Targeted protein degradation can provide advantages over inhibition approaches in the development of therapeutic strategies. Lysosome-targeting chimeras (LYTACs) harness receptors, such as the cation-independent mannose 6–phosphate receptor (CI-M6PR), to direct extracellular proteins to lysosomes. In this work, we used a genome-wide CRISPR knockout approach to identify modulators of LYTAC-mediated membrane protein degradation in human cells. We found that disrupting retromer genes improved target degradation by reducing LYTAC recycling to the plasma membrane. Neddylated cullin-3 facilitated LYTAC-complex lysosomal maturation and was a predictive marker for LYTAC efficacy. A substantial fraction of cell surface CI-M6PR remains occupied by endogenous M6P-modified glycoproteins. Thus, inhibition of M6P biosynthesis increased the internalization of LYTAC-target complexes. Our findings inform design strategies for next-generation LYTACs and elucidate aspects of cell surface receptor occupancy and trafficking.

中文翻译：

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阐明溶酶体靶向嵌合体靶向膜蛋白降解的细胞决定因素


在治疗策略的开发中，靶向蛋白质降解可以提供优于抑制方法的优势。溶酶体靶向嵌合体 (LYTAC) 利用受体，例如不依赖于阳离子的甘露糖 6-磷酸受体 (CI-M6PR)，将细胞外蛋白质引导至溶酶体。在这项工作中，我们使用全基因组 CRISPR 敲除方法来鉴定人类细胞中 LYTAC 介导的膜蛋白降解的调节剂。我们发现，破坏逆转录酶基因可通过减少 LYTAC 循环至质膜来改善靶标降解。 Neddylated cullin-3 促进 LYTAC 复合物溶酶体成熟，是 LYTAC 功效的预测标记。细胞表面 CI-M6PR 的很大一部分仍然被内源性 M6P 修饰的糖蛋白占据。因此，M6P生物合成的抑制增加了LYTAC-靶复合物的内化。我们的研究结果为下一代 LYTAC 的设计策略提供了信息，并阐明了细胞表面受体占据和运输的各个方面。