Sirtuin 5 Is Regulated by the SCFCyclin F Ubiquitin Ligase and Is Involved in Cell Cycle Control.

The ubiquitin-proteasome system is essential for cell cycle progression. Cyclin F is a cell cycle-regulated substrate adapter F-box protein for the Skp1, CUL1, and F-box protein (SCF) family of E3 ubiquitin ligases. Despite its importance in cell cycle progression, identifying cyclin F-bound SCF complex (SCFCyclin F) substrates has remained challenging. Since cyclin F overexpression rescues a yeast mutant in the cdc4 gene, we considered the possibility that other genes that genetically modify cdc4 mutant lethality could also encode cyclin F substrates. We identified the mitochondrial and cytosolic deacylating enzyme sirtuin 5 (SIRT5) as a novel cyclin F substrate. SIRT5 has been implicated in metabolic processes, but its connection to the cell cycle is not known. We show that cyclin F interacts with and controls the ubiquitination, abundance, and stability of SIRT5. We show SIRT5 knockout results in a diminished G1 population and a subsequent increase in both S and G2/M. Global proteomic analyses reveal cyclin-dependent kinase (CDK) signaling changes congruent with the cell cycle changes in SIRT5 knockout cells. Together, these data demonstrate that SIRT5 is regulated by cyclin F and suggest a connection between SIRT5, cell cycle regulation, and metabolism.

APC/C and SCF(cyclin F) Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry.

The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase and core component of the cell-cycle oscillator. During G1 phase, APC/C binds to its substrate receptor Cdh1 and APC/C(Cdh1) plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that cyclin F, a cell-cycle-regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCF(cyclin F). Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell-cycle control. This mutual antagonism could be a feature of other oscillating systems.

Remarkable photocytotoxicity in hypoxic HeLa cells by a dipyridophenazine copper(II) Schiff base thiolate.

Copper(II) complexes [Cu(satp)(L)] (1-3) of a Schiff base thiolate (salicylidene-2-aminothiophenol, H2satp) and phenanthroline bases (L), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq in 2) and dipyrido[3,2-a:2',3'-c]phenazine (dppz in 3), were prepared, characterized and their anaerobic DNA photocleavage activity and hypoxic photocytotoxicity studied. The redox active complexes show the Cu(II)-Cu(I) couple near -0.5 V for 1 and near 0.0 V vs. SCE (saturated calomel electrode) for 2 and 3. The one-electron paramagnetic complexes (~1.85 µB) are avid DNA binders giving Kb values within 1.0×10(5)-8.0×10(5) M(-1). Thermal melting and viscosity data along with molecular docking calculations suggest DNA groove and/or partial intercalative binding of the complexes. The complexes show anaerobic DNA cleavage activity in red light under argon via type-I pathway, while DNA photocleavage in air proceeds via hydroxyl radical pathway. The DFT (density functional theory) calculations reveal a thyil radical pathway for the anaerobic DNA photocleavage activity and suggest the possibility of generation of a transient copper(I) species due to bond breakage between the copper and sulfur to generate the thyil radical. An oxidation of the copper(I) species is likely by oxygen in an aerobic medium or by the buffer medium in an anaerobic condition. Complex 3 exhibits significant photocytotoxicity in HeLa cells (IC50=8.3(±1.0) µM) in visible light, while showing lower dark toxicity (IC50=17.2(±1.0) µM). A significant reduction in the dark toxicity is observed under hypoxic cellular conditions (IC50=30.0(±1.0) µM in dark), while retaining its photocytotoxicity (IC50=8.0(±1.0) µM).

Anaerobic DNA cleavage activity in red light and photocytotoxicity of (pyridine-2-thiol)cobalt(III) complexes of phenanthroline bases.

Cobalt(iii) complexes [Co(pnt)(B)(2)](NO(3))(2) (1-3) of pyridine-2-thiol (pnt) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq in ) and dipyrido[3,2-a:2',3'-c]phenazine (dppz in 3), have been prepared, characterized and their photo-induced anaerobic DNA cleavage activity studied. The crystal structure of 1a as mixed ClO(4)(-) and PF(6)(-) salt of 1 shows a Co(III)N(5)S coordination geometry in which the pnt and phen showed N,S- and N,N-donor binding modes, respectively. The complexes exhibit Co(iii)/Co(ii) redox couple near -0.3 V (vs. SCE) in 20% DMF-Tris-HCl buffer having 0.1 M TBAP. The complexes show binding propensity to calf thymus DNA giving K(b) values within 2.2 x 10(4)-7.3 x 10(5) M(-1). Thermal melting and viscosity data suggest DNA surface and/or groove binding of the complexes. The complexes show significant anaerobic DNA cleavage activity in red light under argon atmosphere possibly involving sulfide anion radical or thiyl radical species. The DNA cleavage reaction under aerobic medium in red light is found to involve both singlet oxygen and hydroxyl radical pathways. The dppz complex shows non-specific BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via both hydroxyl and singlet oxygen pathways. The dppz complex exhibits photocytotoxicity in HeLa cervical cancer cells giving IC(50) values of 767 nM and 19.38 microM in UV-A light of 365 nm and in the dark, respectively. A significant reduction of the dark toxicity of the dppz base (IC(50) = 8.34 microM in dark) is observed on binding to the cobalt(iii) center.

