Name: deacetylation assays to unravel the interplay between sirtuins (sirt2) and specific protein-substrates
Uploaded: 2016-02-27T14:00:00.000+00:00
Duration: 14 min 32 s
Description: Watch this Scientific Journal Video about Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates at JoVE.com

우리는하고 싶은 AV로 Lefkofsky 가족 재단 / 리즈와 에릭 Lefkofsky 혁신 연구 상 - 여기에 설명이 프로젝트는 NIH / NCI (NCI-R01CA182506-01A1)에서 부여에 의해뿐만 아니라 로버트 H. 루리 종합 암 센터 지원 중요한 읽기와이 원고를 편집하기위한 실험실의 구성원 (캐롤 O&#39;Callaghan와 엘리자베스 앤 웨인) 감사합니다.

체외 탈 아세틸 분석 1. <ol><li> SIRT2의 정제 <ol><li> 10 % FBS와 항생제 8 ML의 DMEM에서 배양하고 70 % 합류 다음날로 37 ° C, 5 % CO 2 조직 배양 인큐베이터에서 배양하는 HEK 293T 세포를 10㎝ 배양 플레이트를 준비한다. </li><li> 다음 날, 공동 트랜 4 μg의 pCDH - 순수하지 않아-GFP-SIRT2-국기 (우리의 실험실에서 만든 lentivector), 4 μg의 pCMV- dR8.2 dvpr (포장 벡터) 0.5 μg의을 pCMV-VSV-G (봉투 벡터) 3 μL PEI / μg의 DNA의 비율 18 사용 폴리에틸렌 이민 (PEI). </li><li> 12 시간 후 미디어를 변경합니다. </li><li> 36-48 시간 후 상층 액을 수집합니다. </li><li> 4 ° C에서 5 분 동안 1,000 XG에서 원심 분리하여 이물질을 제거합니다. </li><li> 0.22 μm의 필터를 통해 여과 한 후, SIRT2의 렌티 바이러스 1 ml의 분취 량을 -80 ℃에서 보관하십시오. </li><li> 바이러스를 사용하지 않는 경우에 녹여 얼음 SIRT2 렌티은 (는 -80 ° C에서 바이러스를 저장하는 것이 중요). </li><li> 8 μg의 / ㎖의 폴리 브렌의 존재 렌티 1 ㎖로 80~90% 합류 된 HEK 293T 세포를 6 웰 플레이트를 감염시킨다. </li><li> 다음 날 (24 시간)까지 37 ° C 배양기에서 바이러스에 감염된 세포를 놓습니다. </li><li> 24 시간 후 배지를 교체합니다. </li><li> 감염 후 48 시간은 형광 현미경으로 GFP를 양성 세포를 감지하여 감염의 효율성을 확인합니다. </li><li> 감염 효율이 좋은 경우에 (적어도 40-50% 감염된 세포)은 배양 배지를 세포를 발현하는 안정적인 SIRT2 대한 선택이 μg의 / ml의 퓨로 마이신을 함유하는 배지를 교체한다. 주 :이 약 2 주가 소요 및 배지를 3-4 일마다 변경된다. 비 감염된 세포는 SIRT2의 렌티 바이러스 성장을이 선택 기간에만 감염된 세포를 통해 죽는다. </li><li> 을 정제를 시작하기 전에 10 % SDS-PAGE 겔상에서 총 단백질 40 μg의 실행 후 (1000 희석 1) 항 - 플래그 항체를 사용하여 웨스턴 블롯에 의해 플래그 - 태그 SIRT2 발현 분석ication 과정 (권장) 19, 20. </li><li> 1 활성 SIRT2, 분할 세포를 정화하려면 안정적으로 국기 - SIRT2를 과발현 HEK 293T 세포의 충분한 요리를하기 위해 트립신 3 비율 (텐 10cm 요리 후속 실험에 대한 충분한 단백질의 정제를 수 있습니다). <ol><li> 상기 세포를 Trypsinize 배지를 제거하고 멸균 DPBS로 세포를 세척하여 잔류 혈청을 제거하기 위해. 천천히, 세포 단층을 포함 30 ~ 45 초 동안 상온에서 부화 0.25 % 트립신 - EDTA 용액 2.5 ml를 추가합니다. 세포가 분리 시작할 때까지 트립신 - EDTA 용액을 제거한 후, 37 ° C에서 품어. 