Sirtuin 3 (SIRT3) Protein Regulates Long-chain Acyl-CoA Dehydrogenase by Deacetylating Conserved Lysines Near the Active Site

Long-chain acyl-CoA dehydrogenase (LCAD) is a key mitochondrial fatty acid oxidation enzyme. We previously demonstrated increased LCAD lysine acetylation in SIRT3 knockout mice concomitant with reduced LCAD activity and reduced fatty acid oxidation. To study the effects of acetylation on LCAD and determine sirtuin 3 (SIRT3) target sites, we chemically acetylated recombinant LCAD. Acetylation impeded substrate binding and reduced catalytic efficiency. Deacetylation with recombinant SIRT3 partially restored activity. Residues Lys-318 and Lys-322 were identified as SIRT3-targeted lysines. Arginine substitutions at Lys-318 and Lys-322 prevented the acetylation-induced activity loss. Lys-318 and Lys-322 flank residues Arg-317 and Phe-320, which are conserved among all acyl-CoA dehydrogenases and coordinate the enzyme-bound FAD cofactor in the active site. We propose that acetylation at Lys-318/Lys-322 causes a conformational change which reduces hydride transfer from substrate to FAD. Medium-chain acyl-CoA dehydrogenase and acyl-CoA dehydrogenase 9, two related enzymes with lysines at positions equivalent to Lys-318/Lys-322, were also efficiently deacetylated by SIRT3 following chemical acetylation. These results suggest that acetylation/deacetylation at Lys-318/Lys-322 is a mode of regulating fatty acid oxidation. The same mechanism may regulate other acyl-CoA dehydrogenases.     

The point mutation UCH-L1 C152A protects primary neurons against cyclopentenone prostaglandin-induced cytotoxicity: implications for post-ischemic neuronal injury

Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ^12,14-prostaglandin J₂ (15dPGJ2), are reactive prostaglandin metabolites exerting a variety of biological effects. CyPGs are produced in ischemic brain and disrupt the ubiquitin-proteasome system (UPS). Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain-specific deubiquitinating enzyme that has been linked to neurodegenerative diseases. Using tandem mass spectrometry (MS) analyses, we found that the C152 site of UCH-L1 is adducted by CyPGs. Mutation of C152 to alanine (C152A) inhibited CyPG modification and conserved recombinant UCH-L1 protein hydrolase activity after 15dPGJ2 treatment. A knock-in (KI) mouse expressing the UCH-L1 C152A mutation was constructed with the bacterial artificial chromosome (BAC) technique. Brain expression and distribution of UCH-L1 in the KI mouse was similar to that of wild type (WT) as determined by western blotting. Primary cortical neurons derived from KI mice were resistant to 15dPGJ2 cytotoxicity compared with neurons from WT mice as detected by the WST-1 cell viability assay and caspase-3 and poly ADP ribose polymerase (PARP) cleavage. This protective effect was accompanied with significantly less ubiquitinated protein accumulation and aggregation as well as less UCH-L1 aggregation in C152A KI primary neurons after 15dPGJ2 treatment. Additionally, 15dPGJ2-induced axonal injury was also significantly attenuated in KI neurons as compared with WT. Taken together, these studies indicate that UCH-L1 function is important in hypoxic neuronal death, and the C152 site of UCH-L1 has a significant role in neuronal survival after hypoxic/ischemic injury.Ubiquitin C-terminal hydrolase L1 is a multifunctional protein that is highly expressed in neurons throughout brain.¹ UCH-L1 closely interacts with proteins of the neuronal cytoskeleton and may have an important role in axonal transport and maintaining axonal integrity.^{2, 3} UCH-L1 regulates synaptic function and long-term potentiation (LTP) under normal and pathological conditions and may be involved in memory function.⁴ Mutations and altered function of UCH-L1 have been associated with neurological diseases including Parkinson''s (PD) and Alzheimer''s (AD) diseases and early onset neurodegeneration involving white matter.^{2, 3, 4, 5, 6, 7} However, the role of UCH-L1 function in cerebral ischemic injury and recovery has not been thoroughly investigated.Cyclopentenone prostaglandins (CyPGs) are the reactive metabolites of prostaglandins containing a carbonyl moiety that may covalently modify cysteine in a variety of proteins.^{8, 9, 10} CyPG concentration is dramatically increased in ischemic brain.¹¹ CyPGs such as 15dPGJ2 disrupt the ubiquitin-proteasome system (UPS), resulting in accumulation and aggregation of ubiquitinated (Ub) proteins and neuronal cell death.^{12, 13}UCH-L1 is a target of CyPG modification.^{13, 14, 15} In the current study, mass spectrometry (MS)/MS was used to determine that cysteine152 is the binding site of the CyPG 15dPGJ2 to UCH-L1. We then constructed a knock-in (KI) mouse using the bacterial artificial chromosome (BAC) technique with a cysteine to alanine mutation at this 15dPGJ2 binding site on UCH-L1. Primary neurons derived from KI and wild-type (WT) mice were used to determine the effect of CyPG binding to UCH-L1 on cell death and disruption of the UPS. These studies address a potential role for modification of UCH-L1 by CyPGs and other reactive lipid species in stroke and neurodegenerative diseases.     

