Showing papers by "Emil F. Pai published in 2007"

Atomic model of human Rcd‐1 reveals an armadillo‐like‐repeat protein with in vitro nucleic acid binding properties

Abstract: Cellular differentiation is an essential characteristic of cells required for the formation of multicellular organisms. However, many unicellular organisms, such as yeast, also differentiate in order to survive in hostile environments. Recent studies suggest that the mechanisms and factors involved in differentiation are not very cell type specific, let alone organism specific (Yamamoto et al. 1999; Hiroi et al. 2002). Schizosaccharomyces pombe, a fission yeast, resembles higher eukaryotes in several ways including genetic structure, cell cycle control, and various signaling pathways. It also undergoes cellular differentiation during sexual development involving conjugation, meiosis, and sporulation. Sexual differentiation is controlled primarily through two signals: nutrient starvation and the presence of mating pheromone. Transcriptional factor Ste11 is required and activates several genes involved in conjugation and meiosis (Kelly et al. 1988; Watanabe et al. 1988; Hughes et al. 1990; Sugimoto et al. 1991; Willer et al. 1995). Included are the mei2, rep1, and ste genes (including ste11), and other mating-type genes (Kelly et al. 1988; Watanabe et al. 1988; Hughes et al. 1990; Sugimoto et al. 1991; Miyamoto et al. 1994; Sugiyama et al. 1994; Willer et al. 1995). Nitrogen starvation as well as carbon starvation are two major nutrient-depletion signals triggering sexual development in budding yeast. Pat1/Ran1 kinase plays a unique role in the regulation of sexual development, primarily blocking the onset of meiosis until conjugation occurs by inactivating a key triggering factor, Mei2 (Beach et al. 1985; Iino and Yamamoto 1985a,b; Watanabe et al. 1997). Inactivation of Pat1 in haploid cells induces lethal meiosis that can be suppressed through inactivation of the mei2 gene. As Ste11 is required for the expression of mei2 (Sugimoto et al. 1991), factors inhibiting ste11 expression or function would rescue the pat1 inactivated cells. Rcd1 (required for cell differentiation) was identified as a differentiation-controlling factor crucial for nitrogen starvation-induced sexual development in S. pombe by screening a genetic library for suppressors of pat1 lethality (Okazaki et al. 1998). Surprisingly, cells with nonfunctional rcd1 were also shown to be sterile, despite initial assumptions that these strains would have been highly proficient for sexual development. Rcd-1 is essential for nitrogen starvation-invoked induction of ste11 (Okazaki et al. 1998). Paradoxically, overexpression of Rcd-1 rescued pat1 inactivated cells, supposedly through the inhibiting activity of a dominant active mutant of Mei2 (Y. Watanabe and M. Yamamoto, unpubl.). Homologs of the rcd1 gene from S. pombe are quite conserved throughout eukaryotic genomes (Fig. 1A; Okazaki et al. 1998). One homolog, CAF40, was identified as a component of the CCR4–NOT transcription complex in Saccharomyces cerevisiae (Chen et al. 2001). This complex is evolutionarily conserved in eukaryotes and is involved in initiation (Denis and Malvar 1990; Sakai et al. 1992; Collart and Struhl 1993), elongation (Denis et al. 2001), degradation (Tucker et al. 2001), and deadenylation (Tucker et al. 2001) of mRNA, as well as regulation of TFIID activity (Chen et al. 2002). In the CCR4–NOT complex, only NOT1 is essential (Collart and Struhl 1993; Maillet et al. 2000), although it has no identifiable functional domains, apparently acting as a scaffold. CAF40 associates with NOT1 through the latter's N-terminal 1100 residues, although its presence was not required for CCR4–NOT complex formation (Chen et al. 2001). As CAF40 has also been shown to interact with human NOT1, this suggests that hRcd-1 would be a part of the human CCR4–NOT complex (Chen et al. 2001). Figure 1. Rcd-1 sequences. (A) Alignment of the sequences of Rcd-1 from various organisms as indicated (GenBank accession nos.: Homo sapiens, {"type":"entrez-protein","attrs":{"text":"NP_005435","term_id":"4885579"}}NP_005435; S. pombe, {"type":"entrez-protein","attrs":{"text":"NP_594984","term_id":"19115896"}} ... Mammalian Rcd-1 was investigated for its functional similarity to the yeast homologs using F9 mouse teratocarcinoma cell differentiation, which is regulated by retinoic acid (RA) (Hiroi et al. 2002). F9 cells differentiate into primitive endoderm upon treatment with RA (Hogan et al. 1981; Edwards and McBurney 1983). Inhibition of mouse Rcd-1 (mRcd-1) production through the use of antisense oligonucleotides effectively blocked RA-induced differentiation (Hiroi et al. 2002). As well, overexpression of mRcd-1-induced spontaneous differentiation, apparently sensitizing the F9 cells to rather low levels of RA (Hiroi et al. 2002). Rcd-1 is highly expressed in various mammalian tissues. Antibodies against Rcd-1 were used to probe various tissues from adult and embryonic rat, showing the highest levels of expression in the lung, spleen, testes, and thymus of adult rats, while expressed in virtually all tissues of the early rat embryo (Hiroi et al. 2002). Northern blots using human Rcd-1 as a probe showed significant expression in human testes, ovaries, and thymus (Okazaki et al. 1998). mrcd1 was also isolated as an erythropoietin-responsive gene potentially involved in hematopoetic cell development (Gregory et al. 2000). A recent finding reports that mRcd-1 interacts with the c-Myb protein (Haas et al. 2004), which, in turn, is required for the generation of monocytes from a myeloid progenitor (for review, see Friedman 2002). These results are consistent with Rcd-1’s proposed involvement in differentiation control. Despite the increasing knowledge about the role Rcd-1 plays in cellular differentiation, little is known about the mechanisms by which Rcd-1 may exert its influence. Structural information could provide more insight regarding its actual functioning within cells. We now report the crystal structure of the highly conserved region of human Rcd-1 (a region spanning 90% of the full-length protein). The armadillo-repeat-like structure in conjunction with interesting surface features and charge clustering suggest possible means by which Rcd-1 can mediate its effects on cellular differentiation-related gene control.