그런 문화 매체를 추가하고 새로운 요리에 세포를 전송합니다. </li></ol></li><li> 4 ° C에서 10 분 동안 1,000 XG에서 원심 분리 트립신 처리하여 세포를 PBS로 두 번 세포를 씻어 수집, 배지를 제거합니다. </li><li> 500 ㎕의 용해 완충액 (20 mM의 HEPES pH가 7.9, 18 mM의 KCl을 0.2 mM의 EGTA, 1.5 밀리미터의 MgCl 2를 Lyse 세포 펠렛, 프로테아제 억제제 칵테일과 일정한 회전에서 4 ° C에서 30 분 동안 1 μM TSA를 포함하여 20 % 글리세롤, 0.1 % NP-40). </li><li> 새로운 튜브 15 4 ° C에서 최소 및 전송 상층 액 14,000 XG에 원심 분리기. </li><li> 후술하는 바와 같이 아가 로스 비드를 결합하는 항 - 플래그 항체를 사용하여 면역 침전 법을 수행한다. <ol><li> 단백질 분해물 (10 ~ 20 mg의 총 단백질)에 구슬 아가로 오스 복합 안티 - 플래그 항체의 200 ~ 300 μl를 추가합니다. </li><li> 일정한 교반과 함께 4 ° C에서 밤새 품어. </li><li> 4 ℃에서 5 분 동안 1,000 XG에서 원심 분리하여 immunoprecipitates를 수집합니다. </li><li> 4 ℃에서 5 분 동안 1,000 XG에서 펠렛을 10 ml의 용해 버퍼 5 회 반복한다. 각 세척 한 후, 비드 펠렛 및 버퍼 A.으로 뜨는 교체 </li></ol></li><li> (프로테아제 억제제 칵테일 1 PBS에서 μm의 TSA 포함) 1 배 플래그 펩타이드 솔루션을 준비합니다. </li><li> 1 배 플래그 peptid 500 μl를 추가아가로 오스 비즈에 전자 솔루션 및 일정 회전에서 4 ° C에서 30 분 동안 품어. </li><li> 4 ℃에서 2 분 동안 6,000 XG에서 원심 분리하여 상층 액을 수집합니다. </li><li> 앞의 두 단계를 반복합니다. </li><li> 4 ° C에서 1 분 동안 15,000 XG에 필터 튜브와 원심 분리를 이용하여 상층 액에서 구슬을 제거합니다. </li><li> 최종 단백질 농도를 1 μg의 때까지 / 한외 여과막을 사용하여 용출 μL 샘플을 농축시킨다. </li><li> 용출 된 시료의 1 μg의를 사용하여 수행하는 전기 영동으로 정제 과정을 확인합니다. </li><li> 시중에서 판매하는 염색 액에 얼룩 젤 SIRT2 정화를 확인합니다. </li><li> -80 ° C에서 정제 SIRT2을 유지합니다. </li></ol></li><li> 아세틸 단백질 기판의 정제 <ol><li> 약 70 %의 합류 다음날로 HEK의 293T 세포를 10 ㎝의 배양 접시를 준비합니다. </li><li> 국기의 5 μg의와 다음 날, 공동 트랜 세포는 플라스미드 벡터 expressin 태그g 단백질 기질뿐만 아니라 3 μL PEI / μg의 DNA의 비율로 PEI를 사용하여 단백질의 아세틸 화 수 특정 아세틸 트랜스퍼 5 μg의. 주 : 특정 아세틸 트랜스퍼 라제는 공지되어 있지 않은 경우, 예컨대 P300, CBP, GCN5, Tip60과 PCAF 공지 히스톤 아세틸 트랜스퍼 (HAT들)의 혼합물로 공동 트랜. </li><li> 12 시간 후 매체를 변경합니다. </li><li> 일 세포를 용해시키기 전에, 아세틸 라제를 억제하여 단백질 - 기재의 아세틸 화 수준을 극대화하는 TSA / NAM을 포함하는 배지로 배양액을 대체하여 밤새 1 μM의 TSA 2 mM의 니코 티나 마이드 (NAM)로 세포를 처리한다. </li><li> 48 시간 형질 전환 한 후, 배지를 제거 4 ℃에서 10 분 동안 1,000 XG에서 원심 분리 트립신으로 세포를 PBS로 두 번 세포를 씻어 수집합니다. </li><li> 접시 당 500 ㎕의 용해 완충액에 세포를 Lyse 펠릿 (20 mM의 HEPES pH가 7.9, 18 mM의 KCl을 0.2 mM의 EGTA, 1.5 mM의 MgCl2를, 20 % 글리세롤, 0.1 % NP-40) 프로테아제 I을 포함nhibitors 칵테일, 1 μM의 TSA와 일정한 회전에서 4 ° C에서 30 분 동안 2 MM의 NAM. </li><li> 새로운 튜브 15 4 ° C에서 최소 및 전송 상층 액 14,000 XG에 원심 분리기. </li><li> 후술하는 바와 같이 아가 로스 비드를 결합하는 항 - 플래그 항체를 사용하여 면역 침전 법을 수행한다. <ol><li> 단백질 분해물 (10 ~ 20 mg의 총 단백질)에 구슬 아가로 오스 복합 안티 - 플래그 항체의 200 ~ 300 μl를 추가합니다. </li><li> 일정한 교반과 함께 4 ° C에서 밤새 품어. </li><li> 4 ℃에서 5 분 동안 1,000 XG에서 원심 분리하여 immunoprecipitates를 수집합니다. </li><li> 4 ℃에서 5 분 동안 1,000 XG에서 펠렛을 10 ml의 용해 버퍼 2 회 반복한다. 