Untitled

CC3 (TIP30) is a protein with pro-apoptotic and antimetastatic properties. The tumor suppressor effect of CC3 has been suggested to result from inhibition of nuclear transport by binding to importin ␤s or by regulating transcription through interaction in a complex with co-activator independent of AF-2 function (CIA) and the c-myc gene. Previous biochemical studies indicated that CC3 has protein kinase activity, and a structural similarity to cAMP-dependent protein kinase catalytic subunit was proposed. By contrast, bioinformatics studies suggested a relationship of CC3 to the short chain dehydrogenase reductase family. To clarify details of the CC3 structural family and ligand binding properties, we have determined the crystal structure of CC3 at 1.7-Å resolution. CC3 has a short chain dehydrogenase reductase fold and binding specificity for NADPH, yet it is unlikely to be normally enzymatically active because it is monomeric. These structural results, in conjunction with data from earlier mutagenesis work on the nucleotide binding motif, suggest that NADPH binding is important for the biological activity of CC3, including interaction with importins and with the CIA/c-myc system. CC3 provides an example of the adaptation of a metabolic enzyme fold to include a regulatory role, as also seen in the case of the NADH-binding co-repressor CtBP.Apoptosis or programmed cell death plays an opposing role to cell division in the homeostatic regulation of animal cell populations. As well as being of significance in development, apoptosis has an important place in the pathogenesis of some disease states. Mutated and deleted pro-apoptotic genes can give rise to carcinogenesis and tumor growth (1). CC3 (also known as TIP30) is an anti-tumor protein that predisposes cells to apoptosis, thereby giving properties of metastatic suppression (2). CC3 is an evolutionarily conserved gene ubiquitously expressed as a 27-kDa protein in human tissues. CC3 RNA is absent in variant small cell lung carcinoma cell lines that are derived from tumors with highly aggressive metastatic behavior (2). Significantly, ectopic expression of CC3 in variant small cell lung carcinoma cells induces expression of a number of apoptosis-related genes including Bad and Siva (3) and also attenuates metastasis in vivo (2). Moreover, it has been shown that genetically engineered mice deficient in CC3 have a high incidence of liver cancer and that reduced expression of CC3 is also associated with 33% of human hepatocellular carcinomas (4).A number of studies have been aimed at defining the biochemical properties of CC3, as well as the interactions made with partner proteins that may relate to its pro-apoptotic effects. Recent results have shown that CC3 can bind directly to karyopherins of the importin ␤ family of nuclear transportins in a Ran-GTP-insensitive manner (5). For importin ␤2, the site of interaction with CC3 involves HEAT domain(s) in the Cterminal third of the protein. Binding of CC3 to importins inhibits the nuclear transport of proteins with eith...

Section: Co-crystallization Studies With Potential Cc3 (Tip30)mentioning

confidence: 81%

Section: Cloning Expression and Purification Of Cc3 (Tip30)-mentioning

confidence: 99%

Crystal Structure of CC3 (TIP30)

Omari¹,

Bird

Nichols³

et al. 2005

“…As for the crystal structures of E. coli UDP-galactose 4-epimerase and human biliverdin IX ␤ reductase (BVR-〉), the pyrophosphate group of the modeled cofactor positions itself within hydrogen bonding distance of the amide nitrogens of residues 15 and 16 at the N terminus of the helix, thereby N-capping it and thus compensating for the helix macrodipole (49).…”

Section: Resultsmentioning

confidence: 99%

Crystal Structures of Pinoresinol-Lariciresinol and Phenylcoumaran Benzylic Ether Reductases and Their Relationship to Isoflavone Reductases

Min

Kasahara

Bedgar

et al. 2003

Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio-and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 Å resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous ␣/␤ NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

“…Mutation of one of these three residues, Cys 73 , was reported in one study to cause enzyme inactivation but in another to have no effect on activity (33,34). The crystal structures of rat biliverdin IX␣ reductase (34,35) and human biliverdin IX␤ reductase (36) have been reported. The rat biliverdin IX␣-reductase preferentially binds NADH at lower pH (pH ϳ6.7) but NADPH at higher pH (pH ϳ8.7).…”

mentioning

confidence: 99%

The Binding Sites on Human Heme Oxygenase-1 for Cytochrome P450 Reductase and Biliverdin Reductase

Wang

Montellano

2003

Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000 Heme oxygenase oxidizes heme, 1 a pro-oxidant and toxic species, to biliverdin, CO, and free iron (1). Each of the three metabolites formed in the reaction is thought to be physiologically important. Biliverdin is converted by biliverdin reductase to bilirubin, which must then be conjugated with glucuronic acid to be excreted (2). However, bilirubin, albeit toxic at high concentrations, has potent antioxidant properties that may contribute to the anti-inflammatory activity of the heme oxygenases (3, 4). Although still controversial, CO appears to behave as a gaseous signaling molecule akin to nitric oxide (5-7). Finally, the iron released from heme is largely recycled and is critical for iron homeostasis, since less than 3% of the body's daily iron needs are met by absorption from the diet (8). As a result of these activities, heme oxygenase has been shown to have, inter alia, important anti-inflammatory (9) and antiatherosclerotic (10) functions.Human heme oxygenase-1 (hHO-1) is a 33-kDa membranebound protein of 288 amino acid residues that binds heme and uses the bound entity as both the prosthetic group and substrate. A soluble form of hHO-1 (hHO-1 265 ) has been obtained by heterologous expression in E. coli of a truncated version of 265 amino acids that lacks the membrane binding domain (11). The crystal structures of hHO-1 truncated to a length of 233 amino acids (12), a rat homologue truncated to a length of 267 residues (13), and an inherently soluble full-length microbial heme oxygenase (14) have been determined. As recently reviewed, mechanistic studies have established that the transformation of heme to biliverdin is a three-step process that consumes three molecules of oxygen and seven electrons (15, 16). In the first step, ferric heme is oxidized to ␣-meso-hydroxyheme in a reaction that consumes one molecule of oxygen and two electrons. The ferric ␣-meso-hydroxyheme is then converted to ferrous verdoheme in a reaction that consumes a further molecule of oxygen and two electrons and releases CO. In the final stage of the reaction sequence, the verdoheme is converted to ferrous biliverdin with the consumption of a further molecule of oxygen and three electrons. The ferrous iron is then released, followed by dissociation of the biliverdin. Single turnover studies have shown that the rate-limiting step is the release of biliverdin, but in the presence of biliverdin reductase this step is accelerated, and one of the electron transfer steps becomes rate-limiting (17).The electrons required for the catalytic turnover of heme oxygenase are provided by NADPH-cytochrome P450 reductase (1,19,20), a 78-kDa membrane-bound flavoprotein that incorpo...