Molecular Mechanism of Sirtuin 1 Inhibition by Human Immunodeficiency Virus 1 Tat Protein.

Sirtuins are NAD+-dependent protein lysine deacylases implicated in metabolic regulation and aging-related dysfunctions. The nuclear isoform Sirt1 deacetylates histones and transcription factors and contributes, e.g., to brain and immune cell functions. Upon infection by human immunodeficiency virus 1 (HIV1), Sirt1 deacetylates the viral transactivator of transcription (Tat) protein to promote the expression of the viral genome. Tat, in turn, inhibits Sirt1, leading to the T cell hyperactivation associated with HIV infection. Here, we describe the molecular mechanism of Tat-dependent sirtuin inhibition. Using Tat-derived peptides and recombinant Tat protein, we mapped the inhibitory activity to Tat residues 34-59, comprising Tat core and basic regions and including the Sirt1 deacetylation site Lys50. Tat binds to the sirtuin catalytic core and inhibits Sirt1, Sirt2, and Sirt3 with comparable potencies. Biochemical data and crystal structures of sirtuin complexes with Tat peptides reveal that Tat exploits its intrinsically extended basic region for binding to the sirtuin substrate binding cleft through substrate-like ß-strand interactions, supported by charge complementarity. Tat Lys50 is positioned in the sirtuin substrate lysine pocket, although binding and inhibition do not require prior acetylation and rely on subtle differences to the binding of regular substrates. Our results provide mechanistic insights into sirtuin regulation by Tat, improving our understanding of physiological sirtuin regulation and the role of this interaction during HIV1 infection.

Molecular Mechanism of Sirtuin 1 Modulation by the AROS Protein.

The protein lysine deacylases of the NAD+-dependent Sirtuin family contribute to metabolic regulation, stress responses, and aging processes, and the human Sirtuin isoforms, Sirt1-7, are considered drug targets for aging-related diseases. The nuclear isoform Sirt1 deacetylates histones and transcription factors to regulate, e.g., metabolic adaptations and circadian mechanisms, and it is used as a therapeutic target for Huntington's disease and psoriasis. Sirt1 is regulated through a multitude of mechanisms, including the interaction with regulatory proteins such as the inhibitors Tat and Dbc1 or the activator AROS. Here, we describe a molecular characterization of AROS and how it regulates Sirt1. We find that AROS is a partly intrinsically disordered protein (IDP) that inhibits rather than activates Sirt1. A biochemical characterization of the interaction including binding and stability assays, NMR spectroscopy, mass spectrometry, and a crystal structure of Sirtuin/AROS peptide complex reveal that AROS acts as a competitive inhibitor, through binding to the Sirt1 substrate peptide site. Our results provide molecular insights in the physiological regulation of Sirt1 by a regulator protein and suggest the peptide site as an opportunity for Sirt1-targeted drug development.

A universal stress protein (USP) in mycobacteria binds cAMP.

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as "sinks" for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell.