DNA transcription is initiated by a small regulatory region of transactivators known as the transactivation domain. In contrast to the rapid progress made on the functional aspect of this promiscuous domain, its structural feature is still poorly characterized. Here, our multidimensional NMR study reveals that an unbound full-length p53 transactivation domain, although similar to the recently discovered group of loosely folded proteins in that it does not have tertiary structure, is nevertheless populated by an amphipathic helix and two nascent turns. The helix is formed by residues Thr¹⁸–Leu²⁶(Thr-Phe-Ser-Asp-Leu-Trp-Lys-Leu-Leu), whereas the two turns are formed by residues Met⁴⁰–Met⁴⁴ and Asp⁴⁸–Trp⁵³, respectively. It is remarkable that these local secondary structures are selectively formed by functionally critical and positionally conserved hydrophobic residues present in several acidic transactivation domains. This observation suggests that such local structures are general features of acidic transactivation domains and may represent “specificity determinants” (Ptashne, M., and Gann, A. A. F. (1997),Nature 386, 569–577) that are important for transcriptional activity.

Regulatory T (T_reg) cells act as terminators of T cell immuniy during acute phase of viral infection; however, their role and suppressive mechanism in chronic viral infection are not completely understood. In this study, we compared the phenotype and function of T_reg cells during acute or chronic infection with lymphocytic choriomeningitis virus. Chronic infection, unlike acute infection, led to a large expansion of T_reg cells and their upregulation of programmed death-1 (PD-1). T_reg cells from chronically infected mice (chronic T_reg cells) displayed greater suppressive capacity for inhibiting both CD8⁺ and CD4⁺ T cell proliferation and subsequent cytokine production than those from naive or acutely infected mice. A contact between T_reg and CD8⁺ T cells was necessary for the potent suppression of CD8⁺ T cell immune response. More importantly, the suppression required cell-specific expression and interaction of PD-1 on chronic T_reg cells and PD-1 ligand on CD8⁺ T cells. Our study defines PD-1 upregulated on T_reg cells and its interaction with PD-1 ligand on effector T cells as one cause for the potent T cell suppression and proposes the role of PD-1 on T_reg cells, in addition to that on exhausted T cells, during chronic viral infection.

Hemophilia is a hereditary disease that remains incurable. Although innovative treatments such as gene therapy or bispecific antibody therapy have been introduced, substantial unmet needs still exist with respect to achieving long-lasting therapeutic effects and treatment options for inhibitor patients. Antithrombin (AT), an endogenous negative regulator of thrombin generation, is a potent genome editing target for sustainable treatment of patients with hemophilia A and B. In this study, we developed and optimized lipid nanoparticles (LNPs) to deliver Cas9 mRNA along with single guide RNA that targeted AT in the mouse liver. The LNP-mediated CRISPR-Cas9 delivery resulted in the inhibition of AT that led to improvement in thrombin generation. Bleeding-associated phenotypes were recovered in both hemophilia A and B mice. No active off-targets, liver-induced toxicity, and substantial anti-Cas9 immune responses were detected, indicating that the LNP-mediated CRISPR-Cas9 delivery was a safe and efficient approach for hemophilia therapy.

Chang J. Immune Netw. 2021 Feb;21(1):e6. https://doi.org/10.4110/in.2021.21.e6

Tumor suppressor protein p53 is a potent transcriptional activator and regulates cell growth negatively. To characterize the transcriptional activation domain (TAD) of p53, various point mutants were constructed in the context of Gal4 DNA binding domain and tested for their transactivation ability. Our results demonstrated that the positionally conserved hydrophobic residues shared with herpes simplex virus VP16 and other transactivators are essential for transactivation. Also, the negatively charged residues and proline residues are necessary for full activity, but not essential for the activity of p53 TAD. Deletion analyses showed that p53 TAD can be divided into two subdomains, amino acids 1-40 and 43-73. An in vitro glutathione S-transferase pull-down assay establishes a linear correlation between p53 TAD-mediated transactivation in vivo and the binding activity of p53 TAD to TATA-binding protein (TBP) in vitro. Mutations that diminish the transactivation ability of Gal4-p53 TAD also impair the binding activity to TBP severely. Our results suggest that at least TBP is a direct target for p53 TAD and that the binding strength of TAD to TBP (TFIID) is an important parameter controlling activity of p53 TAD. In addition, circular dichroism spectroscopy has shown that p53 TAD peptide lacks any regular secondary structure in solution and that there is no significant difference between the spectra of the wild type TAD and that of the transactivation-deficient mutant type.