药物滥用继续对艾滋病毒控制工作构成重大挑战。在我们的调查中,我们发现,可卡因不仅上调DNA依赖性蛋白激酶(DNA-PK)的表达,而且通过在S2056增强其磷酸化作用,从而增强DNA-PK的激活.此外,DNA-PK磷酸化触发DNA-PK易位到细胞核中。可卡因促进DNA-PK核易位的发现进一步证实了我们对可卡因暴露后HIV长末端重复序列(LTR)处DNA-PK募集增强的观察。通过激活和促进DNA-PK的核易位,可卡因有效地协调了HIV转录的多个阶段,从而促进HIV复制。此外,我们的研究表明,可卡因诱导的DNA-PK促进RNA聚合酶II(RNAPII)羧基末端结构域(CTD)在Ser5和Ser2位点的过度磷酸化,增强起始和伸长阶段,分别,HIV转录。可卡因通过激活细胞周期蛋白依赖性激酶7(CDK7)和随后的CDK9磷酸化进一步支持其转录起始和延伸的增强,从而促进正转录延伸因子b(P-TEFb)活性。我们第一次证明可卡因,通过DNA-PK激活,促进TRIM28在丝氨酸824的特异性磷酸化(p-TRIM28,S824)。这种修饰将TRIM28从转录抑制剂转化为HIV转录的反式激活因子。此外,我们观察到TRIM28(p-TRIM28,S824)的磷酸化促进HIV转录从暂停阶段到延伸阶段的过渡,从而促进全长HIV基因组转录物的产生。这一发现证实了在Ser2上观察到的增强的RNAPIICTD磷酸化,Ser2是转录延伸的标记,可卡因暴露后。因此,在可卡因治疗后,我们观察到在HIVLTR中p-TRIM28-(S824)的募集增加。总的来说,我们的研究结果揭示了可卡因诱导HIV转录和基因表达的复杂分子机制.这些发现为开发旨在减轻可卡因对HIV感染者的有害影响的高度靶向疗法提供了希望。
■可卡因上调DNA-PK的表达和活性。可卡因在S2056选择性增强DNA-PK的磷酸化,这是一种翻译后修饰,标志着DNA-PK的功能活性形式。可卡因增强DNA-PK的核易位。DNA-PK抑制严重损害HIV转录,复制,和延迟重新激活。可卡因通过分别增强Ser5和Ser2的RNAPIICTD磷酸化来促进HIV的起始和延伸阶段,通过刺激DNA-PK.可卡因还通过刺激CDK7(TFIIH的激酶)和CDK9(P-TEFb的激酶亚基)来支持HIV转录的起始和延伸阶段。分别。可卡因介导的DNA-PK激活通过逆转暂停因子TRIM28的抑制作用并通过催化其在S824位点的磷酸化将其转化为反式激活剂来缓解RNAPII的暂停。因此,可卡因,通过激活DNA-PK,促进HIV转录的多个阶段,即,initiation,RNAPII暂停释放,和伸长率。
Drug abuse continues to pose a significant challenge in HIV control efforts. In our investigation, we discovered that cocaine not only upregulates the expression of DNA-dependent protein kinase (DNA-PK) but also augments DNA-PK activation by enhancing its phosphorylation at S2056. Moreover, DNA-PK phosphorylation triggers the translocation of DNA-PK into the nucleus. The finding that cocaine promotes nuclear translocation of DNA-PK further validates our observation of enhanced DNA-PK recruitment at the HIV long terminal repeat (LTR) following cocaine exposure. By activating and facilitating the nuclear translocation of DNA-PK, cocaine effectively orchestrates multiple stages of HIV transcription, thereby promoting HIV replication. Additionally, our study indicates that cocaine-induced DNA-PK promotes hyper-phosphorylation of RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) at Ser5 and Ser2 sites, enhancing both initiation and elongation phases, respectively, of HIV transcription. Cocaine\'s enhancement of transcription initiation and elongation is further supported by its activation of cyclin-dependent kinase 7 (CDK7) and subsequent phosphorylation of CDK9, thereby promoting positive transcriptional elongation factor b (P-TEFb) activity. We demonstrate for the first time that cocaine, through DNA-PK activation, promotes the specific phosphorylation of TRIM28 at Serine 824 (p-TRIM28, S824). This modification converts TRIM28 from a transcriptional inhibitor to a transactivator for HIV transcription. Additionally, we observe that phosphorylation of TRIM28 (p-TRIM28, S824) promotes the transition from the pausing phase to the elongation phase of HIV transcription, thereby facilitating the production of full-length HIV genomic transcripts. This finding corroborates the observed enhanced RNAP II CTD phosphorylation at Ser2, a marker of transcriptional elongation, following cocaine exposure. Accordingly, upon cocaine treatment, we observed elevated recruitment of p-TRIM28-(S824) at the HIV LTR. Overall, our results have unraveled the intricate molecular mechanisms underlying cocaine-induced HIV transcription and gene expression. These findings hold promise for the development of highly targeted therapeutics aimed at mitigating the detrimental effects of cocaine in individuals living with HIV.