각 세척 한 후, 비드 펠렛 및 용해 버퍼 A.으로 뜨는 교체 </li><li> 4 ° C에서 5 분 동안 1,000 XG에서 NAM없이 펠렛을 10 ml의 용해 버퍼 3 회 반복한다. 각 세척 한 후, 비드 펠렛 및 그것은 importa이다 용해 버퍼 A.으로 뜨는 교체NT를 탈 아세틸 화 반응에 대한 NAM를 이월 할 수 없습니다. </li></ol></li><li> (단백질 분해 효소 억제제와 PBS에서 1 μM TSA 포함) 1 배 플래그 펩타이드 솔루션을 준비합니다. </li><li> 아가로 오스 비즈로 1 배 플래그 펩티드 용액 500 μl를 추가하고 일정한 회전에서 4 ° C에서 30 분 동안 품어. </li><li> 4 ℃에서 2 분 동안 6,000 XG에서 원심 분리하여 상층 액을 수집합니다. </li><li> 앞의 두 단계를 반복합니다. </li><li> 4 ° C에서 1 분 동안 15,000 XG에 필터 튜브와 원심 분리를 이용하여 상층 액에서 구슬을 제거합니다. </li><li> 단백질 농도를 1 μg의 / UL 때까지 한외 여과막을 사용하여 용출 샘플을 농축시킨다. </li><li> 4-12 % SDS-PAGE 겔상에서 용출 시료 1 μL를 실행하여 전기 영동을 수행한다. </li><li> (1,000 희석 1) 단백질 기판 (희석 사용되는 특정 항체에 따라 다름) 서부 AC-K에 대한 항체를 사용하여 블로 팅에 의해 단백질 기판의 아세틸 화를 확인합니다. </li><li> 푸리 유지들이었다는 -80 ° C에서 단백질 기판을 아세틸. </li></ol></li><li> 체외 탈 아세틸 반응 <ol><li> 탈 아세틸 버퍼 B를 준비 (에 50 mM Tris-HCl, pH 7.5, 150 mM의 염화나트륨, 1 ㎜의 MgCl 2 0.5 μM TSA) </li><li> 20 ㎕의 최종 부피 (표 1)에 서로 다른 튜브에 3 개의 다른 반응을 준비합니다. </li><li> (선택 사항) SIRT2에 의해 단백질 기판의 탈 아세틸를 확인하기 위해 추가 반응을 포함합니다. (NAM 대신 아세틸 라제 활성 SIRT2의 반응 (pCDH - 순수하지 않아-GFP-SIRT2 H187Y -flag의 lentivector 사용 후이 1.1에 기술 된 프로토콜을 사용하여 수행 할 수 있습니다) 정제 탈 아세틸 널 (null) 돌연변이 SIRT2의 2 μg의 추가 또는 10 밀리미터에게 니코틴을 추가 ) SIRT2의 탈 아세틸 화 활성을 억제하는 반응 (표 1). </li><li> 일정하게 교반하면서 30 ℃에서 3 시간 동안 반응을 품어. </li><li> F 배 샘플 완충액 20 μl를 첨가하여 반응을 정지하고, 샘플을 비등또는 95 ℃에서 10 분. </li><li> 4-12 % SDS-PAGE 겔상에서 반응 혼합물을 실행하여 전기 영동을 수행하고 항 AC-K 항체 (1 : 1000 희석)를 사용하여 웨스턴 블롯에 의해 단백질 기판의 아세틸 화 상태를 검출 19,20한다. </li></ol></li></ol> 생체 탈 아세틸 분석 2. <ol><li> 배양 된 세포에서 과발현 SIRT2 <ol><li> HEK의 293T 세포와 10cm 배양 접시 안정적으로 pCDH - 순수하지 않아-GFP-빈 벡터를 과발현을 준비 pCDH - 순수하지 않아-GFP-SIRT2 - 플래그 및 pCDH - 순수하지 않아-GFP-SIRT2 H187Y -flag는 (1.1에서 설명) 70 일합니다 %의 합류. </li><li> 대안 적으로, 세포를 약 70 % 합류로 10cm 배양 접시를 준비하고, 3 μL PEI / μg의 DNA의 비율로 PEI를 사용하여 상기 언급 한 벡터 5㎍을 사용하여 일시적 과발현을 수행한다. </li><li> 국기-t의 과발현을 입증 (1,000 희석 1) 안티 - 플래그 항체를 이용하여 총 세포 추출물의 서쪽 블로 팅을 수행SIRT2 19 agged. </li></ol></li><li> 배양 된 세포의 최저 SIRT2에 RNA 간섭 <ol><li> 약 70 % 합류 다음날으로 HEK 293T 세포에의 10㎝ 배양 플레이트를 준비한다. </li><li> 다음 날, 공동 트랜 4 μg의 pLKO1 - 순수하지 않아 - 쉬 SIRT2 (lentivector) 19, 4 μg의 pCMV- dR8.2 dvpr (포장 벡터) 0.5 μg의을 pCMV-VSV-G (봉투 벡터) 18 사용 폴리에틸렌 이민 (PEI) 3 μL PEI / μg의 DNA가 비율. 