Punicafolin,an ellagitannin from the leaves of Punica granatum

A new ellagitannin, punicafolin has been isolated from the leaves of Punica granatum and characterized by physicochemical data and spectral evidence as 1,2,4-tri-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-β-D-glucose. The occurrence in the leaves of the known tannins, granatins A and B, corilagin, strictinin, 1,2,4,6-tetra-O-galloyl-β-D-glucose and 1,2,3,4,6-penta-O-galloyl-β-D-glucose has also been demonstrated.     

Production and purification of a recombinant human 14 kDa β-galactoside-binding lectin

The cDNA for a 14 kDa human β-galactoside-binding lectin was inserted into a plasmid carrying a taq promoter, and the lectin protein was expressed in E. coli cells. The recombinant lectin was extracted from the cells and purified to apparent homogeneity by a single-step chromatography on an asialofetuin-agarose column. Subunit molecular mass (14 kDa), hemagglutinating activity and antigenicity were indistinguishable from those of the human placental lectin. Though the N-terminal of the placental lectin is blocked with an acetyl group, the recombinant lectin was found to have a free amino group. However, the N-terminal amino acid sequences were identical. The recombinant lectin was considered to have the same three-dimensional structure as the placental lectin.     

Stilbene glycoside gallates and proanthocyanidins from Polygonum multiflorum

Two new stilbene glycoside gallates proanthocyanidins have been isolated from Polygonum multiflorum. The stilbenes were shown to be 2″-3″-O-monogalloyl esters of 2,3,5,4′-tetrahydroxystilbene 2-O-β-d-glucopyranoside.     

Bioactive compounds from liverworts: Inhibition of lipopolysaccharide-induced inducible NOS mRNA in RAW 264.7 cells by Herbertenoids and Cuparenoids

The inhibition of lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) by herbertenoids and cuparenoids isolated from liverworts in RAW 264.7 macrophages was evaluated. Among compounds tested, herbertenediol, cuparenediol, 1,2-diacetoxyherbertene and 2-hydroxy-4-methoxycuparene exhibited significant activity. For 2-hydroxy-4-methoxycuparene, chosen as representative compound, the strong inhibitory activity was related to the inhibition on LPS-induced iNOS mRNA. The structure-activity relationship will be discussed.     

