Metabolism and ageing are intimately linked. Compared to ad libitum feeding, dietary restriction (DR) or calorie restriction (CR) consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms1,2. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits3,4. Recently, several metabolites have been identified that modulate ageing5,6 with largely undefined molecular mechanisms. Here we show that the tricarboxylic acid (TCA) cycle intermediate α-ketoglutarate (α-KG) extends the lifespan of adult C. elegans. ATP synthase subunit beta is identified as a novel binding protein of α-KG using a small-molecule target identification strategy called DARTS (drug affinity responsive target stability)7. The ATP synthase, also known as Complex V of the mitochondrial electron transport chain (ETC), is the main cellular energy-generating machinery and is highly conserved throughout evolution8,9. Although complete loss of mitochondrial function is detrimental, partial suppression of the ETC has been shown to extend C. elegans lifespan10–13. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit beta and is dependent on the target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased upon starvation and α-KG does not extend the lifespan of DR animals, indicating that α-KG is a key metabolite that mediates longevity by DR. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator, and DR in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

There is evidence that multi-site phosphorylation of cardiac troponin I (cTnI) by protein kinase C is important in both long-and short-term regulation of cardiac function. To determine the specific functional effects of these phosphorylation sites (Ser-43, Ser-45, and Thr-144), we measured tension and sliding speed of thin filaments in reconstituted preparations in which endogenous cTnI was replaced with cTnI phosphorylated by protein kinase C-⑀ or mutated to cTnI-S43E/S45E/T144E, cTnI-S43E/S45E, or cTnI-T144E. We used detergentskinned mouse cardiac fiber bundles to measure changes in Ca 2؉ -dependence of force. Compared with controls, fibers reconstituted with phosphorylated cTnI, cTnI-S43E/S45E/T144E, or cTnI-S43E/S45E were desensitized to Ca 2؉ , and maximum tension was as much as 27% lower, whereas fibers reconstituted with cTnI-T144E showed no change. In the in vitro motility assay actin filaments regulated by troponin complexes containing phosphorylated cTnI or cTnI-S43E/S45E/T144E showed both a decrease in Ca 2؉ sensitivity and maximum sliding speed compared with controls, whereas filaments regulated by cTnI-S43E/S45E showed only decreased maximum sliding speed and filaments regulated by cTnI-T144E demonstrated only desensitization to Ca 2؉ . Our results demonstrate novel site specificity of effects of PKC phosphorylation on cTnI function and emphasize the complexity of modulation of the actin-myosin interaction by specific changes in the thin filament.

Kuns-Hashimoto R, Kuninger D, Nili M, Rotwein P. Selective binding of RGMc/hemojuvelin, a key protein in systemic iron metabolism, to BMP-2 and neogenin.

The relationship between tropomyosin thermal stability and thin filament activation was explored using two N-domain mutants of ␣-striated muscle tropomyosin, A63V and K70T, each previously implicated in familial hypertrophic cardiomyopathy. Both mutations had prominent effects on tropomyosin thermal stability as monitored by circular dichroism. Wild type tropomyosin unfolded in two transitions, separated by 10°C. The A63V and K70T mutations decreased the melting temperature of the more stable of these transitions by 4 and 10°C, respectively, indicating destabilization of the Ndomain in both cases. Global analysis of all three proteins indicated that the tropomyosin N-domain and Cdomain fold with a cooperative free energy of 1.0 -1.5 kcal/mol. The two mutations increased the apparent affinity of the regulatory Ca 2؉ binding sites of thin filament in two settings: Ca 2؉ -dependent sliding speed of unloaded thin filaments in vitro (at both pH 7.4 and 6.3), and Ca 2؉ activation of the thin filament-myosin S1 ATPase rate. Neither mutation had more than small effects on the maximal ATPase rate in the presence of saturating Ca 2؉ or on the maximal sliding speed. Despite the increased tropomyosin flexibility implied by destabilization of the N-domain, neither the cooperativity of thin filament activation by Ca 2؉ nor the cooperative binding of myosin S1-ADP to the thin filament was altered by the mutations. The combined results suggest that a more dynamic tropomyosin N-domain influences interactions with actin and/or troponin that modulate Ca 2؉ sensitivity, but has an unexpectedly small effect on cooperative changes in tropomyosin position on actin.Contraction of cardiac and skeletal muscle is controlled by the reversible binding of calcium to the N-domain of TnC, 1 which is the regulatory subunit of troponin (for reviews, see Refs. 1 and 2). Troponin and tropomyosin bestow Ca 2ϩ dependence on the productive interactions of actin and myosin: rapid ATPase activity, generation of force, and generation of movement. Three-dimensional, helical reconstructions of thin filament electron micrographs imply three positions of tropomyosin on the actin surface (3). These data, supported by x-ray diffraction of thin filaments (4), indicate that a major component of regulation consists of tropomyosin sterically interfering with myosin binding to actin. In the absence of Ca 2ϩ , much of the myosin-binding site on actin is obscured by tropomyosin. Calcium binding to troponin causes tropomyosin to shift position, exposing much of the myosin-binding site. Strong actinmyosin binding requires a further repositioning of tropomyosin. These findings do not imply that steric interference fully explains regulation. For example, addition of troponin and tropomyosin to bare actin filaments increases actin-myosin affinity, force production, and sliding speed in the presence of Ca 2ϩ (5-7). Nevertheless, as was first proposed 30 years ago (8), most recent reports (albeit not all (e.g. Refs. 9 and 10)) point to the shifting position of tropomyosin ...

The sliding speed of unregulated thin filaments in motility assays is only about half that of the unloaded shortening velocity of muscle fibers. The addition of regulatory proteins, troponin and tropomyosin, is known to increase the sliding speed of thin filaments in the in vitro motility assay. To learn if this effect is related to the rate of MgADP dissociation from the acto-S1 cross-bridge head, the effects of regulatory proteins on nucleotide binding and release in motility assays were measured in the presence and absence of regulatory proteins. The apparent affinity of acto-heavy meromyosin (acto-HMM) for MgATP was reduced by the presence of regulatory proteins. Similarly, the regulatory proteins increase the concentration of MgADP required to inhibit sliding. These results suggest that regulatory proteins either accelerate the rate of MgADP release from acto-HMM-MgADP or slow its binding to acto-HMM. The reduction of temperature also altered the relationship between thin filament sliding speed and the regulatory proteins. At lower temperatures, the regulatory proteins lost their ability to increase thin filament sliding speed above that of unregulated thin filaments. It is hypothesized that structural changes in the actin portion of the acto-myosin interface are induced by regulatory protein binding to actin.