Mitochondrial proteases are key components in mitochondrial stress responses that maintain proteostasis and mitochondrial integrity in harsh environmental conditions, which leads to the acquisition of aggressive phenotypes, including chemoresistance and metastasis. However, the molecular mechanisms and exact role of mitochondrial proteases in cancer remain largely unexplored. Here, we identified functional crosstalk between LONP1 and ClpP, which are two mitochondrial matrix proteases that cooperate to attenuate proteotoxic stress and protect mitochondrial functions for cancer cell survival. LONP1 and ClpP genes closely localized on chromosome 19 and were co-expressed at high levels in most human cancers. Depletion of both genes synergistically attenuated cancer cell growth and induced cell death due to impaired mitochondrial functions and increased oxidative stress. Using mitochondrial matrix proteomic analysis with an engineered peroxidase (APEX)-mediated proximity biotinylation method, we identified the specific target substrates of these proteases, which were crucial components of mitochondrial functions, including oxidative phosphorylation, the TCA cycle, and amino acid and lipid metabolism. Furthermore, we found that LONP1 and ClpP shared many substrates, including serine hydroxymethyltransferase 2 (SHMT2). Inhibition of both LONP1 and ClpP additively increased the amount of unfolded SHMT2 protein and enhanced sensitivity to SHMT2 inhibitor, resulting in significantly reduced cell growth and increased cell death under metabolic stress. Additionally, prostate cancer patients with higher LONP1 and ClpP expression exhibited poorer survival. These results suggest that interventions targeting the mitochondrial proteostasis network via LONP1 and ClpP could be potential therapeutic strategies for cancer.

线粒体蛋白酶是线粒体应激反应中的关键成分，可在恶劣的环境条件下维持蛋白稳定和线粒体完整性，从而导致获得侵袭性表型，包括化学抗性和转移。但是，线粒体蛋白酶在癌症中的分子机制和确切作用仍未得到充分探索。在这里，我们确定了LONP1和ClpP之间的功能性串扰，这是两个线粒体基质蛋白酶，它们协同作用以减轻蛋白毒性压力并保护线粒体功能以维持癌细胞的生存。LONP1和ClpP基因紧密位于19号染色体上，并在大多数人类癌症中以高水平共表达。由于线粒体功能受损和氧化应激增加，这两个基因的耗竭协同减弱了癌细胞的生长并诱导了细胞死亡。使用线粒体基质蛋白质组学分析与工程过氧化物酶（APEX）介导的邻近生物素化方法，我们确定了这些蛋白酶的特定目标底物，它们是线粒体功能的关键组成部分，包括氧化磷酸化，TCA循环以及氨基酸和脂质代谢。此外，我们发现LONP1和ClpP共享许多底物，包括丝氨酸羟甲基转移酶2（SHMT2）。对LONP1和ClpP的抑制都会增加未折叠的SHMT2蛋白的量并增强对SHMT2抑制剂的敏感性，从而导致细胞生长显着减少，并在代谢应激下增加细胞死亡。此外，具有较高LONP1和ClpP表达的前列腺癌患者表现出较差的生存率。这些结果表明，通过LONP1和ClpP靶向线粒体蛋白质稳定网络的干预措施可能是潜在的癌症治疗策略。