Plant carotenoid cleavage oxygenases and their apocarotenoid products

The oxidative cleavage of carotenoids leads to the production of apocarotenoids and is catalyzed by a family of carotenoid cleavage dioxygenases (CCDs). CCDs often exhibit substrate promiscuity, which probably contributes to the diversity of apocarotenoids found in nature. Biologically and commercially important apocarotenoids include the phytohormone abscisic acid, the visual and signaling molecules retinal and retinoic acid, and the aromatic volatile beta-ionone. Unexpected properties associated with the CCD catalytic products emphasize their role in many aspects of plant growth and development. For instance, CCD7 and CCD8 produce a novel, graft-transmissible hormone that controls axillary shoot growth in plants. Here, CCDs are discussed according to their roles in the biosynthesis of these products. Recent studies regarding their mechanism of action are also addressed.     

Post-translational modification of the hepatitis C virus core protein by tissue transglutaminase

The hepatitis C virus (HCV) core protein is a structural protein that packages the viral genomic RNA. In this study, we demonstrate that a stable core protein dimer could be produced in liver cells. The production of this protein could be enhanced by calphostin C and serum deprivation. This protein was determined to be the core protein dimer because of its reactivity with the anti-core antibody, its similar electrophoretic mobility compared with that of the core protein dimer generated by cross-linking with glutaraldehyde, and its increase in size by a hemagglutinin tag fused to the core protein sequence. This core protein dimer was highly stable and resistant to SDS and beta-mercaptoethanol. The enzyme that mediated the formation of this stable core protein dimer was determined to be the tissue transglutaminase (tTG) because, first, tTG could be activated by calphostin C and serum deprivation; second, the formation of this dimer was suppressed by monodansylcadaverine, a tTG inhibitor; and third, the core protein could be cross-linked by tTG in vitro. Thus, the HCV core protein represents the first known viral structural protein substrate of tTG. The post-translational modification by tTG reduced the RNA binding activity of the core protein, raising the possibility that tTG may regulate the biological functions of the HCV core protein.     

Post-translational modification by acetylation regulates the mitochondrial carnitine/acylcarnitine transport protein

The carnitine/acylcarnitine transporter (CACT; SLC25A20) mediates an antiport reaction allowing entry of acyl moieties in the form of acylcarnitines into the mitochondrial matrix and exit of free carnitine. The transport function of CACT is crucial for the β-oxidation pathway. In this work, it has been found that CACT is partially acetylated in rat liver mitochondria as demonstrated by anti-acetyl-lys antibody immunostaining. Acetylation was reversed by the deacetylase Sirtuin 3 in the presence of NAD⁺. After treatment of the mitochondrial extract with the deacetylase, the CACT activity, assayed in proteoliposomes, increased. The half-saturation constant of the CACT was not influenced, while the V _max was increased by deacetylation. Sirtuin 3 was not able to deacetylate the CACT when incubation was performed in intact mitoplasts, indicating that the acetylation sites are located in the mitochondrial matrix. Prediction on the localization of acetylated residues by bioinformatics correlates well with the experimental data. Recombinant CACT treated with acetyl-CoA was partially acetylated by non-enzymatic mechanism with a corresponding decrease of transport activity. The experimental data indicate that acetylation of CACT inhibits its transport activity, and thus may contribute to the regulation of the mitochondrial β-oxidation pathway.     

Post-translational formylglycine modification of bacterial sulfatases by the radical S-adenosylmethionine protein AtsB

C(alpha)-Formylglycine (FGly) is the catalytic residue of sulfatases. FGly is generated by post-translational modification of a cysteine (prokaryotes and eukaryotes) or serine (prokaryotes) located in a conserved (C/S)XPXR motif. AtsB of Klebsiella pneumoniae is directly involved in FGly generation from serine. AtsB is predicted to belong to the newly discovered radical S-adenosylmethionine (SAM) superfamily. By in vivo and in vitro studies we show that SAM is the critical co-factor for formation of a functional AtsB.SAM.sulfatase complex and for FGly formation by AtsB. The SAM-binding site of AtsB involves (83)GGE(85) and possibly also a juxtaposed FeS center coordinated by Cys(39) and Cys(42), as indicated by alanine scanning mutagenesis. Mutation of these and other conserved cysteines as well as treatment with metal chelators fully impaired FGly formation, indicating that all three predicted FeS centers are crucial for AtsB function. It is concluded that AtsB oxidizes serine to FGly by a radical mechanism that is initiated through reductive cleavage of SAM, thereby generating the highly oxidizing deoxyadenosyl radical, which abstracts a hydrogen from the serine-C(beta)H(2)-OH side chain.     

肽酰精氨酸的翻译后修饰

肽酰精氨酸残基甲基化作用是细胞质与细胞核内蛋白质翻译后修饰的普遍方式。精氨酸残基甲基化蛋白质与许多细胞生物学过程有关,包括转录调节、RNA代谢和DNA损伤修复等。生物体内精氨酸N-甲基转移酶类、肽酰精氨酸脱亚氨酶类与JMJD6等催化肽酰精氨酸残基进行甲基化、瓜氨酸化和去甲基化的动态修饰。这种动态修饰对细胞生物学功能有重要调节作用。     

Post-translational modification of proteins

Many proteins, especially those produced by eukaryotic cells, undergo extensive, essentially irreversible, modifications after their synthesis. This review focuses on three classes of such reactions: proteolytic cleavages, formation of S-S cystine bonds, and formation of asparagine-linked carbohydrate chains. Emphasis is placed on the mechanism of these reactions, and on the importance of these modifications for the proper structure, function and stability of the affected proteins. Using recombinant DNA techniques, it is now possible to synthesize the polypeptide portion of many proteins, such as mammalian peptide hormones and enzymes, in bacterial and yeast cells. These host cells, however, may be unable to carry out essential post-translational modifications. Ways in which the properly modified form of these ‘engineered’ proteins can be produced are considered.     

