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The valosin-containing protein (p97) is a ubiquitin-dependent ATPase that plays central roles in ubiquitin proteasome system (UPS)-mediated protein degradation pathways. p97 has been recently identified as a putative substrate of active Caspase-6 (Casp6) in primary human neurons. Since Casp6 is activated in mild cognitive impairment (MCI) and Alzheimer’s disease (AD) patients’ brains, the targeting of p97 by Casp6 may represent an important step that leads to UPS impairment in AD. Here, we show that p97 is a Casp6 substrate in vitro and in vivo. Casp6 cleavage of recombinant p97 generated two N-terminal fragments of 28 and 20 kDa, which were not generated by the other two effector caspases, Caspase-3 and Caspase-7. ATP binding to the D1 ATPase ring of p97 reduced the susceptibility of the N-domain to caspase-mediated proteolysis. Mass spectrometric analysis identified VAPD179 as a Casp6 cleavage site within p97’s N-domain. An anti-neoepitope serum immunohistochemically detected p97 cleaved at VAPD179 in the cytoplasm of the cell soma and neurites of hippocampal neurons in MCI and AD. Overexpression of p97 (1-179) fragment, representing p97 cleaved at D179, impaired the degradation of model substrates in the ubiquitin-fusion degradation and the N-end rule pathways, and destabilized endogenous p97. Collectively, these results show that p97 is cleaved by Casp6 in AD and suggest p97 cleavage as an important mechanism for UPS impairment.

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In plants, O-methylation of phenolic compounds plays an important role in such processes as lignin synthesis, flower pigmentation, chemical defense, and signaling. However, apart from phenylpropanoids and flavonoids, very few enzymes involved in coumarin biosynthesis have been identified. We report here the molecular and biochemical characterization of a gene encoding a novel O-methyltransferase that catalyzes the methylation of 7,8-dihydroxycoumarin, daphnetin. The recombinant protein displayed an exclusive methylation of position 8 of daphnetin. The identity of the methylated product was unambiguously identified as 7-hydroxy-8-methoxycoumarin by co-chromatography on cellulose TLC and coelution from high performance liquid chromatography, with authentic synthetic samples, as well as by UV, mass spectroscopy, 1 H NMR spectral analysis, and NOE correlation signals of the relevant protons. Northern blot analysis and enzyme activity assays revealed that the transcript and corresponding enzyme activity are up-regulated by both low temperature and photosystem II excitation pressure. Using various phenylpropanoid and flavonoid substrates, we demonstrate that cold acclimation of rye leaves increases O-methyltransferase activity not only for daphnetin but also for the lignin precursors, caffeic acid, and 5-hydroxyferulic acid. The significance of this novel enzyme and daphnetin O-methylation is discussed in relation to its putative role in modulating cold acclimation and photosystem II excitation pressure.Low temperature is one of the most important environmental factors limiting the productivity and distribution of plants. Exposure of plants to low, nonfreezing temperatures, a process known as cold acclimation, induces the genetic system required for increased freezing tolerance. Knowledge of the molecular, physiological, and biochemical changes that occur during this process could lead to the improvement of plant productivity.This complex process has been extensively studied and several cold-responsive genes have been isolated from a range of dicotyledonous and monocotyledonous species (1, 2). Although the functions of some of these genes are known (3-6), the details of the processes responsible for their regulation and detection of temperature changes are still incomplete.Previous studies have shown that development of freezing tolerance in winter cereals, such as wheat and rye, is correlated with an increase in their photosynthetic capacity (7). Thus, growth at low temperature not only induces freezing tolerance, but also results in an increased resistance to low temperatureinduced photoinhibition of photosynthesis, and requires adjustment to a combination of light and low temperature. The common photosynthetic response of plants to low temperature and normal light is rationalized in terms of photosystem II (PSII) 1 excitation pressure, which is a measure of the redox state of the first electron acceptor, quinone A (7-9). It has been shown that cold-acclimated rye and wheat grown at 5°C/250 mol m Ϫ2 s Ϫ1

