Miniproteins have a size between that of larger biologics and small molecules and presumably possess the advantages of both; they represent an expanding class of promising scaffolds for the design of affinity reagents, enzymes, and therapeutics. Conventional strategies to promote cellular uptake of miniproteins rely on extensive grafting or embedding of arginine residues. However, the requirement of using cationic arginines would cause problems to the modified miniproteins, for example, low solubility in solutions (proneness of aggregation) and potential toxicity, which are open secrets in the peptide and protein communities. In this work, we report that the cell-permeability of cationic miniproteins can be further markedly increased through appending a magic CXC (cysteine-any-cysteine) motif, which takes advantage of thiol–disulfide exchanges on the cell surface. More importantly, we discovered that the high cell permeability of the CXC-appended miniproteins can still be preserved when the embedded arginines are all substituted with lysine residues, indicating that the “arginine magic” essential to almost all cell-permeable peptides and (mini)proteins is not required for the CXC-mediated cellular uptake. This finding provides a new avenue for designing highly cell-permeable miniproteins without compromise of potential toxicity and stability arising from arginine embedding or grafting.

中文翻译：

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CXC介导的细胞对微蛋白的摄取：抛弃“精氨酸魔术”

小蛋白的大小介于较大的生物制剂和小分子之间，并且可能具有两者的优势。它们代表了用于设计亲和试剂，酶和治疗剂的不断扩展的有前途的支架。促进细胞摄取微蛋白的常规策略依赖于精氨酸残基的广泛接枝或嵌入。但是，使用阳离子精氨酸的要求会给修饰的微蛋白带来问题，例如，在溶液中的溶解度低（聚集的倾向）和潜在的毒性，这是肽和蛋白质群落中的秘密。在这项工作中，我们报告说，通过添加神奇的CXC（半胱氨酸-任何-半胱氨酸）基序，该基序利用了细胞表面硫醇-二硫键的交换。更重要的是，我们发现，当嵌入的精氨酸全部被赖氨酸残基取代时，仍可以保留CXC附加的微蛋白的高细胞通透性，这表明几乎所有细胞可渗透的肽和（微型）必需的“精氨酸魔术” CXC介导的细胞摄取不需要蛋白质。这一发现为设计高细胞渗透性小蛋白提供了一条新途径，而不会损害精氨酸包埋或嫁接所产生的潜在毒性和稳定性。