PTEN蛋白的翻译后修饰

第10号染色体缺失的磷酸酶与张力蛋白同源物(phosphatase and tensin homolog,PTEN)基因所编码的PTEN蛋白兼具有脂质和蛋白磷酸酶活性,它的表达、活性和稳定性受到各种结合蛋白、酶和因子的调节。结合最新研究,本文将集中对PTEN上氨基酸残基位点的各种翻译后修饰进行一综述。     

Post-translational modification of Escherichia coli ribosomal protein S6.

Summary Escherichia coli has multiple forms of ribosomal protein S6, differing in number of glutamyl residues at the C-terminal end. Three forms are revealed when crude cell extracts are fractionated by a two-dimensional gel electrophoresis technique. Pulse-chase experiments show that the shortes and most alkaline form of S6 is the first to appear. In about one doubling time this form reaches equilibrium with the two other forms of S6, implicating the existance of an enzyme, which adds glutamic acid residues to S6. We show that the relative levels of these three S6 forms are not affected by the growth rate of the culture.     

Post-translational modification of apolipoprotein B by transglutaminases.

The major form of cross-link found in apolipoprotein B was identified as N1N12-bis-(gamma-glutamyl)spermine, a product known to be formed through the catalytic action of transglutaminases (EC 2.3.2.13). N1-(gamma-Glutamyl)spermine was present in a trace amount but epsilon-(gamma-glutamyl)lysine cross-links, which are formed during fibrin formation in plasma, were not detected. In the presence of catalytic amounts of plasma Factor XIIIa (a thrombin-dependent extracellular transglutaminase) or cellular transglutaminase (a cytosolic enzyme), apolipoprotein B and other plasma apolipoproteins (A-I, A-II and C) underwent covalently bridged polymerization and served as amine acceptor substrates. These results suggests that transglutaminases may participate in the covalent modification of apolipoproteins, either in the physiological state or during pathogenesis.     

Post-translational modification of Bid has differential effects on its susceptibility to cleavage by caspase 8 or caspase 3

Bid is instrumental in death receptor-mediated apoptosis where it is cleaved by caspase 8 at aspartate 60 and aspartate 75 to generate truncated Bid (tBID) forms that facilitate release of mitochondrial cytochrome c. Bid is also cleaved at these sites by caspase 3 that is activated downstream of cytochrome c release after diverse apoptotic stimuli. In this context, tBid may amplify the apoptotic process. Bid is phosphorylated in vitro by casein kinases that regulate its cleavage by caspase 8 (Desagher, S., Osen-Sand, A., Montessuit, S., Magnenat, E., Vilbois, F., Hochmann, A., Journot, L. Antonsson, A., and Martinou, J.-C. (2001) Mol. Cell 8, 601-611). Using a Bid decapeptide substrate, we observed that phosphorylation at threonine 59 inhibited cleavage by caspase 8. This was also seen when recombinant Bid (rBid) and Bid isolated from murine kidney were incubated with casein kinase II. However, there were differences in the susceptibility of rBid and isolated Bid to cleavage by caspases 3 and 8. Caspase 8 cleaved rBid to generate two C-terminal products, p15 and p13 tBid, but produced only p15 tBid from isolated Bid. Contrary to rBid, isolated Bid was resistant to cleavage by caspase 3, yet was readily cleaved within the cytosolic milieu. Our data suggest that one or more distinct cellular mechanisms regulate Bid cleavage by caspases 8 and 3 in situ.     

Post-translational modification of protein by tyrosine sulfation: active sulfate PAPS is the essential substrate for this modification.

In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form. For this purpose tyrosylprotein sulfotransferase (TPST), the enzyme responsible for tyrosine sulfation of proteins, was characterized from a bovine liver Golgi preparation. TPST was active in a acidic environment with a pH optimum of 6.25, and displayed a stimulation by the Mn2+, with the optimum activity in the presence of 5mM MnCl2. TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15 by using 3'-phosphoadenosine 5'-phosphosulfate (PAPS), indicating its specificity for the naturally sulfated tyrosine 63. Comparison of the reaction kinetics on synthetic peptides showed that the bovine liver TPST has a higher affinity and reaction rates for those peptides with a aspartyl residue on the N-terminal side of the tyrosine when compared with a glutamyl residue.     

Post-translational modification of RAS proteins

Mutations of RAS genes drive cancer more frequently than any other oncogene. RAS proteins integrate signals from a wide array of receptors and initiate downstream signaling through pathways that control cellular growth. RAS proteins are fundamentally binary molecular switches in which the off/on state is determined by the binding of GDP or GTP, respectively. As such, the intrinsic and regulated nucleotide-binding and hydrolytic properties of the RAS GTPase were historically believed to account for the entirety of the regulation of RAS signaling. However, it is increasingly clear that RAS proteins are also regulated by a vast array of post-translational modifications (PTMs). The current challenge is to understand what are the functional consequences of these modifications and which are physiologically relevant. Because PTMs are catalyzed by enzymes that may offer targets for drug discovery, the study of RAS PTMs has been a high priority for RAS biologists.