참고 : SIRT2을 쓰러 뜨린를 들어, 우리가 RNAi의 컨소시엄 (TRC)가 설계 한 pLKO.1의 렌티 바이러스 벡터에 간단한 머리핀 shRNA를 사용합니다. </li><li> shSIRT2의 렌티 바이러스를 생산하고 SIRT2를 넉다운하는 표적 세포를 감염 1.1에 설명 된대로 절차를 따르십시오. </li><li> 10 % SDS-PAGE 겔에 총 단백질의 40 μg의를 실행 한 후 효율적인 SIRT2 최저를 보여 (1,000 희석 1) 항 SIRT2 항체를 이용하여 총 세포 추출물의 웨스턴 블로 팅을 수행합니다. </li><li> 또한,세포를 약 70 %의 합류로 10cm 문화 요리를 준비하고 제조업체의 지침에 따라 siRNA를를 사용 SIRT2의 과도 최저를 수행합니다. </li><li> 10 % SDS-PAGE 겔에 총 단백질의 40 μg의를 실행 한 후 효율적인 SIRT2 최저를 보여 (1,000 희석 1) 항 SIRT2 항체를 이용하여 총 세포 추출물의 웨스턴 블로 팅을 수행합니다. </li></ol></li><li> 면역 및 면역 블 롯팅은 배양 세포에서 아세틸 레벨을 감지하는 <ol><li> 12 시간 SIRT2를 과발현하거나 세포에서 세포 펠렛을 수집하기 전에 비 클래스 III 히스톤 아세틸 라제를 억제하는 배양 배지에 1 ㎛ TSA를 추가, (위의 2.1 참조) SIRT2 세포 (위 2.2 참조)를 무너 뜨렸다. </li><li> 4 ° C에서 10 분 동안 1,000 XG에서 원심 분리 트립신 처리하여 세포를 PBS로 두 번 세포를 씻어 수집, 배지를 제거합니다. </li><li> 10 ml의 용해 완충액 (20 mM의 HEPES pH가 7.9, 18 mM의 KCl을 0.2 mM의 EGTA, 1.5 추가밀리미터의 MgCl 2, 20 % 글리세롤, 프로테아제 억제제 칵테일, 1 μM의 TSA 2 MM의 NAM을 포함하여 0.1 % NP-40). </li><li> 일정한 회전에서 30 분 동안 4 ° C에서 품어. </li><li> 4 ℃에서 15 분 동안 20,000 XG에서 원심 분리하여 상층 액을 수집합니다. </li><li> 브래드 포드 (Bradford) 단백질 분석을 사용하여 단백질 농도를 계산한다. </li><li> 참고로 용해 완충액 A를 사용하여 표 2를 사용하여 1 ㎖의 최종 부피에서 새로운 전송 튜브 전체 단백질 1 mg의 하나 또는 과발현 SIRT2 SIRT2 최저 후의 세포로부터 단백질 샘플을 제조 하였다. </li><li> 단백질 A / G와 함께 특정 단백질 기판에 대한 항체를 사용하여 면역 침전을 수행합니다 (40 μl의 비드 슬러리 권장). 또한, 모든 아세틸 화 단백질을 면역 침전에 구슬을 아가로 오스 복합 항 AC-K 항체 (20 μl의 비드 슬러리는 일반적으로 충분하다)를 사용합니다. </li><li> 정전류 회로에서 4 ° C에서 샘플을 품어NT를 회전 하룻밤. </li><li> 4 ℃에서 2 분 동안 1,000 XG에서 원심 분리하여 immunoprecipitates를 수집합니다. </li><li> 4 ° C에서 2 분 동안 1,000 XG에 구슬 1 ml의 버퍼와 5 회 반복한다. </li><li> 배 샘플 버퍼의 40 μL에 재현 탁 구슬. </li><li> 95 ° C에서 10 분 동안 삶아 샘플. </li><li> 용출 샘플을 전송하는 동안 구슬을 방해하지 않고 4-12 % SDS-PAGE 겔에 용출 된 샘플 (단계 2.3.12에서 40 μl를) 실행하여 전기 영동을 수행합니다. </li><li> 단백질 기질에 특이적인 항체는 면역에 사용 된 경우 (1000 희석 1), 또는 단백질이 기판에 대해 특정 항체 (위한 권장 사항을 방지 AC-K 항체를 이용한 웨스턴 블롯으로 단백질 기판의 아세틸 화 수준을 감지 안티 AC-K 비드 면역에 사용 된 경우에 대하여 특이 적 항체 희석). </li></ol></li></ol>

