Identification of Caspase-6-mediated processing of the valosin containing protein (p97) in Alzheimer's disease: a novel link to dysfunction in ubiquitin proteasome system-mediated protein degradation.

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 VAPD(179) as a Casp6 cleavage site within p97's N-domain. An anti-neoepitope serum immunohistochemically detected p97 cleaved at VAPD(179) 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.

Molecular characterization and functional expression of flavonol 6-hydroxylase.

BACKGROUND: Flavonoids, 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. RESULTS: A 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. CONCLUSIONS: Recombinant 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.

Partial purification, kinetic analysis, and amino acid sequence information of a flavonol 3-O-methyltransferase from Serratula tinctoria.

Serratula tinctoria (Asteraceae) accumulates mainly 3,3'-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 microm, respectively. The 3-OMT had no requirement for Mg(2+), 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.

Daphnetin methylation by a novel O-methyltransferase is associated with cold acclimation and photosystem II excitation pressure in rye.

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.