Neuropeptidomic Analysis of a Genetically Defined Cell Type in Mouse Brain and Pituitary.
Neuropeptidomic Analysis of a Genetically Defined Cell Type in Mouse Brain and Pituitary.
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小鼠脑和垂体中遗传定义的细胞类型的神经肽分析。
DOI：
10.1016/j.chembiol.2020.11.003
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发表时间：
2021-01-21
影响因子：
8.6
通讯作者：
Devi LA
中科院分区：
文献类型：
--
作者：
Fricker LD;Tashima AK;Fakira AK;Hochgeschwender U;Wetsel WC;Devi LA
神经肽和肽类激素是重要的细胞间信号分子，介导许多生理过程。与经典的神经递质不同，肽经历细胞类型特异性的翻译后修饰，从而影响其生物活性。为了能够识别基因定义的细胞类型的肽库，我们产生了有条件破坏羧肽酶E（Cpe）基因的小鼠，这是一种必需的神经肽加工酶。Cpe的缺失导致含有C-末端碱性残基的神经肽前体的积累，所述C-末端碱性残基用作亲和纯化的标签。随后使用定量肽组学鉴定纯化的肽，从而揭示具有破坏的Cpe基因的细胞中神经肽的特定形式。为了验证该方法，我们使用了在阿黑皮素原（Pomc）启动子下表达Cre重组酶的小鼠，并分析了下丘脑和垂体提取物，检测了源自阿黑皮素原的肽（如预期的那样）以及POMC神经元中的proSAAS。该技术能够分析任何表达Cre的细胞类型中的特定形式的肽。我们报告了一种方法，以确定在特定的细胞类型的神经肽，使用小鼠与条件删除羧肽酶E。通过删除阿黑皮素原细胞中的CPE来验证。令人惊讶的是，这些小鼠的促胰岛素生成肽α-MSH水平大大降低，但并不肥胖。
Neuropeptides and peptide hormones are important cell-cell signaling molecules that mediate many physiological processes. Unlike classical neurotransmitters, peptides undergo cell-type-specific post-translational modifications that affect their biological activity. To enable the identification of the peptide repertoire of a genetically-defined cell-type, we generated mice with a conditional disruption of the gene for carboxypeptidase E (Cpe), an essential neuropeptide-processing enzyme. The loss of Cpe leads to accumulation of neuropeptide precursors containing C-terminal basic residues which serve as tags for affinity-purification. The purified peptides are subsequently identified using quantitative peptidomics, thereby revealing the specific forms of neuropeptides in cells with the disrupted Cpe gene. To validate the method, we used mice expressing Cre recombinase under the proopiomelanocortin (Pomc) promoter and analyzed hypothalamic and pituitary extracts, detecting peptides derived from proopiomelanocortin (as expected) and also proSAAS in POMC neurons. This technique enables the analyses of specific forms of peptides in any Cre-expressing cell-type. We report a method to identify neuropeptides in specific cell-types, using mice with a conditional deletion of carboxypeptidase E. It was validated by deleting CPE in proopiomelanocortin cells. Surprisingly, these mice have greatly reduced levels of the anorexigenic peptide α-MSH but are not obese.
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影响因子：
3.5
作者：
Anderson EJ;Çakir I;Carrington SJ;Cone RD;Ghamari-Langroudi M;Gillyard T;Gimenez LE;Litt MJ
通讯作者：
Litt MJ
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Magkos, Faidon
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Bora, Adriana;Annangudi, Suresh P.;Millet, Larry J.;Rubakhin, Stanislav S.;Forbes, Andrew J.;Kelleher, Neil L.;Gillette, Martha U.;Sweedler, Jonathan V.
通讯作者：
Sweedler, Jonathan V.
影响因子：
46.9
作者：
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通讯作者：
Chin JW