Wenta Tomasz, Wang Guanpeng, Van Buren Tessa, Zolkiewski Michal, Zolkiewska Anna
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA.
Department of General and Medical Biochemistry, Faculty of Biology, University of Gdansk, Gdansk, 80-308, Poland.
Apoptosis. 2025 Feb;30(1-2):334-348. doi: 10.1007/s10495-024-02053-1. Epub 2024 Dec 7.
Loss-of-function mutations in the CLPB gene lead to congenital neutropenia due to impaired neutrophil differentiation. CLPB, a member of the AAA+ family of proteins, resides in the intermembrane space of mitochondria. The mechanism by which a loss of CLPB elicits defects in the differentiation program of neutrophil precursor cells is not understood. Here, we used 32D clone 3 (32Dcl3) cells, an interleukin-3 (IL-3)-dependent mouse myeloblastic cell line model, to investigate the effects of CLPB knockout on myeloblast-to-neutrophil differentiation in vitro. We found that CLPB-deficient 32Dcl3 cells showed a decreased mitochondrial membrane potential and increased levels of insoluble HAX1 aggregates in mitochondria, as compared to control cells. Despite those abnormalities, CLPB loss did not affect cell proliferation rates in the presence of IL-3 but it increased apoptosis after IL-3 withdrawal and simultaneous induction of cell differentiation with granulocytic colony stimulating factor (G-CSF). CLPB-deficient cells that survived the stress associated with IL-3 withdrawal/G-CSF treatment expressed the same levels of differentiation markers as control cells. Moreover, we found that increased apoptosis of CLPB-deficient cells is linked to production of reactive oxygen species (ROS). N-acetylcysteine, exogenous free fatty acids, or exogenous citrate protected CLPB-deficient 32Dcl3 cells from apoptosis at the onset of differentiation. The protective effect of citrate was abolished by inhibition of ATP-citrate lyase (ACLY), an enzyme that converts cytosolic citrate into acetyl-CoA, a substrate for protein acetylation. We propose that citrate supplementation may help mitigate the effects of CLPB loss by facilitating ACLY-dependent ROS detoxification in granulocytic precursor cells.
CLPB基因的功能丧失突变由于中性粒细胞分化受损导致先天性中性粒细胞减少。CLPB是AAA + 蛋白家族的成员，位于线粒体的膜间隙中。CLPB缺失引发中性粒细胞前体细胞分化程序缺陷的机制尚不清楚。在这里，我们使用32D克隆3（32Dcl3）细胞，一种依赖白细胞介素-3（IL-3）的小鼠成髓细胞系模型，来研究CLPB基因敲除对体外成髓细胞向中性粒细胞分化的影响。我们发现，与对照细胞相比，CLPB缺陷的32Dcl3细胞线粒体膜电位降低，线粒体中不溶性HAX1聚集体水平升高。尽管存在这些异常，但在有IL-3的情况下，CLPB缺失并不影响细胞增殖率，但在IL-3撤除并同时用粒细胞集落刺激因子（G-CSF）诱导细胞分化后，它会增加细胞凋亡。在与IL-3撤除/G-CSF处理相关的应激中存活下来的CLPB缺陷细胞表达的分化标志物水平与对照细胞相同。此外，我们发现CLPB缺陷细胞凋亡增加与活性氧（ROS）的产生有关。N-乙酰半胱氨酸、外源性游离脂肪酸或外源性柠檬酸盐可保护CLPB缺陷的32Dcl3细胞在分化开始时不发生凋亡。柠檬酸的保护作用通过抑制ATP-柠檬酸裂解酶（ACLY）而被消除，ACLY是一种将胞质柠檬酸转化为乙酰辅酶A的酶，乙酰辅酶A是蛋白质乙酰化的底物。我们提出，补充柠檬酸盐可能有助于减轻CLPB缺失的影响，通过促进粒细胞前体细胞中依赖ACLY的ROS解毒。
