Roy A. Frye

Modulation of NF‐κB‐dependent transcription and cell survival by the SIRT1 deacetylase

NF‐κB is responsible for upregulating gene products that control cell survival. In this study, we demonstrate that SIRT1, a nicotinamide adenosine dinucleotide‐dependent histone deacetylase, regulates the transcriptional activity of NF‐κB. SIRT1, the mammalian ortholog of the yeast SIR2 (Silencing Information Regulator) and a member of the Sirtuin family, has been implicated in modulating transcriptional silencing and cell survival. SIRT1 physically interacts with the RelA/p65 subunit of NF‐κB and inhibits transcription by deacetylating RelA/p65 at lysine 310. Treatment of cells with resveratrol, a small‐molecule agonist of Sirtuin activity, potentiates chromatin‐associated SIRT1 protein on the cIAP‐2 promoter region, an effect that correlates with a loss of NF‐κB‐regulated gene expression and sensitization of cells to TNFα‐induced apoptosis. While SIRT1 is capable of protecting cells from p53‐induced apoptosis, our work provides evidence that SIRT1 activity augments apoptosis in response to TNFα by the ability of the deacetylase to inhibit the transactivation potential of the RelA/p65 protein.

Human SIR2 deacetylates p53 and antagonizes PML/p53-induced cellular senescence

The yeast Sir2 protein mediates chromatin silencing through an intrinsic NAD‐dependent histone deacetylase activity. Sir2 is a conserved protein and was recently shown to regulate lifespan extension both in budding yeast and worms. Here, we show that SIRT1, the human Sir2 homolog, is recruited to the promyelocytic leukemia protein (PML) nuclear bodies of mammalian cells upon overexpression of either PML or oncogenic Ras (Ha‐rasV12). SIRT1 binds and deacetylates p53, a component of PML nuclear bodies, and it can repress p53‐mediated transactivation. Moreover, we show that SIRT1 and p53 co‐localize in nuclear bodies upon PML upregulation. When overexpressed in primary mouse embryo fibroblasts (MEFs), SIRT1 antagonizes PML‐induced acetylation of p53 and rescues PML‐mediated premature cellular senescence. Taken together, our data establish the SIRT1 deacetylase as a novel negative regulator of p53 function capable of modulating cellular senescence.

axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase.

Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.

Acetylation of the C terminus of Ku70 by CBP and PCAF controls bax-mediated apoptosis

Apoptosis is a key tumor suppression mechanism that can be initiated by activation of the proapoptotic factor Bax. The Ku70 DNA end-joining protein has recently been shown to suppress apoptosis by sequestering Bax from mitochondria. The mechanism by which Bax is regulated remains unknown. Here, we identify eight lysines in Ku70 that are targets for acetylation in vivo. Five of these, K539, K542, K544, K533, and K556, lie in the C-terminal linker domain of Ku70 adjacent to the Bax interaction domain. We show that CBP and PCAF efficiently acetylate K542 in vitro and associate with Ku70 in vivo. Mimicking acetylation of K539 or K542 or treating cells with deacetylase inhibitors abolishes the ability of Ku70 to suppress Bax-mediated apoptosis. We demonstrate that increased acetylation of Ku70 disrupts the Ku70-Bax interaction and coincides with cytoplasmic accumulation of CBP. These results shed light on the role of acetyltransferases as tumor suppressors.

The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide–dependent deacetylase

The yeast silent information regulator (Sir)2 protein links cellular metabolism and transcriptional silencing through its nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase activity. We report that mitochondria from mammalian cells contain intrinsic NAD-dependent deacetylase activity. This activity is inhibited by the NAD hydrolysis product nicotinamide, but not by trichostatin A, consistent with a class III deacetylase. We identify this deacetylase as the nuclear-encoded human Sir2 homologue hSIRT3, and show that hSIRT3 is located within the mitochondrial matrix. Mitochondrial import of hSIRT3 is dependent on an NH2-terminal amphipathic α-helix rich in basic residues. hSIRT3 is proteolytically processed in the mitochondrial matrix to a 28-kD product. This processing can be reconstituted in vitro with recombinant mitochondrial matrix processing peptidase (MPP) and is inhibited by mutation of arginines 99 and 100. The unprocessed form of hSIRT3 is enzymatically inactive and becomes fully activated in vitro after cleavage by MPP. These observations demonstrate the existence of a latent class III deacetylase that becomes catalytically activated upon import into the human mitochondria.

SIRT1 regulates HIV transcription via Tat deacetylation.

The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation.

Prodromal bullous pemphigoid

Background  Prodromal bullous pemphigoid (PBP) can be difficult to diagnose. Early recognition in its early stages may decrease the morbidity and progression of the disease. Clinical presentations and current treatments available for PBP will be described.

Evolution of Sirtuins From Archaea to Vertebrates

Most eukaryotic sirtuins (sir2-like proteins) can be grouped into four classes. In deuterostomes such as vertebrates, the urochordate sea squirt Ciona, and the echinoderm sea urchin Strongylocentrotus, there are seven sirtuins. Class I includes SIRT1, SIRT2, and SIRT3. SIRT4 is in class II and SIRT5 is in class III. Class IV includes SIRT6 and SIRT7. Fish have two SIRT5 orthologs while most other vertebrates have only one version of each of the seven sirtuins. Arthropods lack SIRT3, and some arthropods (e.g., Drosophila) lack SIRT5, but other arthropods have SIRT1, SIRT2, SIRT4, SIRT5, SIRT6, and SIRT7. Most prokaryotic sirtuins can be grouped into three categories: the same class II and class III categories as seen in eukaryotes, and a class U, which could be the precursor of the eukaryotic class I and class IV sirtuins. A model is proposed in which the first eukaryote (which resulted from the engulfment of an α-proteobacterium by an archaean) received a class III sirtuin from the archaean parent, while the class II sirtuin and a class U sirtuin came from the α-proteobacterium parent. While most eukaryotic class III sirtuins appear to be derived from an archaeal class III sirtuin, the Kinetoplastida (Leishmania and Trypanosoma) have a class III sirtuin gene that appears to be of γ-proteobacterial origin, possibly an example of lateral gene transfer. The seven mammalian sirtuins are aligned and contrasted with sirtuins from diverse eukaryotic and prokaryotic organisms.