Photocytotoxic lanthanum(III) and gadolinium(III) complexes of phenanthroline bases showing light-induced DNA cleavage activity.

Lanthanide complexes of formulation [La(B)(2)(NO(3))(3)] (1-3) and [Gd(B)(2)(NO(3))(3)] (4-6), where B is a N,N-donor phenanthroline base, namely, 1,10-phenanthroline (phen in 1, 4), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq in 2, 5) and dipyrido[3,2-a:2',3'-c]phenazine (dppz in 3, 6), have been prepared, characterized from physicochemical data, and their photoinduced DNA and protein cleavage activity studied. The photocytotoxicity of the dppz complexes 3 and 6 has been studied using HeLa cancer cells. The complexes exhibit ligand centered bands in the UV region. The dppz complexes show the lowest energy band at 380 nm in N,N-dimethylformamide (DMF). The La(III) complexes are diamagnetic. The Gd(III) complexes (4-6) have magnetic moments that correspond to seven unpaired electrons. The complexes are 1:1 electrolytic in aqueous DMF. The dpq and dppz complexes in DMF show ligand-based reductions. The complexes display moderate binding propensity to calf thymus DNA giving binding constant values in the range of 5.7 x 10(4)-5.8 x 10(5) M(-1) with a relative order: 3, 6 (dppz) > 2, 5 (dpq) > 1, 4 (phen). The binding data suggest DNA surface and/or groove binding nature of the complexes. The complexes do not show any hydrolytic cleavage of plasmid supercoiled pUC19 DNA. The dpq and dppz complexes efficiently cleave SC DNA to its nicked circular form on exposure to UV-A light of 365 nm at nanomolar complex concentration. Mechanistic studies reveal the involvement of singlet oxygen ((1)O(2)) and hydroxyl radical (HO*) as the cleavage active species. The complexes show binding propensity to bovine serum albumin (BSA) protein giving K(BSA) values of approximately 10(5) M(-1). The dppz complexes 3 and 6 show BSA protein cleavage activity in UV-A light of 365 nm. The dppz complexes 3 and 6 exhibit significant photocytotoxicity in HeLa cells giving respective IC(50) values of 341 nM and 573 nM in UV-A light of 365 nm for an exposure time of 15 min (IC(50) > 100 microM in dark for both the complexes). Control experiments show significant dark and phototoxicity of the dppz base alone (IC(50) = 413 nM in light with 4 h incubation in dark and 11.6 microM in dark with 24 h incubation). A significant decrease in the dark toxicity of the dppz base is observed on binding to the lanthanide ions while retaining similar phototoxicity.

Anaerobic photocleavage of DNA in red light by dicopper(II) complexes of 3,3'-dithiodipropionic acid.

Binuclear copper(II) complexes [{(phen)Cu(II)}(2)(mu-dtdp)(2)] (1), [{(dpq)Cu(II)}(2)(mu-dtdp)(2)] (2), [{(phen)Cu(II)}(2)(mu-az)(2)] (3), and [{(dpq)Cu(II)}(2)(mu-az)(2)] (4) and a zinc(II) complex [{(phen)Zn(II)}(2)(mu-dtdp)(2)] (5), having 3,3'-dithiodipropionic acid (H(2)dtdp), azelaic acid (nonanedioic acid), 1,10-phenanthroline (phen), and dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), were prepared and characterized by physicochemical methods. Complex 1 has been structurally characterized by X-ray crystallography. The complexes have each metal center bound to a chelating phenanthroline base and two bridging carboxylate ligands giving a square-planar MN(2)O(2) coordination geometry. The molecular structure of complex 1 shows two sterically constrained disulfide moieties of the dtdp ligands. The complexes show good binding propensity to calf thymus DNA in the major groove. The photoinduced DNA cleavage activity of the complexes has been studied using 365 nm UV light and 647.1 nm and >750 nm red light under both aerobic and anaerobic conditions. The phen complex 1, having dtdp ligand, cleaves supercoiled (SC) DNA to its nicked circular (NC) form. The dpq analogue 2 shows formation of a significant quantity of linear DNA resulting from double-strand breaks (dsb) in air. Mechanistic studies reveal the involvement of HO(*) and (1)O(2) as the reactive species under an aerobic medium. The dsb of DNA is rationalized from the docking studies on 2, showing a close proximity of two photosensitizers, namely, the disulfide moiety of dtdp and the quinoxaline ring of dpq to the complementary strands of DNA. The copper(II) complexes of the dtdp ligand cleave SC DNA to its NC form upon exposure to UV or red light under an argon atmosphere. An enhancement of the DNA cleavage activity under argon has been observed upon increasing the concentration of the DMF solvent in the DMF-Tris buffer medium. Theoretical studies suggest the possibility of sulfide anion radical formation from a copper(II)-bound dtdp ligand in >750 nm red light, which further cleaves the DNA. The copper(II) azelate complexes are inactive under similar reaction conditions. The azelate complex of the dpq ligand cleaves DNA in air following the (1)O(2) pathway. The zinc(II) complex of the dtdp ligand (5) does not show any photoinduced DNA cleavage activity in red light.