Serratula tinctoria (Asteraceae) accumulates mainly 3,39-dimethylquercetin and small amounts of 3-methylquercetin as an intermediate. The fact that 3-methylquercetin rarely accumulates in plants in significant amounts, and given its important role as an antiviral and antiinflammatory agent that accumulates in response to stress conditions, prompted us to purify and characterize the enzyme involved in its methylation. The flavonol 3-O-methyltransferase (3-OMT) was partially purified by ammonium sulfate precipitation and successive chromatography on Superose-12, Mono-Q, and adenosine-agarose affinity columns, resulting in a 194-fold increase of its specific activity. The enzyme protein exhibited an expressed specificity for the methylation of position 3 of the flavonol, quercetin, although it also utilized kaempferol, myricetin, and some monomethyl flavonols as substrates. It exhibited a pH optimum of 7.6, a pI of 6.0, and an apparent molecular mass of 31 kD. Its K m values for quercetin as the substrate and S-adenosyl-L-Met (AdoMet) as the cosubstrate were 12 and 45 mM, respectively. The 3-OMT had no requirement for Mg 21 , but was severely inhibited by p-chloromercuribenzoate, suggesting the requirement for SH groups for catalytic activity. Quercetin methylation was competitively inhibited by S-adenosyl-L-homo-Cys with respect to the cosubstrate AdoMet, and followed a sequential bi-bi reaction mechanism, where AdoMet was the first to bind and S-adenosyl-L-homo-Cys was released last. In-gel trypsin digestion of the purified protein yielded several peptides, two of which exhibited strong amino acid sequence homology, upon protein identification, to a number of previously identified Group II plant OMTs. The availability of peptide sequences will allow the design of specific nucleotide probes for future cloning of the gene encoding this novel enzyme for its use in metabolic engineering.Flavonoid compounds constitute one of the most ubiquitous groups of natural plant products. They exhibit a wide range of functions and play important roles in the biochemistry, physiology, and ecology of plants. These include their contribution to flower color, protection against UV radiation and pathogenic organisms, promotion of pollen germination and pollen fertility, and activation of Rhizobium nodulation genes. They also act as growth regulators, enzyme inhibitors, insect antifeedants, and antioxidants, and are of potential benefit to human health (Bohm, 1998, and references therein). Flavonoids owe their structural biodiversity to a number of enzyme-catalyzed substitution reactions (Ibrahim and Anzellotti, 2003).Of these, enzymatic O-methylation, which is catalyzed by a family of S-adenosyl-L-Met (AdoMet)-dependent O-methyltransferases (OMTs; Ibrahim and Muzac, 2000), involves the transfer of the methyl group of AdoMet to the hydroxyl groups of an acceptor molecule, with the concomitant formation of the corresponding methyl ether derivative and S-adenosyl-Lhomo-Cys (AdoHcy) as products. O-Methylation of flavonoids neutralizes th...

BackgroundFlavonoids, one of the major groups of secondary metabolites, play important roles in the physiology, ecology and defence of plants. Their wide range of activities is the result of their structural diversity that encompasses a variety of functional group substitutions including hydroxylations. The aromatic hydroxylation at position 6 of flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase (ODD), rather than a cytochrome P450-dependent monooxygenase. ODDs catalyze a variety of enzymatic reactions implicated in secondary metabolite biosynthesis.ResultsA cDNA fragment encoding an ODD involved in the 6-hydroxylation of partially methylated flavonols, flavonol 6-hydroxylase (F6H), was isolated and characterized from Chrysosplenium americanum using internal peptide sequence information obtained from the native plant protein. This novel clone was functionally expressed in both prokaryotic and eukaryotic expression systems and exhibited ODD activity. The cofactor and cosubstrate requirements of the recombinant proteins are typical for ODDs, and the recombinant enzymes utilize 3,7,4'-trimethylquercetin as the preferred substrate. The genomic region encoding this enzyme possesses two introns at conserved locations for this class of enzymes and is present as a single copy in the C. americanum genome.ConclusionsRecombinant F6H has been functionally expressed and characterized at the molecular level. The results demonstrate that its cofactor dependence, physicochemical characteristics and substrate preference compare well with the native enzyme. The N-terminal region of this protein is believed to play a significant role in catalysis and may explain the difference in the position specificity of the 6-hydroxylation reaction.

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