Name: preparation and in vivo use of an activity-based probe for n-acylethanolamine acid amidase
Uploaded: 2016-11-23T13:00:00
Description: Watch this Scientific Journal Video about Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase at JoVE.com

The authors thank the Nikon Imaging Center at Istituto Italiano di Tecnologia, Genova, Italy (NIC@IIT).

주의 : 모든 화학 반응이 환기 흄 후드 및 실험실 코트, 장갑 및 보호 고글의 사용으로 수행되어야한다. 반응은 질소 환경에서 수행되어야한다. 윤리 문 : 동물을 포함하는 우리의 절차는 실험과 기타 과학적 목적 (DM 116192), 유럽 경제 공동체 규정에 사용되는 동물의 보호에 이탈리아의 규정에 따라 수행된다 (OJ EC의 L 1분의 358 1986년 12월 18일 ). 참고 합성 [(3- S) -2- oxoazetidin -3- 일] 아세트산 암모?...

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효소 활성은 미세 RNA 전사, 단백질 합성, 단백질 전좌, 번역 후 변형 및 단백질 - 단백질 상호 작용을 포함하여 다양한 수준에서 조절된다. 종종, 효소의 발현 혼자의 활동을 설명하지 않습니다. ABPP는 천연 상태의 단백질의 활성을 연구하기 위해 개발되었다. 두 가지 기능이 요구된다 : 공유 관심 효소 리포터 태그의 활성 부위에 결합하는 화학 프로브는 프로브 - 표지 효소를 검출. <p class="jove_co...

일반적 산탄 분석 1,2- 효모 용 액체 크로마토 그래피 질량 분석 플랫폼을 포함하는 발현 패턴의 상호 작용 및 기능 단백질을 조사하는 방법을 사용하는 두 개의 하이브리드 방법 3,4 및 시험 관내 분석에서 그들이 것을 한정 그 나라의 상태에서 단백질의 활성을 평가할 수 없습니다. 활동 기반 단백질 프로파일 (ABPP)이 갭을 채우기 위해 사용될 수있다. 이 방식에서, 작은 분자에 공유 표적 탐지 가능 리포터 그룹에 결합되는 관심있는 효소의 활성 부위에 결합 할 수있는 프로브. 클릭 화학 (CC)를 사용하여 리포터 프로브에 통합 될 수 있거나 또는 표적 결합이 5,6 발생한 후에 도입 될 수있다. 후자의 방법은 SUC 바이오 직교 반응을 통해 리포터 시약 다수 개질 될 수있는 단자 알킨 또는 아 지드와 같은 적합한 화학 그룹을 포함하는 프로브를 사용해야구리 (I)와 같은 시간 [3 + 2] 사이클로 7-9 또는 슈타 우 딩거 결찰 10, 11 Huisgen을 촉매 반응. 최근에는 체외위한 제 ABP와 같은 시스테인 가수 분해 효소, NAAA (12)의 생체 검출의 화합물 ARN14686 개시된. NAAA는 항 염증 핵 수용체 PPAR 알파 13 ~ 15의 내인성 작용제이다 oleoylethanolamide (OEA) 및 팔미 토일 (PEA)을 포함하여 포화 및 불포화 FAES,의 가수 분해 비활성화를 촉매한다. NAAA 주로 선천성 면역 반응의 조절에 중요한 역할을 시사뿐만 B 림프구 (14,16)와 같이, 대 식세포 및 다른 단핵구 유래 된 세포에서 발현된다. 효소는 비활성 형태로 거친 소포체에서 합성 및 autoproteolytic기구 (17)에 의해 세포의 산성 구획에 활성화됩니다. autoproteolytic 분열은 (C13를 새 N 말단 시스테인을 생성마우스 및 쥐에서 1, 인간의 C126), 즉 FAE의 친핵체 책임은 (18, 19)을 가수 분해한다. NAAA 활동의 약리 억제는 FAES 16,20,21 증가 세포 수준에 찬성 FAE 합성 / 분해의 균형을 변경합니다. 여러 β-락톤 및 β - 락탐 유도체는 높은 효능 및 선택성 16,22-26와 NAAA 활성을 억제하는 것으로 나타났다. 이러한 억제제는 촉매 시스테인 16,27,28의 S의 -acylation을 통해 작용한다. (- [(S) -2- oxoazetidin -3- 일] 카르 바 메이트 4- cyclohexylbutyl- N) 16 화합물 ARN14686은 전신적 활성 세린 유래 β 락탐 NAAA 억제제의 화학 구조, ARN726에 기초하여 설계되었다. ARN726의 4 부틸 사이클로 헥실 그룹은 아 지드 베어링 기자 태그 이후 CC의 접합을위한 터미널 알킨 태그를 베어링 C9 포화 지방족 체인으로 대체되었다. 우리 minimall하는 두 단계 ABP 디자인을 선택따라서 Y NAAA 용 프로브의 친화력을 유지하면서 원래의 지지체의 구조를 변화. 또한, 부피가 태그의 도입을 피하는 등의 프로브가 직접 ABP보다 생체 내 치료에 적합 할 수있다. ARN14686 높은 효능으로 NAAA 억제 효소 촉매 (12)의 시스테인과 공유 부가 물을 형성함으로써 (hNAAA IC 50 = 6 nM의, rNAAA IC 50 = 13 ㎚). 살아있는 쥐의 실험 프로브가 NAAA가 폐에 표현 캡처 선택적 있음을 보여 주었다. 높은 프로브 농도 (시험 관내에서 10 μM, 10 밀리그램 / 정맥 주사, 정맥 ㎖) (12)를 사용할 때 산성 세라 미다, NAAA와 33~34% ID를 공유하는 다른 시스테인 아미 다제는 또한 낮은 친 화성 대상으로 확인되었다. 우리는 또한 완전한 프로 인트 보조제 (CFA) (29)의 투여 후 염증이 쥐의 조직에서 활동 NAAA의 존재를 연구하는 ARN14686을 사용했다. 여기에서는 preparati위한 프로토콜 개요ARN14686 (그림 1)과 NAAA 활성화 생체의 조사에의 응용의에. 예를 들어, 우리는 CFA 투여 후 쥐의 발에 NAAA을 시각화하는 실험 절차를 설명합니다. 이 실험에서, 단백질은 프로브의 IV 주입 후 발 조직으로부터 추출되고, ABP 표지 프로테옴 비오틴 지드와 CC 받는다. 비오틴 샘플을 스트렙 타비 딘 비드를 사용하여 농축하고, 단백질 블롯 행한다. 다른 응용 프로그램에서, 우리는 프로브 처리 된 마우스에서 마우스의 폐에서 형광 현미경으로 활성화 NAAA의 현지화를 설명합니다. 이 경우, 조직을 절단하고, 절편을 로다 민 첨가 CC 실시된다. 워크 플로 방식은도 2에 도시되어있다.