Evolutionary repair reveals an unexpected role of the tRNA modification m1G37 in aminoacylation

The tRNA modification m¹G37, introduced by the tRNA methyltransferase TrmD, is thought to be essential for growth in bacteria because it suppresses translational frameshift errors at proline codons. However, because bacteria can tolerate high levels of mistranslation, it is unclear why loss of m¹G37 is not tolerated. Here, we addressed this question through experimental evolution of trmD mutant strains of Escherichia coli. Surprisingly, trmD mutant strains were viable even if the m¹G37 modification was completely abolished, and showed rapid recovery of growth rate, mainly via duplication or mutation of the proline-tRNA ligase gene proS. Growth assays and in vitro aminoacylation assays showed that G37-unmodified tRNA^Pro is aminoacylated less efficiently than m¹G37-modified tRNA^Pro, and that growth of trmD mutant strains can be largely restored by single mutations in proS that restore aminoacylation of G37-unmodified tRNA^Pro. These results show that inefficient aminoacylation of tRNA^Pro is the main reason for growth defects observed in trmD mutant strains and that proS may act as a gatekeeper of translational accuracy, preventing the use of error-prone unmodified tRNA^Pro in translation. Our work shows the utility of experimental evolution for uncovering the hidden functions of essential genes and has implications for the development of antibiotics targeting TrmD.     

A novel method for the three-dimensional (3-D) analysis of orthodontic tooth movement-calculation of rotation about and translation along the finite helical axis.

The purpose of this study was to establish a novel method for evaluating orthodontic tooth movement in three-dimensional (3-D) space. The present system consisted of the following procedures at a given treatment period: (1) 3-D tooth positions were measured with a 3-D surface-scanning system using a slit laser beam; (2) the 3-D shape data were registered automatically at the maxillary first molars, and the coordinate systems were normalized; (3) the rotation matrix and translation vector were calculated from the automatic registration of the two position data for a given tooth; (4) the finite helical axes of teeth were calculated as the locus of zero rotational displacement; and (5) tooth movement was presented as rotation about and translation along the finite helical axis. To test this system, a male patient (age 22 yr 2 months) with Angle Class III malocclusion and moderate crowding of the anterior teeth, who had been treated using a standard multi-bracket appliance, was used as a model case in this study. Impressions for a dental cast model were taken at five phases; immediately before and after application of the appliance, and 10 days, 1 month and 2 months after beginning treatment. The results demonstrated that the present analytical method can more simply describe the movement of a given tooth by rotation about and translation along the finite helical axis, and provides quantitative visual 3-D information on complicated tooth movement during orthodontic treatment.     

Prevention of heart failure in mice by an antiviral agent that inhibits type 5 cardiac adenylyl cyclase

Despite numerous discoveries from genetically engineered mice, relatively few have been translated to the bedside, mainly because it is difficult to translate from genes to drugs. This investigation examines an antiviral drug, which also has an action to selectively inhibit type 5 adenylyl cyclase (AC5), a pharmaceutical correlate of the AC5 knockout (KO) model, which exhibits longevity and stress resistance. Our objective was to examine the extent to which pretreatment with this drug, adenine 9-β-d-arabinofuranoside (Ara-A), favorably ameliorates the development of heart failure (HF). Ara-A exhibited selective inhibition for AC5 compared with the other major cardiac AC isoform, AC6, i.e., it reduced AC activity significantly in AC5 transgenic (Tg) mice, but not in AC5KO mice and had little effect in either wild-type or AC6Tg mice. Permanent coronary artery occlusion for 3 wk in C57Bl/6 mice increased mortality and induced HF in survivors, as reflected by reduced cardiac function, while increasing cardiac fibrosis. The AC5 inhibitor Ara-A significantly improved all of these end points and also ameliorated chronic isoproterenol-induced cardiomyopathy. As with the AC5KO mice, Ara-A increased mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation. A MEK inhibitor abolished the beneficial effects of the AC5 inhibitor in the HF model, indicating the involvement of the downstream MEK-ERK pathway of AC5. Our data suggest that pharmacological AC5 inhibition may serve as a new therapeutic approach for HF.