Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.
BAF250a and BAF250b are subunits of the SWI/SNF chromatin-remodeling complex that recruit the complex to chromatin allowing transcriptional activation of several genes. Despite being the central subunits of the SWI/SNF complex, the structural and functional annotation of BAF250a/b remains poorly understood. BAF250a (nearly 2200 residues protein) harbors an N-terminal DNA binding ARID (~110 residues) and a C-terminal folded region (~250 residues) of unknown structure and function, recently annotated as BAF250_C. Using hydrophobic core analysis, fold prediction and comparative modeling, here we have defined a domain boundary and associate a β-catenin like ARM-repeat fold to the C-terminus of BAF250a that encompass BAF250_C. The N-terminal DNA-binding ARID is found in diverse domain combinations in proteins imparting unique functions. We used a comparative sequence analysis based approach to study the ARIDs from diverse domain contexts and identified conserved residue positions that are important to preserve its core structure. Supporting this, mutation of one such conserved residue valine, at position 1067, to glycine, resulted in destabilization, loss of structural integrity and DNA binding affinity of ARID. Additionally, we identified a set of conserved and surface-exposed residues unique to the ARID when it co-occurs with the ARM repeat containing BAF250_C in BAF250a. Several of these residues are found mutated in somatic cancers. We predict that these residues in BAF250a may play important roles in mediating protein-DNA and protein-protein interactions in the BAF complex.
AT‐rich interaction domain (ARID)‐containing BAF250a protein is a central DNA‐binding subunit of the SWI/SNF chromatin‐remodeling complex. ARIDs are found in several eukaryotic proteins that play roles in different aspects of cellular physiology. However, despite their biological importance, ARIDs remain relatively uncharacterized for their dynamics and DNA binding. Here, we have probed the structure and DNA‐binding properties of BAF250a ARID. We show that the core BAF250a ARID interacts with DNA sequences with low micromolar affinities. NMR chemical shift perturbation (CSP) results reveal a number of conserved residues in ARID that are involved in DNA binding. An NMR CSP‐based docking model of ARID–DNA complexes reveals that BAF250a ARID possesses necessary determinants of specific DNA binding.
The AT-rich interaction domain (ARID) containing BAF250a is a subunit of the BAF-A class of SWI/SNF chromatin remodeling complexes. The ARID belongs to a family of conserved DNA binding domains found in several eukaryotic proteins; however, its exact contribution to BAF250a function and the mechanism of its DNA binding are not well understood. Here we have probed the interaction of the BAF250a ARID with three different double-stranded DNA (dsDNA) sequences to understand its DNA binding properties. A comprehensive biophysical and thermodynamic study using nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry revealed the complex nature of BAF250a ARID–DNA interactions. The thermodynamic signatures of the BAF250a ARID with 12 A-T bp dsDNA (AT-12) are distinct from those of 12 G-C bp dsDNA (GC-12) or 12 bp Dickerson dodecamer DNA (DD-12) sequences. We observed that the binding of the BAF250a ARID with AT-12 DNA is enthalpically driven in a tested temperature range of 5–25 °C. BAF250a ARID/AT-12 DNA interaction exhibited a larger negative calorimetric specific heat change (ΔC p ) compared to that of BAF250a ARID/GC-12 DNA or BAF250a ARID/DD-12 DNA interactions. In the presence of salt (NaCl), ARID/AT-12 DNA binding was less perturbed than ARID/GC-12 DNA or ARID/DD-12 DNA binding. Overall, these results show that BAF250a ARID/AT-12 DNA interaction has signatures of “specific” binding. Furthermore, using NMR chemical shift perturbation experiments, we have identified DNA binding residues on the BAF250a ARID and generated a data-driven HADDOCK model of the ARID/DNA complex that was further supported by mutating key lysine residues that were found to be important for DNA binding.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.