<ol>
	<li>Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+age+of+crosstalk:+phosphorylation,+ubiquitination,+and+beyond.">The age of crosstalk: phosphorylation, ubiquitination, and beyond.</a> Molecular Cell. 28, 730-738 (2007).</li><li>Gao, M., Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+regulators:+control+of+protein+ubiquitination+and+ubiquitin-like+modifications+by+extracellular+stimuli.">Regulating the regulators: control of protein ubiquitination and ubiquitin-like modifications by extracellular stimuli.</a> Molecular Cell. 19, 581-593 (2005).</li><li>Philp, A., Rowland, T., Perez-Schindler, J., Schenk, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+acetylome:+translating+targeted+proteomics+into+meaningful+physiology.">Understanding the acetylome: translating targeted proteomics into meaningful physiology.</a> American Journal of Physiology. Cell Physiology. 307, C763-C773 (2014).</li><li>Allfrey, V. G., Faulkner, R., Mirsky, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+and+Methylation+of+Histones+and+Their+Possible+Role+in+the+Regulation+of+Rna+Synthesis.">Acetylation and Methylation of Histones and Their Possible Role in the Regulation of Rna Synthesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 51, 786-794 (1964).</li><li>Shahbazian, M. D., Grunstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+site-specific+histone+acetylation+and+deacetylation.">Functions of site-specific histone acetylation and deacetylation.</a> Annual Review of Biochemistry. 76, 75-100 (2007).</li><li>L'Hernault, S. W., Rosenbaum, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlamydomonas+alpha-tubulin+is+posttranslationally+modified+in+the+flagella+during+flagellar+assembly.">Chlamydomonas alpha-tubulin is posttranslationally modified in the flagella during flagellar assembly.</a> The Journal of Cell Biology. 97, 258-263 (1983).</li><li>Kim, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+and+functional+diversity+of+lysine+acetylation+revealed+by+a+proteomics+survey.">Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.</a> Molecular Cell. 23, 607-618 (2006).</li><li>Choudhary, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysine+acetylation+targets+protein+complexes+and+co-regulates+major+cellular+functions.">Lysine acetylation targets protein complexes and co-regulates major cellular functions.</a> Science. 325, 834-840 (2009).</li><li>Smith, K. T., Workman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+the+acetylome.">Introducing the acetylome.</a> Nature Biotechnology. 27, 917-919 (2009).</li><li>Roth, S. Y., Denu, J. M., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetyltransferases.">Histone acetyltransferases.</a> Annual Review of Biochemistry. 70, 81-120 (2001).</li><li>Ozden, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+of+MnSOD+directs+enzymatic+activity+responding+to+cellular+nutrient+status+or+oxidative+stress.">Acetylation of MnSOD directs enzymatic activity responding to cellular nutrient status or oxidative stress.</a> Aging. 3, 102-107 (2011).</li><li>Anderson, K. A., Hirschey, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein+acetylation+regulates+metabolism.">Mitochondrial protein acetylation regulates metabolism.</a> Essays in Biochemistry. 52, 23-35 (2012).</li><li>Schwer, B., Bunkenborg, J., Verdin, R. O., Andersen, J. S., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+lysine+acetylation+controls+the+activity+of+the+mitochondrial+enzyme+acetyl-CoA+synthetase+2.">Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 10224-10229 (2006).</li><li>Haberland, M., Montgomery, R. L., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+roles+of+histone+deacetylases+in+development+and+physiology:+implications+for+disease+and+therapy.">The many roles of histone deacetylases in development and physiology: implications for disease and therapy.</a> Nature Reviews. Genetics. 10, 32-42 (2009).</li><li>Finkel, T., Deng, C. X., Mostoslavsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+biology+and+physiology+of+sirtuins.">Recent progress in the biology and physiology of sirtuins.</a> Nature. 460, 587-591 (2009).</li><li>Houtkooper, R. H., Pirinen, E., Auwerx, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirtuins+as+regulators+of+metabolism+and+healthspan.">Sirtuins as regulators of metabolism and healthspan.</a> Nature reviews. Molecular Cell Biology. 13, 225-238 (2012).</li><li>Choudhary, C., Weinert, B. T., Nishida, Y., Verdin, E., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+landscape+of+lysine+acetylation+links+metabolism+and+cell+signalling.">The growing landscape of lysine acetylation links metabolism and cell signalling.</a> Nature Reviews. Molecular Cell Biology. 15, 536-550 (2014).</li><li>Stewart, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus-delivered+stable+gene+silencing+by+RNAi+in+primary+cells.">Lentivirus-delivered stable gene silencing by RNAi in primary cells.</a> RNA. 9, 493-501 (2003).</li><li>Kim, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRT2+maintains+genome+integrity+and+suppresses+tumorigenesis+through+regulating+APC/C+activity.">SIRT2 maintains genome integrity and suppresses tumorigenesis through regulating APC/C activity.</a> Cancer Cell. 20, 487-499 (2011).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRT2+directs+the+replication+stress+response+through+CDK9+deacetylation.">SIRT2 directs the replication stress response through CDK9 deacetylation.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 13546-13551 (2013).</li><li>Weinert, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome-wide+mapping+of+the+Drosophila+acetylome+demonstrates+a+high+degree+of+conservation+of+lysine+acetylation.">Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation.</a> Science Signaling. 4, ra48 (2011).</li><li>Still, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+mitochondrial+acetylation+dynamics+highlights+prominent+sites+of+metabolic+regulation.">Quantification of mitochondrial acetylation dynamics highlights prominent sites of metabolic regulation.</a> The Journal of Biological Chemistry. 288, 26209-26219 (2013).</li><li>Sadoul, K., Wang, J., Diagouraga, B., Khochbin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tale+of+protein+lysine+acetylation+in+the+cytoplasm.">The tale of protein lysine acetylation in the cytoplasm.</a> Journal of Biomedicine &amp; Biotechnology. 2011, 970382 (2011).</li><li>Preble, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+and+purification+of+mitochondria+for+transplantation+by+tissue+dissociation+and+differential+filtration.">Rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration.</a> Journal of Visualized Experiments : JoVE. , e51682 (2014).</li><li>Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> Journal of Visualized Experiments : JoVE. , (2011).</li><li>He, W., Newman, J. C., Wang, M. Z., Ho, L., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+sirtuins:+regulators+of+protein+acylation+and+metabolism.">Mitochondrial sirtuins: regulators of protein acylation and metabolism.</a> Trends in Endocrinology and Metabolism: TEM. 23, 467-476 (2012).</li></ol>

