Amyloid-␤ peptide (A␤) is the toxic agent in Alzheimer's disease (AD), although the mechanism causing the neurodegeneration is not known. We previously proposed a mechanism in which excessive A␤ binds to regulatory heme, triggering functional heme deficiency (HD), causing the key cytopathologies of AD. We demonstrated that HD triggers the release of oxidants (e.g., H 2O2) from mitochondria due to the loss of complex IV, which contains heme-a. Now we add more evidence that A␤ binding to regulatory heme in vivo is the mechanism by which A␤ causes HD. Heme binds to A␤, thus preventing A␤ aggregation by forming an A␤-heme complex in a cell-free system. We suggest that this complex depletes regulatory heme, which would explain the increase in heme synthesis and iron uptake we observe in human neuroblastoma cells. The A␤-heme complex is shown to be a peroxidase, which catalyzes the oxidation of serotonin and 3,4-dihydroxyphenylalanine by H 2O2. Curcumin, which lowers oxidative damage in the brain in a mouse model for AD, inhibits this peroxidase. The binding of A␤ to heme supports a unifying mechanism by which excessive A␤ induces HD, causes oxidative damage to macromolecules, and depletes specific neurotransmitters. The relevance of the binding of regulatory heme with excessive A␤ for mitochondrial dysfunction and neurotoxicity and other cytopathologies of AD is discussed.curcumin ͉ heme deficiency ͉ mitochondria ͉ regulatory heme ͉ serotonin

Methylene blue (MB) has been used clinically for about a century to treat numerous ailments. We show that MB and other diaminophenothiazines extend the life span of human IMR90 fibroblasts in tissue culture by >20 population doubling (PDLs). MB delays senescence at nM levels in IMR90 by enhancing mitochondrial function. MB increases mitochondrial complex IV by 30%, enhances cellular oxygen consumption by 37-70%, increases heme synthesis, and reverses premature senescence caused by H2O2 or cadmium. MB also induces phase-2 antioxidant enzymes in hepG2 cells. Flavin-dependent enzymes are known to use NAD(P)H to reduce MB to leucomethylene blue (MBH2), whereas cytochrome c reoxidizes MBH2 to MB. Experiments on lysates from rat liver mitochondria suggest the ratio MB/cytochrome c is important for the protective actions of MB. We propose that the cellular senescence delay caused by MB is due to cycling between MB and MBH2 in mitochondria, which may partly explain the increase in specific mitochondrial activities. Cycling of MB between oxidized and reduced forms may block oxidant production by mitochondria. Mitochondrial dysfunction and oxidative stress are thought to be key aberrations that lead to cellular senescence and aging. MB may be useful to delay mitochondrial dysfunction with aging and the decrease in complex IV in Alzheimer disease.

Protein–protein interactions (PPIs) governing the recognition of substrates by E3 ubiquitin ligases are critical to cellular function. There is significant therapeutic potential in the development of small molecules that modulate these interactions; however, rational design of small molecule enhancers of PPIs remains elusive. Herein, we report the prospective identification and rational design of potent small molecules that enhance the interaction between an oncogenic transcription factor, β-Catenin, and its cognate E3 ligase, SCF β-TrCP . These enhancers potentiate the ubiquitylation of mutant β-Catenin by β-TrCP in vitro and induce the degradation of an engineered mutant β-Catenin in a cellular system. Distinct from PROTACs, these drug-like small molecules insert into a naturally occurring PPI interface, with contacts optimized for both the substrate and ligase within the same small molecule entity. The prospective discovery of ‘molecular glue’ presented here provides a paradigm for the development of small molecule degraders targeting hard-to-drug proteins.

