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.

Loss of Peter Pan (PPAN) Affects Mitochondrial Homeostasis and Autophagic Flux.

Nucleolar stress is a cellular response to inhibition of ribosome biogenesis or nucleolar disruption leading to cell cycle arrest and/or apoptosis. Emerging evidence points to a tight connection between nucleolar stress and autophagy as a mechanism underlying various diseases such as neurodegeneration and treatment of cancer. Peter Pan (PPAN) functions as a key regulator of ribosome biogenesis. We previously showed that human PPAN localizes to nucleoli and mitochondria and that PPAN knockdown triggers a p53-independent nucleolar stress response culminating in mitochondrial apoptosis. Here, we demonstrate a novel role of PPAN in the regulation of mitochondrial homeostasis and autophagy. Our present study characterizes PPAN as a factor required for maintaining mitochondrial integrity and respiration-coupled ATP production. PPAN interacts with cardiolipin, a lipid of the inner mitochondrial membrane. Down-regulation of PPAN enhances autophagic flux in cancer cells. PPAN knockdown promotes recruitment of the E3-ubiquitin ligase Parkin to damaged mitochondria. Moreover, we provide evidence that PPAN knockdown decreases mitochondrial mass in Parkin-expressing cells. In summary, our study uncovers that PPAN knockdown is linked to mitochondrial damage and stimulates autophagy.