STAM, AMSH와 Ubiquitin 사이의 상호작용에 대한 분자적 특성 : Molecular Characterization of Interaction among STAM, AMSH, and Ubiquitin
- 발행기관 고려대학교
- 발행년도 2006
- 학위수여년월 2006. 2
- 학위명 석사
- 학과 대학원 분자세포생물학과 분자생물학전공
- 식별자(기타) DL:000016999554
- 서지제어번호 000045281851
초록/요약
STAM (signal trandusing adaptor molecule) protein은 VHS(Vps27/Hrs/STAM)와 ubiquitin interacting motif를 가지고 있어 ubiquitin 과 ubiquitination 된 protein과 binding 한다. 그래서 STAM 은 endosome 내에서 ubiquitin과 관련된 receptor regulation sorting에 중요한 역할을 한다. AMSH(Associated Molecule with the SH3 domain of STAM) protein은 STAM 의 SH3 domain 과 binding 하고 JAMM(JAB1/MPN/Mov34 metalloenzyme) domain 을 가지고 있어 선택적으로 K63으로 linked된 polyubiquitin chain을 monoubiquitin으로 만드는 isopeptidase activity를 가지고 있는 것으로 최근 보고되었다. 먼저 STAM 과 AMSH의 interaction을 cell based system을 통해 확인하였고, in vitro 에서도 binding 함을 증명하였고, AMSH의 2nd PXXP motif 와 STAM 의 SH3 domain을 통해서 이러한 interaction이 이루어짐을 확인하였다. 아울러 우리는 AMSH의 isopeptidase activity를 확인하였고 N-terminal part를 제거한 C-term part의 AMSH 만으로도 full length AMSH 와 비슷한 정도의 activity 가지고 있음을 찾아내었다. 또한 STAM은 K63 linked 된 polyubiquitin chain과도 binding함을 확인하였다. 그러나 monoubiquitin 에 대해서는 아주 약한 binding activity를 보였다. 우리는 또한 AMSH가 STAM에 binding 된 K63 linked polyubiquitin chain에 대해서도 isopeptidase activity를 증가시킨 확인할 수 있었다. 그러나 STAM binding에 관여하는 2nd PXXP motif 가 deletion 된 AMSH에 경우에는 STAM–K63 linked polyubiquitin chain에 대해서isopeptidase activity 를 나타내지 않았다. STAM에 binding 한 K63 tetra 나 di ubiquitin chain 에서도 동일한 결과를 얻을 수 있었다. 이러한 결과로 보아 STAM의 SH3 domain과 AMSH의 2nd PXXP motif 에 의한 binding을 통해서 AMSH는 STAM에 binging 된 K63 linked polyubiquitin chain에 증가된 isopeptidase activity를 나타낸다.
more초록/요약
STAM (signal transducing adaptor molecule) proteins play a role in the regulation of receptor sorting in the endosome, by binding to ubiqutin and ubiquitinated proteins through the tandemly located VHS (Vps27/Hrs/STAM) domain and ubiquitin- interacting-motif. AMSH (Associated Molecule with the SH3 domain of STAM), a JAMM (JAB1/MPN/Mov34metalloenzyme) domain–containing protein that associates with the SH3-domain of STAM, has been reported to have the isopeptidase activity against K63-linked polyubiquitin chains to generate free ubiquitin. I have checked the interactions between STAM and AMSH in cell-based system and in vitro, and confirmed that the 2nd PXXP motif in AMSH binds to the SH3 domain of STAM. It is also found that the mutant lacking N-terminal half of AMSH has the deubiquitination activity to K63-polyubiquitin chain in vitro, similar to the full-length protein. STAM was able to bind to K63-linked polyubiquitin chains (di- to hepta-ubiquitin), however showed a very weak binding activity to mono-ubiquitin. AMSH also has the deubiquitination activity to STAM bound K63-ployubiquitin (di- to hepta-ubiquitin) chain even at the low temperature but, when the 2nd PXXP motif was deleted from AMSH, it lost that activity. The same result for STAM bound-K63 tetra- and di-ubiquitin chain has been observed. These results indicate that AMSH has the isopeptidase activity toward STAM bound-K63 ubiquitins enhanced by binding to the SH3 domain of STAM through the 2nd PXXP motif.
more목차
Table of Contents
List of Figures
Abbreviations
Abstract
1. Introduction
1.1 Multivesicular body pathway
1.1.1 Regulation of cargo entry into the multivesicular-body sorting pathway
1.1.2 Monoubiquitination as the MVB sorting signal
삭제됨: Schmatic structure
1.2 STAM in signal transduction삭제됨:
1.2.1 Structure and localization of the Hrs/STAM complex
1.2.2 The Hrs/STAM complex as the ub receptor in MVB sorting
삭제됨: The
1.3 Functions of AMSHdeubnat enzymes to STAM bpartner
1.3.1 Deubiquitinating enzyme (DUB) UBPY
1.3.2 AMSH (associated molecule with the SH3 domain of STAM)
2. Materials and Methods
2.1 Methods
2.1.1 Preparation of plasmid DNA
2.1.2 Ligation and Transformation
2.1.3 Construction of STAM and STAM mutant
2.1.4 Construction of AMSH and AMSH mutant
2.1.5 Purification of STAM-His
2.1.6 Purified Glutathione S Transferase (GST) fus prote
2.1.7 Gel electrophoresis
2.1.8 Deubiquitination assay
2.1.9 Cell culture and transfection
2.1.10 Pull down assay with GST fusion protein
2.1.11 Immunoprecipitation and immunoblotting
2.2 Materials
2.2.1 E.coli strains and plasmids
2.2.2 Cell line and plasmid for mammalian cell expression
2.2.3 Chemicals and enzymes
3. Results
3.1 Interaction of STAM and AMSH
3.1.1 STAM-AMSH interaction
3.1.2 STAM-AMSH interaction
3.1.3 STAM-ubiquitin interaction
3.1.4 AMSH-ubiquitin interaction
3.2 Deubiquitination by AMSH
3.2.1 AMSH shows deubiquitinating activity
3.2.2 AMSH deubiquitinating activity to STAM-K63 polyubiquitin
4. Discussion
5. References
6. 국문요약
List of Figures
Figure 1. Membrane traffic via the endosome
Figure 2. Schematic domain organizations of the Hrs/STAM1
Figure 3. Schematic domain organizations of the AMSH
Figure 4. Schematic domain of the STAM mutants and AMSH mutants
Figure 5. Co-immunoprecipitation between AMSH and STAM
Figure 6. Binding of STAM to AMSH in vitro
Figure 7. STAM bind K63 linked polyubiquitin chain
Figure 8. AMSHUbn eract ro
삭제됨: s
Figure 9. Deubiquitinating activity of AMSH and mutant AMSHs
Figure 10. AMSH deubiquitinating activity to STAM-ubiquitin chain
Figure 11. AMSH deubiquitinating activity to STAM mutant-ubiquitin chain -
Figure 12. AMSH deubiquitinating activity to STAM- di-and tetra-ubiquitin