저자는 공개 아무것도 없어.

Recent high throughput proteomic studies have established acetylation as a widespread PTM found not only in nucleus but also in cytoplasm and mitochondria 7,8,21-23. Taking into account the likelihood that many more acetylated proteins and sites might have been not detected due to several reasons, such as specificity of the anti-Ac-K antibodies used, the low abundance of the acetylated proteins, and the transient nature of the PTM, it is safe to predict that more acetylated proteins remain to be discovered in ...

Post-translational modifications (PTMs) regulate cell signaling networks allowing cells to rapidly respond to internal and external signals. Over the last few decades, many different PTMs playing a pivotal role in diverse processes have been identified but only a few have been studied extensively, such as phosphorylation, acetylation and ubiquitination 1-3. Focusing on acetylation, Allfrey et al. were the first to propose a role for histone acetylation in regulating gene transcription about 50 years ago 4. Research in this field has revealed that histone lysine acetylation modulates chromatin condensation and it is considered to be an epigenetic mark as part of the histone code 5. Although it took a long time until the discovery of tubulin as the first non-histone acetylation target 6, it is well established now that hundreds of eukaryotic proteins beyond histones can be acetylated and lysine acetylation has been recognized as a wide-spread PTM that may rival phosphorylation and ubiquitination in its prevalence 7-9. Interestingly, non-histone acetylated proteins can be signaling molecules in the cytoplasm, transcription factors in the nucleus, and metabolic enzymes in mitochondria, highlighting the significance of acetylation in regulating a plethora of cellular processes. 
The acetylation status of a protein depends on the coordinated and opposing function of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) which add and remove acetyl groups from proteins. The reversible acetylation of lysine, which involves neutralization of a positive charge 10, alters protein structure and it seems very likely to also alter enzymatic function in several cases 11-13. Focusing on KDACs, 18 proteins have been identified in the human and mouse genomes 14-16. Among them, mammalian sirtuins (also called class III histone lysine deacetylases) which are distinct from other members as they require NAD+ for their enzymatic function, have attracted extensive interest in this research field 16. In mammals, seven sirtuins (SIRT1-7) have been identified, each of them sharing a conserved 275-amino-acid catalytic core domain, which are mainly categorized according to their subcellular localization to the nucleus (SIRT1, 6, and 7), mitochondria (SIRT3, 4, and 5), or cytoplasm (SIRT2). SIRT1-3 have a robust deacetylation activity, while SIRT4 is reported to display ADP-ribosyltransferase activity, SIRT5 may function as a protein desuccinylase and demalonylase, and SIRT6 and SIRT7 display weak deacetylase activity but are involved in other types of acylations 17. In accordance with the significance of acetylation as a regulatory PTM modification involved in several cellular functions, sirtuins have also been implicated in a wide range of processes. After the first breakthrough studies establishing the role of sirtuins in life span extension, it has been shown that they are involved in diverse cellular functions including DNA repair, maintenance of genomic instability, apoptosis, response to stress and inflammation, control of energy efficiency, circadian clocks and metabolism, as well as contributing to the initiation and/or progression of age-related diseases such as cancer, neurodegeneration and type 2 diabetes 15,16.
Despite the significant progress in the field of sirtuin biology, more work remains to unravel undiscovered roles and functions through the identification of novel substrates. This is evidenced more emphatically by recent advances in high-resolution mass spectrometry (MS) based proteomics which have significantly increased the number of proteins found to be acetylated but most importantly have identified several different acetylated lysines in each protein, arguing that acetylation may be as wide-spread as other PTMs such as phosphorylation 7,8,17. Taking into consideration that specific deacetylases have not yet been identified for most of these acetylated proteins-substrates, it is reasonable to suggest that both in vitro and in vivo deacetylation assays are needed to confirm and establish an acetylated protein as a legitimate substrate of a specific deacetylase. In the experimental protocols described below, details will be given on how to perform both in vitro and in vivo deacetylation assays using SIRT2 as the specific deacetylase.

