proteins are targeted to a specific organelle is called protein sorting.
Protein sorting can be of two types: signal-based sorting and vesicle-based trafficking. In signal-based sorting, specific amino acid sequences called sorting signals target proteins to the proper location inside the cell either via gated transport or by protein translocation. In gated transport, folded...
Protein sorting can be of two types: signal-based sorting and vesicle-based trafficking. In signal-based sorting, specific amino acid sequences called sorting signals target proteins to the proper location inside the cell either via gated transport or by protein translocation. In gated transport, folded...
01:41Signal Sequences and Sorting Receptors
Signal sequences are short amino acid sequences that guide newly synthesized proteins to their proper location within the cell. Classical signal sequences are fifteen to sixty amino acids long and present at the N-terminus of a polypeptide chain. Each signal sequence has a conserved segment of basic residues towards their N terminus, a hydrophobic core, and a C-terminus rich in polar residues. The C-terminus also contains a signal cleavage site and features a -3 -1 sequence motif. The -3-1...
01:20Cotranslational Protein Translocation
Translocation of proteins across membranes is an ancient process that occurs even in bacteria and archaebacteria. In fact, the components of the translocation machinery are still conserved between prokaryotes and eukaryotes.
Sec61 channel partners for cotranslational translocation
During cotranslational translocation, the Sec61 channel partners with the signal recognition particle (SRP), the signal recognition particle receptor (SR), and the ribosomes to transport the nascent polypeptide chain...
Sec61 channel partners for cotranslational translocation
During cotranslational translocation, the Sec61 channel partners with the signal recognition particle (SRP), the signal recognition particle receptor (SR), and the ribosomes to transport the nascent polypeptide chain...
01:26Insertion of Single-pass Transmembrane Proteins in the RER
Integral membrane proteins are proteins adhered to the lipid bilayer of a cell organelle or membrane. They can be of two types: transmembrane integral proteins that span the lipid bilayer and monotopic proteins that are attached to either side of the membrane but do not pass through it.
Integral transmembrane proteins possess transmembrane and extra membrane domains. The transmembrane domains are primarily made of 20-25 hydrophobic amino acids arranged in a helical secondary confirmation. These...
Integral transmembrane proteins possess transmembrane and extra membrane domains. The transmembrane domains are primarily made of 20-25 hydrophobic amino acids arranged in a helical secondary confirmation. These...