The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control

Circadian rhythms govern a large array of metabolic and physiological functions. The central clock protein CLOCK has HAT properties. It directs acetylation of histone H3 and of its dimerization partner BMAL1 at Lys537, an event essential for circadian function. We show that the HDAC activity of the NAD(+)-dependent SIRT1 enzyme is regulated in a circadian manner, correlating with rhythmic acetylation of BMAL1 and H3 Lys9/Lys14 at circadian promoters. SIRT1 associates with CLOCK and is recruited to the CLOCK:BMAL1 chromatin complex at circadian promoters. Genetic ablation of the Sirt1 gene or pharmacological inhibition of SIRT1 activity lead to disturbances in the circadian cycle and in the acetylation of H3 and BMAL1. Finally, using liver-specific SIRT1 mutant mice we show that SIRT1 contributes to circadian control in vivo. We propose that SIRT1 functions as an enzymatic rheostat of circadian function, transducing signals originated by cellular metabolites to the circadian clock.     

Structural basis of inhibition of the human NAD+-dependent deacetylase SIRT5 by suramin

Sirtuins are NAD(+)-dependent protein deacetylases and are emerging as molecular targets for the development of pharmaceuticals to treat human metabolic and neurological diseases and cancer. To date, several sirtuin inhibitors and activators have been identified, but the structural mechanisms of how these compounds modulate sirtuin activity have not yet been determined. We identified suramin as a compound that binds to human SIRT5 and showed that it inhibits SIRT5 NAD(+)-dependent deacetylase activity with an IC(50) value of 22 microM. To provide insights into how sirtuin function is altered by inhibitors, we determined two crystal structures of SIRT5, one in complex with ADP-ribose, the other bound to suramin. Our structural studies provide a view of a synthetic inhibitory compound in a sirtuin active site revealing that suramin binds into the NAD(+), the product, and the substrate-binding site. Finally, our structures may enable the rational design of more potent inhibitors.     

Nucleocytoplasmic shuttling of the splicing factor SIPP1

SIPP1 (splicing factor that interacts with PQBP1 and PP1) is a widely expressed protein of 70 kDa that has been implicated in pre-mRNA splicing. It interacts with protein Ser/Thr phosphatase-1 (PP1) and with the polyglutamine-tract-binding protein 1 (PQBP1), which contributes to the pathogenesis of X-linked mental retardation and neurodegenerative diseases caused by polyglutamine tract expansions. We show here that SIPP1 is a nucleocytoplasmic shuttling protein. Under basal circumstances SIPP1 was largely nuclear, but it accumulated in the cytoplasm following UV- or X-radiation. Nuclear import was mediated by two nuclear localization signals. In addition, SIPP1 could be piggy-back transported to the nucleus with its ligand PQBP1. In the nucleus SIPP1 and PQBP1 formed inclusion bodies similar to those detected in polyglutamine diseases. SIPP1 did not function as a nuclear targeting subunit of PP1 but re-localized nuclear PP1 to storage sites for splicing factors. The C-terminal residues of SIPP1, which do not conform to a classic nuclear export signal, were required for its nuclear export via the CMR-1 pathway. Finally, SIPP1 activated pre-mRNA splicing in intact cells, and the extent of splicing activation correlated with the nuclear concentration of SIPP1. We conclude that SIPP1 is a positive regulator of pre-mRNA splicing that is regulated by nucleocytoplasmic shuttling. These findings also have potential implications for a better understanding of the pathogenesis of X-linked mental retardation and polyglutamine-linked neurodegenerative disorders.     

Sir2-Related Protein 1 from Leishmania amazonensis is a glycosylated NAD+-dependent deacetylase

Sirtuin proteins form a family of NAD+-dependent protein deacetylases that are considered potential drug targets against parasites. Here, we present the first characterization of a sirtuin orthologue from Leishmania amazonensis, an aetiological agent of American tegumentary leishmaniasis that has been the subject of many studies focused in the development of therapeutic approaches. The protein has high sequence identity with other Kinetoplastid Silent information regulator 2 Related Protein 1 (Sir2RP1) and was named LaSir2RP1. The gene exists as a single copy, encoding a monomeric protein (LaSir2RP1) of approximately 41 kDa that has NAD+-dependent deacetylase activity. LaSir2RP1 was immunodetected in total protein extracts, in cytoplasmic granules, and in the secreted material of both promastigotes and lesion-derived amastigotes. Analysis of both lectin?affinity purified promastigote and amastigote extracts revealed the presence of a major enriched protein of approximately 66 kDa that was recognized by an anti-LaSir2RP1 serum, suggesting that a parasite sirtuin could be glycosylated in vivo.     

