Lysine acetylation regulates the activity of nuclear Pif1

In Saccharomyces cerevisiae, the Pif1 helicase functions in both nuclear and mitochondrial DNA replication and repair processes, preferentially unwinding RNA:DNA hybrids and resolving G-quadruplex structures. We sought to determine how the various activities of Pif1 are regulated in vivo Here, we report lysine acetylation of nuclear Pif1 and demonstrate that it influences both Pif1's cellular roles and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 and deacetylase Rpd3 are primarily responsible for the dynamic acetylation of nuclear Pif1. MS analysis revealed that Pif1 was modified in several domains throughout the protein's sequence on the N terminus (Lys-118 and Lys-129), helicase domain (Lys-525, Lys-639, and Lys-725), and C terminus (Lys-800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase, ATPase, and DNA-binding activities, whereas maintaining its substrate preferences. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose that acetylation is involved in regulating of Pif1 activities, influencing a multitude of DNA transactions vital to the maintenance of genome integrity

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The Biochemical Activities of the <i>Saccharomyces cerevisiae</i> Pif1 Helicase Are Regulated by Its N-Terminal Domain

Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1&#916;N) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1&#916;N was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity

Dynamic regulation of Pif1 acetylation is crucial to the maintenance of genome stability

PIF1 family helicases are evolutionarily conserved among prokaryotes and eukaryotes. These enzymes function to support genome integrity by participating in multiple DNA transactions that can be broadly grouped into DNA replication, DNA repair, and telomere maintenance roles. However, the levels of PIF1 activity in cells must be carefully controlled, as Pif1 over-expression in Saccharomyces cerevisiae is toxic, and knockdown or over-expression of human PIF1 (hPIF1) supports cancer cell growth. This suggests that PIF1 family helicases must be subject to tight regulation in vivo to direct their activities to where and when they are needed, as well as to maintain those activities at proper homeostatic levels. Previous work shows that C-terminal phosphorylation of S. cerevisiae Pif1 regulates its telomere maintenance activity, and we recently identified that Pif1 is also regulated by lysine acetylation. The over-expression toxicity of Pif1 was exacerbated in cells lacking the Rpd3 lysine deacetylase, but mutation of the NuA4 lysine acetyltransferase subunit Esa1 ameliorated this toxicity. Using recombinant proteins, we found that acetylation stimulated the DNA binding affinity, ATPase activity, and DNA unwinding activities of Pif1. All three domains of the helicase were targets of acetylation in vitro, and multiple lines of evidence suggest that acetylation drives a conformational change in the N-terminal domain of Pif1 that impacts this stimulation. It is currently unclear what triggers lysine acetylation of Pif1 and how this modification impacts the many in vivo functions of the helicase, but future work promises to shed light on how this protein is tightly regulated within the cell

The WYL Domain of the PIF1 Helicase from the Thermophilic Bacterium Thermotoga elfii is an Accessory Single-Stranded DNA Binding Module

PIF1 family helicases are conserved from bacteria to man. With the exception of the well-studied yeast PIF1 helicases (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding of the protein domains found N- and C-terminal to the characteristic central PIF1 helicase domain in these proteins. Here, using chimeric constructs, we show that the ScPif1 and ScRrm3 helicase domains are interchangeable and that the N-terminus of ScRrm3 is important for its function in vivo. This suggests that PIF1 family helicases evolved functional modules fused to a generic motor domain. To investigate this hypothesis, we characterized the biochemical activities of the PIF1 helicase from the thermophilic bacterium Thermotoga elfii (TePif1), which contains a C-terminal WYL domain of unknown function. Like helicases from other thermophiles, recombinant TePif1 was easily prepared, thermostable in vitro, and displayed activities similar to its eukaryotic homologues. We also found that the WYL domain was necessary for high-affinity single-stranded DNA (ssDNA) binding and affected both ATPase and helicase activities. Deleting the WYL domain from TePif1 or mutating conserved residues in the predicted ssDNA binding site uncoupled ATPase activity and DNA unwinding, leading to higher rates of ATP hydrolysis but less efficient DNA helicase activity. Our findings suggest that the domains of unknown function found in eukaryotic PIF1 helicases may also confer functional specificity and additional activities to these enzymes, which should be investigated in future work

The WYL Domain of the PIF1 Helicase from the Thermophilic Bacterium Thermotoga elfii is an Accessory Single-Stranded DNA Binding Module

PIF1 family helicases are conserved from bacteria to man. With the exception of the well-studied yeast PIF1 helicases (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding of the protein domains found N- and C-terminal to the characteristic central PIF1 helicase domain in these proteins. Here, using chimeric constructs, we show that the ScPif1 and ScRrm3 helicase domains are interchangeable and that the N-terminus of ScRrm3 is important for its function <i>in vivo</i>. This suggests that PIF1 family helicases evolved functional modules fused to a generic motor domain. To investigate this hypothesis, we characterized the biochemical activities of the PIF1 helicase from the thermophilic bacterium Thermotoga elfii (TePif1), which contains a C-terminal WYL domain of unknown function. Like helicases from other thermophiles, recombinant TePif1 was easily prepared, thermostable <i>in vitro</i>, and displayed activities similar to its eukaryotic homologues. We also found that the WYL domain was necessary for high-affinity single-stranded DNA (ssDNA) binding and affected both ATPase and helicase activities. Deleting the WYL domain from TePif1 or mutating conserved residues in the predicted ssDNA binding site uncoupled ATPase activity and DNA unwinding, leading to higher rates of ATP hydrolysis but less efficient DNA helicase activity. Our findings suggest that the domains of unknown function found in eukaryotic PIF1 helicases may also confer functional specificity and additional activities to these enzymes, which should be investigated in future work