A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity.

Abstract: Orotidine 5‘-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5‘-monophosphate without any covalent intermediates. Active site residues in ODCase are in...

Two mutations convert mammalian xanthine oxidoreductase to highly superoxide-productive xanthine oxidase.

Abstract: Reactive oxygen species are generated by various systems, including NADPH oxidases, xanthine oxidoreductase (XOR) and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide in a molar ratio of about 1:3, depending upon the conditions. Here, we present a mutant of rat XOR that displays mainly XO activity with a superoxide:hydrogen peroxide production ratio of about 6:1. In the mutant, tryptophan 335, which is a component of the amino acid cluster crucial for switching from the XDH to the XO conformation, was replaced with alanine, and phenylalanine 336, which modulates FAD's redox potential through stacking interactions with the flavin cofactor, was changed to leucine. When the mutant was expressed in Sf9 cells, it was obtained in the XO form, and dithiothreitol treatment only partially restored the pyridine nucleotide-binding capacity. The crystal structure of the dithiothreitol-treated mutant at 2.3 Angstroms resolution showed the enzyme's two subunits to be quite similar, but not identical: the cluster involved in conformation-switching was completely disrupted in one subunit, but remained partly associated in the other one. The chain trace of the active site loop in this mutant is very similar to that of the bovine XO form. These results are consistent with the idea that the XDH and XO forms of the mutant are in an equilibrium that greatly favours the XO form, but the equilibrium is partly shifted towards the XDH form upon incubation with dithiothreitol.

Enzyme-Driven Speciation: Crystallizing Archaea via Lipid Capture

Abstract: As the origin(s) of life on Earth remains an open question, detailed characteristics about the “last universal ancestor” (LUA) continue to be obscured. Here we provide arguments that strengthen the bacterial-like nature of the LUA. Our view attempts to recreate the evolution of archaeal lipids, the major components of the distinctive membrane that encapsulates these ancient prokaryotes. We show that (S)- 3-O-geranylgeranylglyceryl phosphate synthase (GGGPS), a TIM-barrel protein that performs the committed step in archaeal lipid synthesis, likely evolved from the duplication and fusion of a (βα)4 half-barrel ancestor. By comparison to the well-characterized HisA and HisF TIM-barrel proteins, we propose a time line for the invention of this diagnostic archaeal biosynthetic pathway. After excluding the possibility of horizontal gene transfer, we conclude that the evolutionary history of GGGPS mirrors the emergence of Archaea from the LUA. We illustrate aspects of this “lipid capture” model that support its likelihood in recreating key evolutionary events and, as our hypothesis is built on a single initiating event, we suggest that the appearance of GGGPS represents an example of enzyme-driven speciation.

Quantifying the intrinsic effects of two point mutation models of proline-proline diamino acid diamide: a first-principle computational study.

Abstract: Stabilities and conformational properties of two Pro → Thr point mutation models were computed at the B3LYP/6-31G(d) level of theory for the parent triamino acid diamide Pro-Pro-Pro (HCO-Pro-Pro-Pro-NH2). Geometrical parameters for the amino acid sequences, used in the molecular orbital computations for Pro-Pro-Thr and Pro-Thr-Pro, were retrieved from the Protein Data Bank. Thermodynamic functions (S, H, G) were computed for the fully optimized geometries. To assess the stabilization energetics of these mutant models, relative to the parent Pro-Pro-Pro reference conformer eLeLγL, isodesmic reactions were constructed to calculate ΔS, ΔH, and ΔG. The importance of intramolecular hydrogen bonds involving the −OH group of the Thr side chain, which emerged after the point mutations, was also examined to determine the internal stabilization of these peptide models. This study describes an approach to analyzing a point mutation at the center of a peptide chain and compares its stability to that of a point mutati...

Transition state infrared spectra for the trans-->cis isomerization of a simple peptide model.

Abstract: Trans-->cis isomerization of N-methylacetylamide (MeCO-NHMe) has been studied at the G3MP2B3 level of theory and the vibration spectrum has been calculated as a function of the torsional mode of motion along the peptide bond. Noticeable spectral differences have been observed for the transition state interconnecting the cis and trans isomers.