). To complement this biochemical analysis, we undertook a genetic approach to the analysis of the structure and function of the A20 protein. Here we report the application of clustered charge-to-alanine mutagenesis of the A20 gene. Eight mutant viruses containing altered A20 alleles were isolated using this approach; two of these, tsA20-6 and tsA20-ER5, have tight temperature-sensitive phenotypes. At the nonpermissive temperature, neither virus forms macroscopic plaques and the yield of infectious virus is <1% of that obtained at the permissive temperature. Both viruses show a profound defect in the accumulation of viral DNA at the nonpermissive temperature, although both the A20 protein and DNA polymerase accumulate to wild-type levels. Cytoplasmic extracts prepared from cells infected with the tsA20 viruses show a defect in processive polymerase activity; they are unable to direct the formation of RFII product using a singly primed M13 template. In sum, these data indicate that the A20 protein plays an essential role in the viral life cycle and that viruses with A20 lesions exhibit a DNA ؊ phenotype that is correlated with a loss in processive polymerase activity as assayed in vitro. The vaccinia virus A20 protein can, therefore, be considered a new member of the family of proteins (E9, B1, D4, and D5) with essential roles in vaccinia virus DNA replication.Vaccinia virus, the prototypic member of the poxvirus family, displays a great deal of genetic and physical autonomy from the host. The virus replicates solely within the cytoplasm of the host, and the 192-kb genome is thought to encode most if not all of the functions required for genome replication, gene expression, and virion morphogenesis. The centerpiece of the replication apparatus is the E9 DNA polymerase, which displays significant homology to the ␣ and ␦ families of eucaryotic replicative polymerases as well as the polymerases encoded by herpesviruses. We and others have characterized the polymerase both genetically and biochemically (4, 5,9, 10, 12, 29-31, 36, 38, 39, 41). Temperature-sensitive (ts) alleles, mutator and anti-mutator alleles, and mutants conferring resistance to aphidicolin, phosphonoacetic acid, and cytosine arabinoside have been isolated and studied. The polymerase has been overexpressed and purified and shown to have both polymerase and proofreading exonuclease activities. We have also shown that the enzyme is inherently distributive in vitro, being able to catalyze the addition of Ͻ10 nucleotides (nt) per binding event when moderate levels of salt (40 mM NaCl) or divalent cations (8 mM MgCl 2 ) are present (31). In sharp contrast, the cytoplasmic lysates of infected cells are able to catalyze the addition of as many as 7,000 nt in a single binding event under the same reaction conditions (29). We demonstrated that the protein(s) responsible for conferring processivity on the viral polymerase was present in extracts prepared from infected cells in which only early proteins were present but not in extracts prepared from uninfect...

In vitro analysis of the catalytic DNA polymerase encoded by vaccinia virus has demonstrated that it is innately distributive, catalyzing the addition of <10 nucleotides per primer-template binding event in the presence of 8 mM MgCl 2 or 40 mM NaCl (W. F. McDonald and P. Traktman, J. Biol. Chem. 269:31190-31197, 1994). In contrast, cytoplasmic extracts isolated from vaccinia virus-infected cells contain a highly processive form of DNA polymerase, able to catalyze the replication of a 7-kb template per binding event under similar conditions. To study this holoenzyme, we were interested in purifying and characterizing the vaccinia virus processivity factor (VPF). Our previous studies indicated that VPF is expressed early after infection and has a native molecular mass of ϳ48 kDa (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). Using these criteria, we established a six-step chromatographic purification procedure, in which a prominent ϳ45-kDa band was found to copurify with processive polymerase activity. This species was identified as the product of the A20 gene. By use of recombinant viruses that direct the overexpression of A20 and/or the DNA polymerase, we verified the physical interaction between the two proteins in coimmunoprecipitation experiments. We also demonstrated that simultaneous overexpression of A20 and the DNA polymerase leads to a specific and robust increase in levels of processive polymerase activity. Taken Vaccinia virus exhibits a high degree of genetic and physical autonomy from the host cell, possessing a genome that encodes more than 200 genes and directing a replicative cycle in which DNA replication, gene expression, and morphogenesis take place solely within the cytoplasm of the infected cell. It is therefore likely that the trans-acting functions required for DNA replication are virally encoded, and several laboratories have engaged in studies aimed at identifying and characterizing these functions. The catalytic DNA polymerase of vaccinia virus is encoded by the E9 gene (6,20,31). The 116-kDa enzyme possesses both polymerase and proofreading exonuclease activity and retains many of the conserved domains that are found within the replicative polymerases of mammalian and yeast cells, as well as those encoded by other large DNA viruses. In addition, the vaccinia virus enzyme, like the catalytic subunit of these other replicative polymerases, is inherently distributive. Our studies on the purified E9 protein indicate that in the presence of 40 mM NaCl or 8 mM MgCl 2 , the enzyme catalyzes the addition of Ͻ10 nucleotides (nt) per primer-template binding event (21). Such distributive behavior would be incompatible with efficient replication of a large genome in vivo, and it is therefore no surprise that a highly processive form of the vaccinia virus enzyme is found in unfractionated cytoplasmic extracts of infected cells (19). This processive enzyme is capable of catalyzing the addition of Ͼ7,000 nt per primer-template binding event.This dichotomy between the dis...