纯化膜隔间跨演示文稿中的外源抗原内质网关联降解
概要
这里介绍的方法是一种新的囊泡隔离协议，允许纯化细胞的车厢外源性抗原处理由内质网关联退化跨演示文稿中的地方。
Abstract
树突状细胞 (Dc) 是高度能够处理和后主要组织相容性类 (MHC) 的内化外源抗原呈递我分子也被称为跨演示文稿 (CP)。CP 扮演重要的角色，不仅在天真 CD8 的刺激+ T 细胞和 CD8 记忆+ T 细胞的传染性，而且在自立式灭活肿瘤免疫 t 细胞的 T 细胞无能或 T 细胞删除。虽然 CP 的关键分子机制仍有待澄清，越来越多的证据表明外源抗原进行处理通过内质网关联降解 (ERAD) 后从非古典出口内吞隔间。直到最近，刻画这些细胞内吞的车厢都有限，因为有没有除了外源性抗原的特异分子标记。这里介绍的方法是一种新的囊泡隔离协议，允许纯化这些细胞内吞的车厢。我们使用此纯化的微粒，重组 ERAD 像运输、 泛素化和处理的外源性抗原体外，暗示泛素-蛋白酶体系统后从这出口加工外源性抗原细胞的隔间。该协议可以进一步应用于其他细胞类型以澄清 CP 的分子机制。
Introduction
MHC 我用内源性抗原，由泛素-蛋白酶体系统在胞浆1处理短抗原肽表达上所有的有核细胞表面的分子。经处理后，抗原肽被送入内质网 (ER) 腔通过肽转运蛋白水龙头。在 ER 腔，一系列特定的分子伴侣的协助肽加载和正确折叠的 MHC I 复合。这一系列的分子称为肽加载复杂 (PLC)，指示 ER 车厢中部为肽加载在 MHC2。后加载，多肽 MHC 我分子被运送到细胞表面，在发挥关键作用自适应免疫系统自我标记，以及使 CD8+细胞毒性 T 淋巴细胞 (Ctl) 来检测癌细胞或传染性病原体的抗原从非自我蛋白质3肽。
在抗原提呈细胞 (APC)，从外源抗原的抗原肽也出示 MHC 我4，，56，7，8 通过CP，其中主要进行由 DCs9，，1011。CP 是必不可少的两个为激活天真 CD8+ T 细胞和 CD8 记忆+ T 细胞抗感染和抗肿瘤 Ctl12，13，和在维持免疫耐受的失活自署理幼稚 T 细胞14，15。CP 许多重要作用中自适应的免疫系统，然而 CP 的分子机制尚未予以详细说明。以前对 CP 的研究揭示了外源性抗原被本地化在急诊室和内体和通过 ERAD，暗示外源抗原从内体运到 ERAD 样加工和肽加载16 ER 进行处理.然而，越来越多的证据表明 CP 的肽加载进行不在急诊室，而在于非经典的内吞车厢，也有特色的 ER (图 1)17，18 ，19，，2021。通过来避免退化的抗原肽前体肽氨基肽酶活性较高22胞液、 加工和多肽在 CP 中加载发生在近端地区的这些非经典的内吞车厢 (图 1)。虽然这些细胞内吞的车厢的刻画是有争议的没有现有的特定分子，除了这个包间的外源性抗原。
ERAD 是细胞通路，专门从急诊室中移除错误折叠的蛋白质。ERAD 通路中错误折叠的蛋白是逆行通过对细胞质 ER 膜运输和处理由泛素-蛋白酶体系统23，，2425。大分子，比如蛋白质，运输时通过脂质双分子层，这些分子通过分子的装置称为解，如 Sec61 复杂和德林在 ER26，和汤姆复杂复杂和蒂姆在复杂线粒体27。当外部添加抗原是通过内质网膜，他们必须穿透脂质双分子层中复杂的 translocons，例如 Sec61 复杂。这里介绍的方法通过利用这些膜穿透的分子标志物在细胞内吞的隔间作为纯化有针对性的囊泡。
这里介绍的方法是一种新的使用作为外源性抗原的树突状细胞线 DC2.428和生物素化的卵清蛋白 （宝物） 的囊泡净化协议。内吞的车厢进行纯化的链霉亲和 (SA)-磁珠作为制造商使用膜穿透宝物。在这纯净的微粒，一些外部添加宝物仍保持膜组分中但被运到外面的微粒体，然后泛素化和处理体外29。这纯净的微粒体载不仅吞隔间特异性蛋白二村居民蛋白质 ERAD 和肽加载复杂;建议细胞隔间 CP29前瞻性吞车厢。本议定书依赖外源性抗原，这种并不是也适用于其他 DC 亚群和其他细胞类型，如巨噬细胞、 B 细胞和内皮细胞，以澄清精通 CP DCs 的精确分子机制。
Protocol
Representative Results
Discussion
在以往研究中的 CP，注册外源抗原在禁区的晚体膜或 ER 通过免疫荧光显微镜16，30，，3132积累。据估计在这些专业领域的 ER 或晚体膜进行 ERAD 样运输和加工的外源性抗原细胞室是由蔗糖或碘密度梯度离心法分离使用标识泛素化作为一种 ERAD 样处理标记的宝物。后刺激他们先天免疫，CP 效率显著提高，…
開示
The authors have nothing to disclose.
Acknowledgements
这项工作被支持由高崎大学健康和福利。
Materials
| RPMI 1640 | gibco by life technologies | 11875-093 | |
| Fetal bovine serum | Equitech bio | SFB30 | |
| Sodium pyruvate | gibco by life technologies | 11360-070 | |
| MEM non-essential amino acids | gibco by life technologies | 11140-050 | |
| HEPES | gibco by life technologies | 15630-080 | |
| 2-mercaptoethanol | gibco by life technologies | 21985-023 | |
| L-glutamine | gibco by life technologies | 25030-164 | |
| Penisicillin-Sreptomycin | gibco by life technologies | 15140-122 | |
| DMEM | gibco by life technologies | 12100-46 | |
| OVA | SIGMA | A5503 | |
| Biotin-protein labelling kit | Thermo Fisher Scientific | F6347 | |
| MG-132 | Santa Cruz Biotechnology | 201270 | |
| lactacystin | SIGMA | L6785 | |
| Dounce homogenizer | IUCHI | 131703 | |
| protease inhibitor cocktails | SIGMA | P8340 | |
| iodixanol | Cosmo bio | 1114542 | |
| SA-magnetic beads | New England Biolabs | 201270 | |
| control magnetic beads | Chemagen | M-PVA012 | |
| magnetic stand | BD Biosciences | 552311 | |
| BCA protein assay kit | Thermo Fisher Scientific | 23225 | |
| silver staining kits | Cosmo bio | 423413 | |
| Reticulocyte Lysate | Promega | 1730714 | |
| Flag-tagged ubiquitin | SIGMA | U5382 | |
| anti-ovalbumin (OVA,mouse) | Antibody Shop | HYB 094-06 | |
| ant-multi-ubiquitin (mouse) | MBL | D058−3 | |
| anti-Flag (mouse) | SIGMA | F3165 | |
| trypsin | SIGMA | 85450C |
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