Nonhemolytic Cell-Penetrating Peptides: Site Specific Introduction of Glutamine and Lysine Residues into the α-Helical Peptide Causes Deletion of Its Direct Membrane Disrupting Ability but Retention of Its Cell Penetrating Ability
Nonhemolytic Cell-Penetrating Peptides: Site Specific Introduction of Glutamine and Lysine Residues into the α-Helical Peptide Causes Deletion of Its Direct Membrane Disrupting Ability but Retention of Its Cell Penetrating Ability
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DOI：
10.1021/acs.biomac.6b00874
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发表时间：
2016-09-01
影响因子：
6.2
通讯作者：
Yu, Jaehoon
中科院分区：
文献类型：
--
作者：
Kim, Seoyeon;Hyun, Soonsil;Yu, Jaehoon
细胞穿透肽 (CPP) 通常具有与 α 螺旋肽相关的阳离子和两亲特性。这些特性赋予 CPP 兼具破膜和渗透能力。为了使 CPP 在生物医学应用中安全，必须消除其膜破坏能力所产生的毒性，同时必须保留其细胞穿透能力。在这项研究中，我们系统地构建了两亲性α螺旋模型肽（LKKLLKLLKKLLK-LAG，LK肽）的突变体。 LK肽中的疏水性氨基酸亮氨酸被亲水性氨基酸取代，以减少溶血或细胞毒性。大多数突变体被发现膜破坏能力减弱，但细胞穿透能力也减弱。然而，L8Q和L8K突变体被发现具有低微摩尔范围的细胞穿透能力，并且几乎没有膜破坏能力。这些选定的突变体利用能量依赖性内吞机制而不是能量独立的直接细胞穿透机制来进入细胞。此外，突变体可用于将抗癌药物甲氨蝶呤（MTX）递送至细胞，从而克服对该药物的耐药性。为了确定这些突变对膜破坏和细胞穿透能力的影响是否具有普遍性，引入了天然两亲性 α-螺旋抗菌肽 (AMP) LL37 的 Q 和 K 突变。 LL37 的特定位置 Q 和 K 突变体被发现具有较低的溶血毒性，并保留了穿透真核细胞（如 MDA-MB-231 细胞）的能力。总而言之，这项工作中的观察结果表明，通过用 Q 和 K 等轻度亲水性氨基酸替换疏水性残基来中断两亲性 α 螺旋 CPP 和 AMP 的整体疏水性，可能是构建具有强细胞穿透能力和弱细胞膜破坏能力的肽的理想策略。
Cell-penetrating peptides (CPPs) often have cationic and amphipathic characteristics that are commonly associated with alpha-helical peptides. These features give CPPs both membrane demolishing and penetrating abilities. To make CPPs safe for biomedical applications, their toxicities resulting from their membrane demolishing abilities must be removed while their cell penetrating abilities must be retained. In this study, we systematically constructed mutants of the amphipathic alpha-helical model peptide (LKKLLKLLKKLLK-LAG, LK peptide). The hydrophobic amino acid leucine in the LK peptide was replaced with hydrophilic amino acids to reduce hemolytic or cell toxicity. Most of the mutants Were found to have weakened membrane disrupting abilities, but their cell penetrating abilities were also weakened. However, the L8Q and L8K mutants were found to have low micromolar range cell penetrating ability and almost no membrane disrupting ability. These selected mutants utilize energy-dependent endocytosis mechanisms instead of an energy-independent direct cell penetrating mechanism to enter cells. In addition, the mutants can be used to deliver the anticancer drug methotrexate (MTX) to cells, thereby overcoming resistance to this drug. To determine if the effect of these mutations on the membrane disrupting and cell penetrating abilities is general, Q and K mutations of the natural amphipathic alpha-helical antimicrobial peptide (AMP), LL37, were introduced. Specific positional Q and K mutants of LL37 were found to have lower hemolytic toxicities and preserved the ability to penetrate eukaryotic cells such as MDA-MB-231 cells. Taken together, observations made in this work Suggest that interrupting the global hydrophobicity of amphipathic alpha-helical CPPs and AMPs, by replacing hydrophobic residues with mildly hydrophilic amino acids such as Q and K, might be an ideal strategy for constructing peptides that have strong cell penetrating abilities and weak cell membrane disrupting abilities.