Evidence that S6K1, but not 4E-BP1, mediates skeletal muscle pathology associated with loss of A-type lamins.

The mechanistic target of rapamycin (mTOR) signaling pathway plays a central role in aging and a number of different disease states. Rapamycin, which suppresses activity of the mTOR complex 1 (mTORC1), shows preclinical (and sometimes clinical) efficacy in a number of disease models. Among these are Lmna-/- mice, which serve as a mouse model for dystrophy-associated laminopathies. To confirm that elevated mTORC1 signaling is responsible for the pathology manifested in Lmna-/- mice and to decipher downstream genetic mechanisms underlying the benefits of rapamycin, we tested in Lmna-/- mice whether survival could be extended and disease pathology suppressed either by reduced levels of S6K1 or enhanced levels of 4E-BP1, two canonical mTORC1 substrates. Global heterozygosity for S6K1 ubiquitously extended lifespan of Lmna-/- mice (Lmna-/-S6K1+/- mice). This life extension is due to improving muscle, but not heart or adipose, function, consistent with the observation that genetic ablation of S6K1 specifically in muscle tissue also extended survival of Lmna-/- mice. In contrast, whole-body overexpression of 4E-BP1 shortened the survival of Lmna-/- mice, likely by accelerating lipolysis. Thus, rapamycin-mediated lifespan extension in Lmna-/- mice is in part due to the improvement of skeletal muscle function and can be phenocopied by reduced S6K1 activity, but not 4E-BP1 activation.

Rapamycin Reverses Metabolic Deficits in Lamin A/C-Deficient Mice.

The role of the mTOR inhibitor, rapamycin, in regulation of adiposity remains controversial. Here, we evaluate mTOR signaling in lipid metabolism in adipose tissues of Lmna-/- mice, a mouse model for dilated cardiomyopathy and muscular dystrophy. Lifespan extension by rapamycin is associated with increased body weight and fat content, two phenotypes we link to suppression of elevated energy expenditure. In both white and brown adipose tissue of Lmna-/- mice, we find that rapamycin inhibits mTORC1 but not mTORC2, leading to suppression of elevated lipolysis and restoration of thermogenic protein UCP1 levels, respectively. The short lifespan and metabolic phenotypes of Lmna-/- mice can be partially rescued by maintaining mice at thermoneutrality. Together, our findings indicate that altered mTOR signaling in Lmna-/- mice leads to a lipodystrophic phenotype that can be rescued with rapamycin, highlighting the effect of loss of adipose tissue in Lmna-/- mice and the consequences of altered mTOR signaling.

Expression and crystallization of SeDsbA, SeDsbL and SeSrgA from Salmonella enterica serovar Typhimurium.

Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 A and belonged to space groups P2(1), P2(1)2(1)2 and C2, respectively.

Crystallization and preliminary diffraction analysis of a DsbA homologue from Wolbachia pipientis.

alpha-DsbA1 is one of two DsbA homologues encoded by the Gram-negative alpha-proteobacterium Wolbachia pipientis, an endosymbiont that can behave as a reproductive parasite in insects and as a mutualist in medically important filarial nematodes. The alpha-DsbA1 protein is thought to be important for the folding and secretion of Wolbachia proteins involved in the induction of reproductive distortions. Crystals of native and SeMet alpha-DsbA1 were grown by vapour diffusion and belong to the monoclinic space group C2, with unit-cell parameters a = 71.4, b = 49.5, c = 69.3 A, beta = 107.0 degrees and one molecule in the asymmetric unit (44% solvent content). X-ray data were recorded from native crystals to a resolution of 2.01 A using a copper anode and data from SeMet alpha-DsbA1 crystals were recorded to 2.45 A resolution using a chromium anode.

Expression and crystallization of DsbA from Staphylococcus aureus.

Bacterial Dsb proteins catalyse the in vivo formation of disulfide bonds, a critical step in the stability and activity of many proteins. Most studies on Dsb proteins have focused on Gram-negative bacteria and thus the process of oxidative folding in Gram-positive bacteria is poorly understood. To help elucidate this process in Gram-positive bacteria, DsbA from Staphylococcus aureus (SaDsbA) has been focused on. Here, the expression, purification, crystallization and preliminary diffraction analysis of SaDsbA are reported. SaDsbA crystals diffract to a resolution limit of 2.1 A and belong to the hexagonal space group P6(5) or P6(1), with unit-cell parameters a = b = 72.1, c = 92.1 A and one molecule in the asymmetric unit (64% solvent content).