<table><tbody><tr><td>cell culture dishes</td><td>Denville Scientific Inc.</td><td>T1110 and T1115</td><td></td></tr><tr><td>pCDH-puro-GFP lentiviral vector</td><td>System Biosciences</td><td>CD513B-1</td><td></td></tr><tr><td>pCMV- dR8.2 dvpr (packaging vector)</td><td>Addgene</td><td>8455</td><td></td></tr><tr><td>pCMV-VSV-G (envelope vector)</td><td>Addgene</td><td>8454</td><td></td></tr><tr><td>polyethylenimine (PEI)&amp;nbsp;</td><td>Polysciences Inc.</td><td>24885</td><td>other transfection reagents can be used as well. PEI is cost effective and very efficient in transfecting 293T cells</td></tr><tr><td>0.22 &amp;mu;m filters</td><td>Denville Scientific Inc.</td><td>F5512</td><td></td></tr><tr><td>polybrene</td><td>Sigma</td><td>H9268</td><td></td></tr><tr><td>fluorescent microscope</td><td>Carl Zeiss MicroImaging Inc.</td><td>Axiovert 200</td><td></td></tr><tr><td>puromycin</td><td>Invivogen</td><td>A11138-03</td><td></td></tr><tr><td>PBS</td><td>Corning</td><td>21-031-CM</td><td></td></tr><tr><td>anti-Flag antibody</td><td>Sigma</td><td>F3165</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>NP-40</td><td>Sigma</td><td>74385</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M9272</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>34596</td><td></td></tr><tr><td>protease inhibitors coctail 100x</td><td>Biotool</td><td>B14001</td><td></td></tr><tr><td>Trichostatin A (TSA)</td><td>Sigma</td><td>T8552</td><td>selective inhibitor of class I and II histone deacetylases (HDACs) but not class III HDACs (sirtuins)</td></tr><tr><td>anti-Flag agarose beads</td><td>Sigma</td><td>A2220</td><td></td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>rotator</td><td>Thermo Scientific</td><td>415220Q</td><td></td></tr><tr><td>filter tubes</td><td>Millipore</td><td>UFC30HV00</td><td></td></tr><tr><td>Flag peptide&amp;nbsp;</td><td>Sigma</td><td>F3290</td><td></td></tr><tr><td>Vivaspin Centrifugal Concentrator</td><td>Sartorius Stedim Biotech S.A.</td><td>VS0102</td><td></td></tr><tr><td>SimplyBlue SafeStain solution&amp;nbsp;</td><td>Invitrogen</td><td>LC6060</td><td></td></tr><tr><td>NuPAGE LDS sample buffer (4x)</td><td>Life Techologies</td><td>NP0007</td><td></td></tr><tr><td>Tris-HCl pH 7.5</td><td>Sigma</td><td>T5941</td><td></td></tr><tr><td>NAD&lt;sup&gt;+&lt;/sup&gt;</td><td>Sigma</td><td>N0632</td><td>required cofactor for sirtuins</td></tr><tr><td>nicotinamide (NAM)</td><td>Sigma</td><td>72340</td><td>selective inhibitor class III HDACs (sirtuins)</td></tr><tr><td>pLKO.1 lentiviral vector&amp;nbsp;</td><td>Addgene</td><td>8453</td><td></td></tr><tr><td>SIRT2 si RNA&amp;nbsp;</td><td>Qiagen</td><td>GS22933</td><td></td></tr><tr><td>anti-SIRT2 antibody</td><td>Proteintech</td><td>15345-1-AP</td><td></td></tr><tr><td>Bradford protein assay</td><td>BIO-RAD</td><td>500-0006</td><td></td></tr><tr><td>anti Ac-K agarose beads</td><td>Immunechem</td><td>ICP0388</td><td></td></tr></tbody></table>