<table border="0" cellpadding="0" cellspacing="0" width="1209">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">1,1&#8217;-sulfonyldiimidazole&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">367818</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">2-dipyridylcarbonate</td>
			<td style="width:205px;">Fluorochem</td>
			<td style="width:111px;">11331</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">2-Methylbutan</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">M32631</td>
			<td>Flamable, toxic,hazardous to the aquatic environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">4-(Dimethylamino)pyridine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">107700</td>
			<td style="width:471px;">Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Acetic acid</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">695092</td>
			<td style="width:471px;">Flammable, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Acetonitrile&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34998</td>
			<td style="width:471px;">Flammable, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Activated charcoal</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">161551</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ammonium chloride</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A9434</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Azide-PEG3-Biotin</td>
			<td style="width:205px;">Jena Biosciences</td>
			<td style="width:111px;">CLK-AZ104P4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Azide-PEG3-Fluor 545</td>
			<td style="width:205px;">Jena Biosciences</td>
			<td style="width:111px;">CLK-AZ109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">BCA protein assay kit</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bio-spin columns</td>
			<td style="width:205px;">Biorad</td>
			<td style="width:111px;">732-6204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Biotin</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">B4501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Blocking buffer</td>
			<td style="width:205px;">Li-Cor Biosciences</td>
			<td style="width:111px;">927-40000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">b-mercaptoethanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">M6250</td>
			<td>Higly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bovin serum albumine (BSA)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bromophenol blue</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">B0126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Bruker Avance III 400</td>
			<td style="width:205px;">Bruker</td>
			<td style="width:111px;"></td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Celite</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">419931</td>
			<td style="width:471px;">Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ceric ammonium nitrate&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">22249</td>
			<td style="width:471px;">Oxidizing, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Chloral hydrate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">C8383</td>
			<td>Higly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">CuSO4.5H2O&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">209198</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Cyclohexadiene</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">125415</td>
			<td style="width:471px;">Flammable, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Cyclohexane</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34855</td>
			<td style="width:471px;">Flammable, Harmful, Health hazard, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dichloromethane</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34856</td>
			<td style="width:471px;">Harmful, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Diethyl ether</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">296082</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:205px;">Acros Organics</td>
			<td style="width:111px;">348441000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Dimethyl sulfoxide d6 (DMSO-d6)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">175943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ethanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">2860</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ethyl acetate</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34858</td>
			<td style="width:471px;">Flammable, Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Glycerol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Irdye 680-LT Streptavidin</td>
			<td style="width:205px;">Li-Cor Biosciences</td>
			<td style="width:111px;">925-68031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">IRDye680-LT Streptavidin&#160;</td>
			<td style="width:205px;">Licor</td>
			<td style="width:111px;">925-68031</td>
			<td>Briefly centrifuge before use to precipitate protein complexes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Methanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34966</td>
			<td>Highly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Methanol</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">34860</td>
			<td style="width:471px;">Flammable, Toxic, Health hazard</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">E7750</td>
			<td style="width:471px;">Harmful, Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N,N-diisopropylethylamine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">D125806</td>
			<td style="width:471px;">Flammable, Corrosive, Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N,N-dimethylformamide</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">227056</td>
			<td style="width:471px;">Flammable, Harmful, Health hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">N-Cbz-L-Serine</td>
			<td style="width:205px;">Fluorochem&#160;</td>
			<td style="width:111px;">M03053</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Nikon A1 confocal microscopy</td>
			<td style="width:205px;">Nikon</td>
			<td style="width:111px;"></td>
			<td>Read&#160; the user manual</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">NuPAGE 4-12% Bis-Tris gel</td>
			<td style="width:205px;">Thermo Fisher Scientific</td>
			<td style="width:111px;">NP0335BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Palladium on carbon</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">330108</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">p-anisidine</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">A88255</td>
			<td style="width:471px;">Toxic, Health hazard, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Paraformaldehyde</td>
			<td style="width:205px;">sigma Aldrich</td>
			<td style="width:111px;">441244</td>
			<td>Toxic, respiratory harmful, corrosive, falmable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(ethylene glycol)&#160;</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P3265</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
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			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P8340</td>
			<td></td>
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		<tr height="21">
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			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">360538</td>
			<td>Toxic, flammable</td>
		</tr>
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			<td style="width:471px;">Flammable, Harmful, Health hazard</td>
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			<td>Toxic, warm at 50 &#176;C to dissolve</td>
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			<td style="width:205px;">Sigma Aldrich</td>
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		</tr>
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			<td height="21" style="height:21px;">Tris(2-carboxyethyl)phosphine (TCEP)</td>
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			<td height="21" style="height:21px;">Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">678937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Triton-x100</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">X100</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tween-20</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P9416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Tween-80</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">P1754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Ultra turrax IKA T18 basic tissue homogenizer</td>
			<td style="width:205px;">IKA</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Undec-10-yn-1-ol</td>
			<td style="width:205px;">Fluorochem</td>
			<td style="width:111px;">13739</td>
			<td style="width:471px;">Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Urea</td>
			<td style="width:205px;">Sigma Aldrich</td>
			<td style="width:111px;">U5378</td>
			<td>Toxic, warm at 50 &#176;C to dissolve</td>
		</tr>
	</tbody>
</table>

