Production and Assay of Recombinant Multisubunit Chromatin Remodeling Complexes.

We have developed a novel system to facilitate the rapid and easy cloning of multiple genes (>10) in under a week. Using this system we have been able to successfully clone, overexpress, and purify a number of large multimeric proteins from insect cells, including the chromatin remodeling complexes SWR1 and INO80. Using Förster resonance energy transfer (FRET)-based assays we have demonstrated that our overexpressed enzymes have activities comparable to those purified from sources where the proteins are expressed under their endogenous promoters.

Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex.

We have prepared recombinant fourteen subunit yeast SWR1 complex from insect cells using a modified MultiBac system. The 1.07 MDa recombinant protein complex has histone-exchange activity. Full exchange activity is realized with a single SWR1 complex bound to a nucleosome. We also prepared mutant complexes that lack a variety of subunits or combinations of subunits and these start to reveal roles for some of these subunits as well as indicating interactions between them in the full complex. Complexes containing a series of N-terminally and C-terminally truncated Swr1 subunits reveal further details about interactions between subunits as well as their binding sites on the Swr1 subunit. Finally, we present electron microscopy studies revealing the dynamic nature of the complex and a 21 Å resolution reconstruction of the intact complex provides details not apparent in previously reported structures, including a large central cavity of sufficient size to accommodate a nucleosome.

SIRT3 deacetylates ATP synthase F1 complex proteins in response to nutrient- and exercise-induced stress.

AIMS: Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. RESULTS: By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCP(K139) directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. INNOVATION: This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. CONCLUSION: Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins.

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F1-ATPase from bovine heart mitochondria.

The molecular description of the mechanism of F(1)-ATPase is based mainly on high-resolution structures of the enzyme from mitochondria, coupled with direct observations of rotation in bacterial enzymes. During hydrolysis of ATP, the rotor turns counterclockwise (as viewed from the membrane domain of the intact enzyme) in 120° steps. Because the rotor is asymmetric, at any moment the three catalytic sites are at different points in the catalytic cycle. In a "ground-state" structure of the bovine enzyme, one site (ß(E)) is devoid of nucleotide and represents a state that has released the products of ATP hydrolysis. A second site (ß(TP)) has bound the substrate, magnesium. ATP, in a precatalytic state, and in the third site (ß(DP)), the substrate is about to undergo hydrolysis. Three successive 120° turns of the rotor interconvert the sites through these three states, hydrolyzing three ATP molecules, releasing the products and leaving the enzyme with two bound nucleotides. A transition-state analog structure, F(1)-TS, displays intermediate states between those observed in the ground state. For example, in the ß(DP)-site of F(1)-TS, the terminal phosphate of an ATP molecule is undergoing in-line nucleophilic attack by a water molecule. As described here, we have captured another intermediate in the catalytic cycle, which helps to define the order of substrate release. In this structure, the ß(E)-site is occupied by the product ADP, but without a magnesium ion or phosphate, providing evidence that the nucleotide is the last of the products of ATP hydrolysis to be released.

The structure of the membrane extrinsic region of bovine ATP synthase.

The structure of the complex between bovine mitochondrial F(1)-ATPase and a stator subcomplex has been determined at a resolution of 3.2 A. The resolved region of the stator contains residues 122-207 of subunit b; residues 5-25 and 35-57 of F(6); 3 segments of subunit d from residues 30-40, 65-74, and 85-91; and residues 1-146 and 169-189 of the oligomycin sensitivity conferral protein (OSCP). The stator subcomplex represents its membrane distal part, and its structure has been augmented with an earlier structure of a subcomplex containing residues 79-183, 3-123, and 5-70 of subunits b, d, and F(6), respectively, which extends to the surface of the inner membrane of the mitochondrion. The N-terminal domain of the OSCP links the stator with F(1)-ATPase via alpha-helical interactions with the N-terminal region of subunit alpha(E). Its C-terminal domain makes extensive helix-helix interactions with the C-terminal alpha-helix of subunit b from residues 190-207. Subunit b extends as a continuous 160-A long alpha-helix from residue 188 back to residue 79 near to the surface of the inner mitochondrial membrane. This helix appears to be stiffened by other alpha-helices in subunits d and F(6), but the structure can bend inward toward the F(1) domain around residue 146 of subunit b. The linker region between the 2 domains of the OSCP also appears to be flexible, enabling the stator to adjust its shape as it passes over the changing profile of the F(1) domain during a catalytic cycle. The structure of the membrane extrinsic part of bovine ATP synthase is now complete.

Radiation exposure during elective coronary angioplasty: the effect of flat-panel detection.

BACKGROUND: Coronary angiography and angioplasty have to date been performed using digital angiography and fluoroscopic systems which incorporate an image intensifier (II). More recently flat-panel (FP) detectors have been introduced which are thought to improve spatial resolution. However, there is limited data on the effect of flat-panel detection on radiation exposure. We sought to determine the impact of flat-panel on cumulative radiation exposure in patients undergoing elective coronary angioplasty at our institution. METHODS: Patients who underwent elective coronary angioplasty in the six months prior to and following upgrade of our Toshiba catheterisation laboratory from image intensifier to flat-panel were included. Demographic and radiation data were collected prospectively and the same five operators performed interventions during the 12-month period. Radiation data was obtained from the dose-area product meter intrinsic to the fluoroscopy system. RESULTS: One hundred and thirty seven patients underwent elective angioplasty over the 12-month period (68 II, 69 FP). Cumulative radiation exposure was increased in flat-panel cases (99, 129 Gy cm(2) versus 71, 77 Gy cm(2), p=0.001). This increase was independent of patient weight (78+/-15 kg versus 78+/-17 kg, p=NS), screening time (19+/-12 min versus 18+/-13 min, p=NS) and total number of digital acquisitions (1475, 820 versus 1668, 1365, p=NS). The total amount of contrast dye did not differ between flat-panel and image intensifier cases (195+/-76 ml versus 194+/-79 ml, p=NS). CONCLUSIONS: Adoption of flat-panel detector technology increases radiation exposure. This may have important safety implications for catheterisation laboratory staff and patients undergoing multiple interventional procedures.