deacetylation assays to unravel the interplay between sirtuins (sirt2) and specific protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

단백질을 위해 시험 관내 및 생체 내 탈 아세틸 분석 모두에서 탈 아세틸 화 활성을 갖는 임의의 효소를 탈 아세틸 적법한 대상으로서 취급 할 아세틸 라제 및 기판 사이의 상호 작용을 확립하기 위해 수행 될 필요가있다. 분석이 완료되기 전에 시험 관내 아세틸 라제 분석을 위해, 아세틸 라제 및 아세틸 단백질 기질 모두의 정제가 필요하다. 여...

Watch this Scientific Journal Video about Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates at JoVE.com

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

탈 아세틸 화 분석법은 시르 투 인 사이의 상호 작용 (SIRT2) 및 특정 단백질 기판을 해명

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

deacetylation-assays-to-unravel-interplay-between-sirtuins-sirt2

Research

JoVE Journal

Chemistry

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

8.2K 조회수.  Feinberg School of Medicine, Northwestern University. This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

비디오: Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

체외 유비퀴틴화 및 뉴클레오소말 히스톤의 이중화 율법의 비퀴틴화 어설션

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

연구

생화학

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

효소 활성 분석을 위한 사상 균 류 아 스퍼 질러 스 니 둘 란 스의 탭 태그 Histone de살리실산의 단일 단계 농축

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Assays for Validating Histone Acetyltransferase Inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

히스톤 아세틸트랜스퍼효소 억제제 검증을 위한 아세서

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

암 연구학

Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.

Acyl-RAC (Acyl-Resin Assisted Capture) is a highly sensitive, reliable and easy to perform method to detect reversible lipid modification of cysteine residues (S-acylation) in a variety of biological samples.

아실-레신 보조 포획을 이용한 단백질 S-아실레이션 검출

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

dNTPase SAMHD1과의 저분자 상호작용을 프로브하기 위한 고처리량 효소 결합 활성 분석

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

HAT1 효소 활성을 스크리닝하기 위한 클릭 기반 분석 개발

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

저자 스포트라이트: HAT1 Inhibitor Screening을 위한 Acetyl-Click Assay 개발

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

히스톤 아세틸전이효소 1 아세틸화를 측정하기 위한 아세틸 클릭 화학 분석

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

<meta charset="utf8"></head><body>단백질-리간드 상호작용을 위한 High-Throughput Thermal Shift Assay

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding. 
Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.

<meta charset="utf8"></head><body>저자 스포트라이트: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays (열 이동 분석을 통한 단백질의 구조 및 생화학적 연구 발전)

Here, we present the thermal shift assay, a high-throughput, fluorescence-based technique used to investigate the binding of small molecules to proteins of interest.

탈 아세틸 화 분석법은 시르 투 인 사이의 상호 작용 (SIRT2) 및 특정 단백질 기판을 해명

요약

더 많은 비디오 탐색

이 비디오의 챕터

체외 유비퀴틴화 및 뉴클레오소말 히스톤의 이중화 율법의 비퀴틴화 어설션

효소 활성 분석을 위한 사상 균 류 아 스퍼 질러 스 니 둘 란 스의 탭 태그 Histone de살리실산의 단일 단계 농축

히스톤 아세틸트랜스퍼효소 억제제 검증을 위한 아세서

아실-레신 보조 포획을 이용한 단백질 S-아실레이션 검출

dNTPase SAMHD1과의 저분자 상호작용을 프로브하기 위한 고처리량 효소 결합 활성 분석

히스톤 아세틸전이효소 1 아세틸화를 측정하기 위한 아세틸 클릭 화학 분석

히스톤 아세틸전이효소 1 아세틸화를 측정하기 위한 아세틸 클릭 화학 분석

히스톤 아세틸전이효소 1 아세틸화를 측정하기 위한 아세틸 클릭 화학 분석

Selenoprotein O에 대한 기질 결합을 프로브하기 위해 Thermal Shift Assay를 활용

Selenoprotein O에 대한 기질 결합을 프로브하기 위해 Thermal Shift Assay를 활용