Plasmodium falciparum Sir2 is an NAD+-dependent deacetylase and an acetyllysine-dependent and acetyllysine-independent NAD+ glycohydrolase

Sirtuins are NAD (+)-dependent enzymes that deacetylate a variety of cellular proteins and in some cases catalyze protein ADP-ribosyl transfer. The catalytic mechanism of deacetylation is proposed to involve an ADPR-peptidylimidate, whereas the mechanism of ADP-ribosyl transfer to proteins is undetermined. Herein we characterize a Plasmodium falciparum sirtuin that catalyzes deacetylation of histone peptide sequences. Interestingly, the enzyme can also hydrolyze NAD (+). Two mechanisms of hydrolysis were identified and characterized. One is independent of acetyllysine substrate and produces alpha-stereochemistry as established by reaction of methanol which forms alpha-1- O-methyl-ADPR. This reaction is insensitive to nicotinamide inhibition. The second solvolytic mechanism is dependent on acetylated peptide and is proposed to involve the imidate to generate beta-stereochemistry. Stereochemistry was established by isolation of beta-1- O-methyl-ADPR when methanol was added as a cosolvent. This solvolytic reaction was inhibited by nicotinamide, suggesting that nicotinamide and solvent compete for the imidate. These findings establish new reactions of wildtype sirtuins and suggest possible mechanisms for ADP-ribosylation to proteins. These findings also illustrate the potential utility of nicotinamide as a probe for mechanisms of sirtuin-catalyzed ADP-ribosyl transfer.     

Nucleocytoplasmic shuttling of phospholipase C-delta1: a link to Ca2+

Phosphoinositides (PIs) and proteins involved in the PI signaling pathway are distributed in the nucleus as well as at the plasma membrane and in the cytoplasm, although their nuclear localization mechanisms have not been clarified in detail. Generally, proteins that shuttle between the cytoplasm and nucleus contain nuclear localization signal (NLS) and nuclear export signal (NES) sequences for nuclear import and export, respectively. They bind to specific carrier proteins of the importin/exportin family and are transported to and from the nucleus. Thus there is a steady state shuttling of the cargo molecules to and from the nucleus, and the shift in equilibrium determines their nuclear or cytoplasmic localization. Our previous studies have shown that phospholipase C (PLC)-delta1, regarded as having cytoplasmic- or plasma membrane-bound localization, accumulates in the nucleus when its NES sequence is disrupted. In addition, a cluster of positively charged residues on the surface of the catalytic barrel is important for nuclear import. In quiescent cells, the shuttling equilibrium seems to be shifted to the nuclear export of PLCdelta1. In this review, recent findings regarding the molecular machineries and mechanisms of the nucleocytoplasmic shuttling of PLCdelta1 will be discussed. It is important to know when and how they are regulated. A shift in the equilibrium in a certain stage of the cell cycle or by external stimuli is possible and resulting changes in the intra-nuclear environments (or architectures) may alter proliferation and differentiation patterns. Evidences support the idea that an increase in the levels of intracellular Ca2+ shifts the equilibrium to the nuclear import of PLCdelta1. A myriad of external stimuli have also been reported to change the nuclear PI metabolism following accelerated accumulation in the nucleus of other phospholipases such as phospholipase A2 and phospholipase D in addition to PLC isoforms such as PLCbeta1 and PLCgamma1. The consequence of the nuclear accumulation of PLC is also discussed.     

Nucleocytoplasmic shuttling of the progesterone receptor.

The nuclear localization of the progesterone receptor is mediated by two signal sequences: one is constitutive and lies in the hinge region (between the DNA and steroid binding domains), the other is hormone dependent and is localized in the second zinc finger of the DNA binding domain. The use of various inhibitors of energy synthesis in cells expressing permanently or transiently the wild-type receptor or a receptor mutated within the nuclear localization signals, demonstrated that the nuclear residency of the receptor reflects a dynamic situation: the receptor diffusing into the cytoplasm and being constantly and actively transported back into the nucleus. The existence of this nucleo-cytoplasmic shuttle mechanism was confirmed by receptor transfer from one nucleus to the other in heterokaryons. Preliminary evidence was obtained, using oestrogen receptor, that this phenomenon may be of general significance for steroid receptors.     

Nucleocytoplasmic shuttling by human immunodeficiency virus type 1 Vpr

Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin alpha/beta-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich alpha-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.