Gao, Ming

ABSTRACT Lactylation, a protein post-translational modification driven by lactate, plays crucial roles in various physiological and pathological processes. However, the function and underlying mechanisms of lactylation in infection and pathogenesis of influenza A virus (IAV) remain poorly understood. Here, we demonstrated that sirtuin 1 (SIRT1) suppressed IAV replication by mediating delactylation of viral proteins. We found that IAV infection significantly downregulated the expression of SIRT1. Functional studies revealed that depletion of SIRT1 markedly enhanced replication of IAV, whereas overexpression of SIRT1 attenuated the viral replication. Mechanistically, we observed that SIRT1 interacted with IAV NP, PA, PB1, and PB2 proteins, components of the viral ribonucleoprotein (vRNP) complex, and restrained the activity of the vRNP complex, thereby repressing transcription and replication of the IAV genome. Furthermore, we identified that viral NP, PA, PB1, and PB2 proteins underwent lactylation during IAV infection, and SIRT1 facilitated the delactylation of NP, PA, and PB2. Loss of SIRT1 enhanced the lactylation of NP, PA, and PB2, while overexpression of SIRT1 impaired lactylation of these viral proteins during IAV infection. Moreover, inhibition or depletion of lactate dehydrogenase A (LDHA) robustly reduced the lactylation of NP, PA, and PB2, thereby abolishing the promotion of IAV replication caused by SIRT1 deficiency, suggesting that SIRT1 repressed IAV replication via delactylation of viral NP, PA, and PB2 proteins. Together, these results uncover a mechanism that SIRT1 suppresses IAV replication via delactylation of the vRNP components and elicit a critical role for the lactylation of viral proteins in regulating IAV infection and pathogenesis. IMPORTANCE Lactylation plays vital roles in diverse pathological processes. However, the relationship between lactylation and influenza A virus (IAV) infection remains largely unexplored. Particularly, little information is available about the lactylation of viral proteins and their roles in the IAV pathogenesis. Here, viral proteins (NP, PA, PB1, and PB2) were identified to become lactylated during IAV infection. SIRT1 interacted with and promoted the delactylation of viral NP, PA, and PB2 proteins, thereby restraining the vRNP activity and inhibiting the viral transcription and replication. Interestingly, the expression of SIRT1 decreased following IAV infection, suggesting that IAV had evolved a mechanism to downregulate the SIRT1 expression, thereby ensuring appropriate lactylation of viral proteins and facilitating the IAV replication. Together, these findings reveal a critical role for the lactylation of viral proteins in regulating the replication of IAV and provide an important insight into the complicated interplay between the host and IAV.

Abstract Enhanced DNA repair is an important mechanism of inherent and acquired resistance to DNA targeted therapies, including poly ADP ribose polymerase inhibition. Spleen associated tyrosine kinase (Syk) is a non-receptor tyrosine kinase known to regulate immune cell function, cell adhesion, and vascular development. Here, we report that Syk can be expressed in high grade serous ovarian cancer and triple negative breast cancers and promotes DNA double strand break resection, homologous recombination (HR) and therapeutic resistance. We found that Syk is activated by ATM following DNA damage and is recruited to DNA double strand breaks by NBS1. Once at the break site, Syk phosphorylates CtIP, a key mediator of resection and HR, at Thr-847 to promote repair activity, specifically in Syk expressing cancer cells. Syk inhibition or genetic deletion abolished CtIP Thr-847 phosphorylation and overcame the resistant phenotype. Collectively, our findings suggest that Syk drives therapeutic resistance by promoting DNA resection and HR through a novel ATM-Syk-CtIP pathway, and that Syk is a new tumor-specific target to sensitize Syk-expressing tumors to PARPi and other DNA targeted therapy.

Abstract RPA is a critical factor for DNA replication and replication stress response. Surprisingly, we found that chromatin RPA stability is tightly regulated. We report that the GDP/GTP exchange factor DOCK7 acts as a critical replication stress regulator to promote RPA stability on chromatin. DOCK7 is phosphorylated by ATR and then recruited by MDC1 to the chromatin and replication fork during replication stress. DOCK7-mediated Rac1/Cdc42 activation leads to the activation of PAK1, which subsequently phosphorylates RPA1 at S135 and T180 to stabilize chromatin-loaded RPA1 and ensure proper replication stress response. Moreover, DOCK7 is overexpressed in ovarian cancer and depleting DOCK7 sensitizes cancer cells to camptothecin. Taken together, our results highlight a novel role for DOCK7 in regulation of the replication stress response and highlight potential therapeutic targets to overcome chemoresistance in cancer.

Abstract PrimPol has been recently identified as a DNA damage tolerant polymerase that plays an important role in replication stress response. However, the regulatory mechanisms of PrimPol are not well defined. In this study, we identify that the deubiquitinase USP36 interferes with degradation of PrimPol to regulate the replication stress response. Mechanistically, USP36 is deubiquitinated following DNA replication stress, which in turn facilitates its upregulation and interaction with PrimPol. USP36 deubiquitinates K29-linked polyubiquitination of PrimPol and increases its protein stability. Depletion of USP36 results in replication stress-related defects and elevates cell sensitivity to DNA-damage agents, such as cisplatin and olaparib. Moreover, USP36 expression positively correlates with the level of PrimPol protein and poor prognosis in patient samples. These findings indicate that the regulation of PrimPol K29-linked ubiquitination by USP36 plays a critical role in DNA replication stress and chemotherapy response.