Departments of Molecular Pharmacology and Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue F248, Bronx, NY 10461, USA.
Department of Biochemistry, Escola Paulista de Medicina, Federal University of Sao Paulo, Sao Paulo, SP 04023-901, Brazil.
Cell Chem Biol. 2021 Jan 21;28(1):105-112.e4. doi: 10.1016/j.chembiol.2020.11.003. Epub 2020 Nov 19.
Neuropeptides and peptide hormones are important cell-cell signaling molecules that mediate many physiological processes. Unlike classic 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.
神经肽和肽类激素是重要的细胞间信号分子，它们介导许多生理过程。与经典神经递质不同，肽类经历细胞类型特异性的翻译后修饰，从而影响其生物学活性。为了能够鉴定特定基因细胞类型的肽组，我们生成了条件性敲除羧肽酶 E (Cpe)基因的小鼠，Cpe 是一种重要的神经肽加工酶。Cpe 的缺失导致含有 C 末端碱性残基的神经肽前体积累，这些残基可作为亲和纯化的标签。随后使用定量肽组学来鉴定纯化的肽，从而揭示具有 Cpe 基因缺失的细胞中的特定神经肽形式。为了验证该方法，我们使用了在 proopiomelanocortin (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，检测到源自 proopiomelanocortin（如预期的那样）和 POMC 神经元中的 proSAAS 的肽。该技术可用于分析任何表达 Cre 的细胞类型中的特定形式的肽。
