Abstract：Objective To investigate the role of cytosolic carboxypeptidase 1(CCP1) in the proliferation of mouse testicular primary spermatocytes (GC-2) and its molecular mechanism. Methods GC-2 cells of mouse were cultured in vitro, the cells of negative control group (NC group) and CCP1 overexpression group(GC-2+CCP1 group) were constructed by infecting GC-2 cells with lentivirus containing empty plasmid and CCP1 recombinant plasmid. The influence of CCP1 overexpression on the proliferation and cycle of GC-2 cells were detected by CCK-8 and flow cytometry; the changes of cycle key regulatory proteins in GC-2+CCP1 cells, including cyclin-dependent kinase 2(Cdk2), cyclin B1(Ccnb1) and cyclin D1(Ccnd1) were detected by real-time fluorescence quantitative PCR; the changes of Cdk2 in GC-2+CCP1 cells were analyzed by Western blot. Results CCK-8 and flow cytometry showed that the proliferation ability of GC-2+CCP1 cells in vitro was lower than NC group and cycle of GC-2+CCP1 cells arrest in G2/M phase, the differences were statistically significant (P ＜ 0.05). The mRNA and protein of Cdk2 in GC-2+CCP1 cells was higher than NC group and Cyclin B1 and Cyclin D1 mRNA were lower, the differences were statistically significant (P ＜ 0.05). Conclusion Overexpression of CCP1 can block GC-2 cells in G2/M phase, inhibit the proliferation of GC-2 cells in vitro and affect the expression of Cdk2, Cyclin B1, and Cyclin D1.
[1] Fainberg J，Kashanian JA. Recent Advances in Understanding and Managing Male Infertility [J]. F1000Res，2019，8：F1000.
[2] Hou CC，Yang WX. New Insights to the Ubiquitin-Proteasome Pathway （Upp） Mechanism During Spermatogenesis [J]. Mol Biol Rep，2013，40（4）：3213-3230.
[3] Jan SZ，Vormer TL，Jongejan A，et al. Unraveling Transcriptome Dynamics in Human Spermatogenesis [J]. Development，2017，144（20）：3659-3673.
[4] Harris A，Morgan JI，Pecot M，et al. Regenerating Motor Neurons Express Nna1，a Novel Atp/Gtp-Binding Protein Related to Zinc Carboxypeptidases [J]. Mol Cell Neurosci，2000， 16（5）：578-596.
[5] Rodriguez de la Vega M，Sevilla RG，Hermoso A，et al. Nna1- Like Proteins Are Active Metallocarboxypeptidases of a New and Diverse M14 Subfamily [J]. FASEB J，2007，21（3）：851-865.
[6] Tanco S，Tort O，Demol H，et al. C-Terminomics Screen for Natural Substrates of Cytosolic Carboxypeptidase 1 Reveals Processing of Acidic Protein C Termini [J]. Mol Cell Proteomics，2015，14（1）：177-190.
[7] Rogowski K，van Dijk J，Magiera MM，et al. A Family of Protein-Deglutamylating Enzymes Associated with Neurodegeneration [J]. Cell，2010，143（4）：564-578.
[8] 刘婧，张传领，肖瑞.胞浆羧肽酶1的研究进展[J].生命的化学，2017，37（4）：488-494.
[9] 张婷，肖瑞.胞质羧肽酶家族的分布、结构与功能[J].生命的化学，2021，41（3）：506-511.
[10] Rodriguez de la Vega Otazo M，Lorenzo J，Tort O，et al. Functional Segregation and Emerging Role of Cilia-Related Cytosolic Carboxypeptidases （Ccps） [J]. FASEB J，2013， 27（2）：424-431.
[11] Li J，Gu X，Ma Y，et al. Nna1 Mediates Purkinje Cell Dendritic Development Via Lysyl Oxidase Propeptide and Nf-Kappab Signaling [J]. Neuron，2010，68（1）：45-60.
[12] Ford GD，Ford BD，Steele EC，et al. Analysis of Transcriptional Profiles and Functional Clustering of Global Cerebellar Gene Expression in Pcd3j Mice [J]. Biochem Biophys Res Commun，2008，377（2）：556-561.
[13] Fernandez-Gonzalez A，La Spada AR，Treadaway J，et al. Purkinje Cell Degeneration （Pcd） Phenotypes Caused by Mutations in the Axotomy-Induced Gene，Nna1 [J]. Science，2002，295（5561）：1904-1906.
[14] Covaleda G，Gallego P，Vendrell J，et al. Synthesis and Structural/Functional Characterization of Selective M14 Metallocarboxypeptidase Inhibitors Based on Phosphinic Pseudopeptide Scaffold：Implications on the Design of Specific Optical Probes [J]. J Med Chem，2019，62（4）：1917- 1931.
[15] Greer CA，Shepherd GM. Mitral Cell Degeneration and Sensory Function in the Neurological Mutant Mouse Purkinje Cell Degeneration（Pcd）[J]. Brain Res，1982，235（1）：156- 161.
[16] O’Gorman S，Sidman RL. Degeneration of Thalamic Neurons in “Purkinje Cell Degeneration” Mutant Mice. I. Distribution of Neuron Loss [J]. J Comp Neurol，1985，234（3）：277-297.
[17] Blanks JC，Spee C. Retinal Degeneration in the Pcd/Pcd Mutant Mouse：Accumulation of Spherules in the Interphotoreceptor Space [J]. Exp Eye Res，1992，54（5）：637-644.
[18] Das V，Kanakkanthara A，Chan A，et al. Potential Role of Tubulin Tyrosine Ligase-Like Enzymes in Tumorigenesis and Cancer Cell Resistance [J]. Cancer Lett，2014，350（1/2）：1-4.
[19] Soucek K，Kamaid A，Phung AD，et al. Normal and Prostate Cancer Cells Display Distinct Molecular Profiles of Alpha-Tubulin Posttranslational Modifications [J]. Prostate，2006，66（9）：954-965.
[20] Kwak HJ，Gil M，Chae HS，et al. Expression of Atp/Gtp Binding Protein 1 Has Prognostic Value for the Clinical Outcomes in Non-Small Cell Lung Carcinoma [J]. J Pers Med，2020，10（4）：263.
[21] Chocu S，Calvel P，Rolland AD，et al. Spermatogenesis in Mammals：Proteomic Insights [J]. Syst Biol Reprod Med，2012，58（4）：179-190.
[22] Li Y，Yan H，Yu Y，et al. Bisphenol B Stimulates Leydig Cell Proliferation but Inhibits Maturation in Late Pubertal Rats [J]. Food Chem Toxicol，2021，153：112248.
[23] Duan PJ，Zhao JH，Xie LL. Cul4b Promotes the Progression of Ovarian Cancer by Upregulating the Expression of Cdk2 and Cyclind1 [J]. J Ovarian Res，2020，13（1）：76.
[24] Satyanarayana A，Kaldis P. Mammalian Cell-Cycle Regulation：Several Cdks，Numerous Cyclins and Diverse Compensatory Mechanisms [J]. Oncogene，2009，28（33）：2925- 2939.
[25] Tang JX，Li J，Cheng JM，et al. Requirement for Ccnb1 in Mouse Spermatogenesis [J]. Cell Death Dis，2017，8（10）：e3142.
京公网安备 11010502046598号