Acetylation of yeast AMPK controls intrinsic aging independently of caloric restriction

Acetylation of histone and nonhistone proteins is an important posttranslational modification affecting many cellular processes. Here, we report that NuA4 acetylation of Sip2, a regulatory β subunit of the Snf1 complex (yeast AMP-activated protein kinase), decreases as cells age. Sip2 acetylation, controlled by antagonizing NuA4 acetyltransferase and Rpd3 deacetylase, enhances interaction with Snf1, the catalytic subunit of Snf1 complex. Sip2-Snf1 interaction inhibits Snf1 activity, thus decreasing phosphorylation of a downstream target, Sch9 (homolog of Akt/S6K), and ultimately leading to slower growth but extended replicative life span. Sip2 acetylation mimetics are more resistant to oxidative stress. We further demonstrate that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. We propose a protein acetylation-phosphorylation cascade that regulates Sch9 activity, controls intrinsic aging, and extends replicative life span in yeast.     

Structural and thermodynamic basis for weak interactions between dihydrolipoamide dehydrogenase and subunit-binding domain of the branched-chain alpha-ketoacid dehydrogenase complex

The purified mammalian branched-chain α-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain α-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-Å resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other α-ketoacid dehydrogenase complexes.     

Cyclophilin A (CyPA) induces chemotaxis independent of its peptidylprolyl cis-trans isomerase activity: direct binding between CyPA and the ectodomain of CD147

Cyclophilin A (CyPA) is a ubiquitously distributed peptidylprolyl cis-trans isomerase (PPIase) that possesses diverse biological functions. Extracellular CyPA is a potent chemokine, which can directly induce leukocyte chemotaxis and contribute to the pathogenesis of inflammation-mediated diseases. Although it has been identified that the chemotaxis activity of CyPA is mediated through its cell surface signaling receptor CD147, the role of CyPA PPIase activity in this process is disputable, and the underlying molecular mechanism is still poorly understood. In this study, we present the first evidence that CyPA induces leukocyte chemotaxis through a direct binding with the ectodomain of CD147 (CD147(ECT)), independent of its PPIase activity. Although NMR study indicates that the CD147(ECT) binding site on CyPA overlaps with the PPIase active site, the PPIase inactive mutant CyPA(R55A) exhibits similar CD147(ECT) binding ability and chemotaxis activity to those of CyPA(WT). Furthermore, we have identified three key residues of CyPA involved in CD147(ECT) binding and found that mutations H70A, T107A, and R69A result in similar levels of reduction in CD147(ECT) binding ability and chemotaxis activity for CyPA, without affecting the PPIase activity. Our findings indicate that there exists a novel mechanism for CyPA to regulate cellular signaling processes, shedding new light on its applications in drug development and providing a new targeting site for drug design.     

SDF-1α inhibits hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells through PI3K/Akt and ERK1/2 signaling pathways

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated to be a promising cell sources for cardiac regeneration. Poor survival rate of transplanted BMSCs in infarcted myocardium attenuated its clinical application. It’s reported that stromal-derived factor-1 (SDF-1) could protect progenitor cells including endothelial progenitor cells and embryonic stem cells from apoptosis. But little is known whether SDF-1α protein has the same protective effects on BMSCs under conditions of hypoxia and serum deprivation (hypoxia/SD). In present study, we verified that SDF-1α (0.50–2.0 μg/ml) inhibited hypoxia/SD induced apoptosis of BMSCs through mitochondrial pathway. After administration of SDF-1α, the loss of mitochondrial membrane potential and cytochrome c released from mitochondria to cytosol were significantly inhibited, and caspase 3 activity also declined. Furthermore, the effect of SDF-1α on mitochondrial pathway was neutralized by using PI3K inhibitor (Wortmannin) and ERK1/2 inhibitor (U0126). Our observations suggested that SDF-1α inhibits hypoxia/SD induced BMSCs apoptosis through PI3K/Akt and ERK1/2 signaling pathways. These data also imply that the anti-apoptotic effect mediated by SDF-1α may enhance cell survival after cell transplantation.     

Differential expression of porcine testis proteins during postnatal development

The development of the testes includes changes in cell morphology and endocrine levels that are essential for the maturation of males. A large number of novel proteins are expressed throughout testis development and play important roles in spermatogenesis. Differences in protein expressions during the development of porcine testes have not been systematically studied. The purpose of this study was to investigate differential protein expression in porcine testes during postnatal development. Testes from four pigs each at 1wk, 3mo, and 1yr of age were used for a proteomic analysis. Expression levels of 264 protein spots were quantified using the Melanie 3 software. In total, 108 protein spots showed more than 2-fold differences (P<0.05) among developmental stages, and 90 of them were successfully identified by mass spectrometry. The proteins were sorted based on whether the expression levels increased with age (36.1%), decreased with age (38.0%), or fluctuated among different developmental stages (25.9%). In total, 69 unique gene products were further classified according to their gene ontology annotations. A majority of the proteins are organelle proteins (41%) with the nucleus and mitochondria being the main organelles. About 45% of the proteins have a protein binding domain and are likely involved in protein-protein interactions. Finally, a large proportion of these differentially expressed proteins are involved in cellular (25%) and metabolic (22%) processes. Identifying these differentially expressed proteins should be valuable for exploring developmental biology and the pathology of male reproduction.     

Genetic requirements and meiotic function of phosphorylation of the yeast axial element protein Red1

The synaptonemal complex (SC) is a meiosis-specific tripartite structure that forms between two homologous chromosomes; it consists of a central region and two parallel lateral elements. Lateral elements also are called axial elements prior to synapsis. In Saccharomyces cerevisiae, Red1, Hop1, and Mek1 are structural components of axial/lateral elements. The red1/mek1/hop1 mutants all exhibit reduced levels of interhomolog recombination and produce no viable spores. Red1 is a phosphoprotein. Several earlier reports proposed that phosphorylated Red1 plays important roles in meiosis, including in signaling meiotic DNA damage or in preventing exit from the pachytene chromosomes. We report here that the phosphorylation of Red1 is carried out in CDC28-dependent and CDC28-independent manners. In contrast to previous results, we found Red1 phosphorylation to be independent of meiotic DNA recombination, the Mec1/Tel1 DNA damage checkpoint kinases, and the Mek1 kinase. To functionally validate the phosphorylation of Red1, we mapped the phosphorylation sites on this protein. A red1(14A) mutant showing no detectable Red1 phosphorylation did not exhibit decreased sporulation efficiency, defects in viable spore production, or defects in meiotic DNA damage checkpoints. Thus, our results suggest that the phosphorylation of Red1 is not essential for its functions in meiosis.     

Purification of a toxic metalloprotease produced by the pathogenic Photobacterium damselae subsp. piscicida isolated from cobia (Rachycentron canadum)

The aim of the present study was to purify and characterize a toxic protease secreted by the pathogenic Photobacterium damselae subsp. piscicida strain CP1 originally isolated from diseased cobia (Rachycentron canadum). The toxin isolated by anion exchange chromatography, was a metalloprotease, inhibited by L-cysteine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 1,10-phenanthroline, N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0-8.0 and an apparent molecular mass of about 34.3 kDa. The toxin was also completely inhibited by HgCl2, and partially by sodium dodecyl sulfate (SDS) and CuCl2. The extracellular products and the partially purified protease were lethal to cobia with LD50 values of 1.26 and 6.8 microg protein/g body weight, respectively. The addition of EDTA completely inhibited the lethal toxicity of the purified protease, indicating that this metalloprotease was a lethal toxin produced by the bacterium.