preparation and in vivo use of an activity-based probe for n-acylethanolamine acid amidase

활동 기반 단백질 프로파일 (ABPP)는 효소의 활성 부위를 타겟팅하는 화학 프로브의 사용을 통해 복잡한 프로테옴 관심 효소의 동정하는 방법이다. 프로브에 도입 된 리포터 태그의 겔 형광 검사, 단백질 오, 형광 현미경, 액체 크로마토 그래피 질량 분석에 의해 효소 표지의 검출을 허용한다. 여기, 우리는 준비 및 복합 ARN14686의 사용, 선택적으로 효소 N -acylethanolamine 산 아미 다제 (NAAA)를 인식하는 클릭 화학의 활동 기반 프로브 (CC-ABP)을 설명합니다. NAAA는 팔미 토일 (PEA)과 oleoylethanolamide (OEA)과 같은 내인성 페 록시 솜 증식 제 - 활성화 수용체 (PPAR) - 알파 작용제를 비활성화하여 염증을 촉진 시스테인 가수 분해 효소이다. NAAA은 리소좀의 산성 pH에서 autoproteolysis에 의해 활성화되는 비활성 전체 길이 proenzyme으로 합성된다. 현지화 연구 시간NAAA은 주로 B 림프구뿐만 아니라, 대 식세포 및 다른 단핵구 유래 된 세포에서 발현되는 것으로 나타 집결지. 우리는 ARN14686 감지하고 단백질 얼룩과 형광 현미경으로 설치류 조직에서 활동 NAAA 생체을 정량화 할 수있는 방법의 예를 제공합니다.

ARN14686는 NAAA 억제제 ARN726의 발판을 기반으로 설계되었다. ARN726의 4-tert- 부틸 시클로 헥 실기가 C9 포화 지방족 쇄 말단 알킨 태그 (도 1) 방위로 치환 하였다. 알킨 태그는 형광 또는 CC 통해 비오틴 분자를 추가하는 두 단계의 표시 절차의 이용을 허용하기 위해 도입되었다. 이 특징은 생체 외 및 생체 내 NAAA을 프로빙 매우 다용도 공구 ARN14686 렌더?...

Watch this Scientific Journal Video about Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase at JoVE.com

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

여기서는 활동 기반 프로브의 제조 및 사용을 설명 (ARN14686, 운데 10 ynyl- N - [(3- S) -2- oxoazetidin -3- 일] 카르 바 메이트)의 검출 및 정량화를 허용 염증성 효소 N -acylethanolamine 산 아미 다 아제의 활성 형태 (NAAA), 모두 생체 외 및 생체있다.

준비 및<em&gt; 생체</em&gt; 액티비티 기반 프로브의 사용을위한<em&gt; N</em&gt; -acylethanolamine 산 아미 다제

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

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JoVE Journal

Biochemistry

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

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