Evolutionary conservation and over-representation of functionally enriched network patterns in the yeast regulatory network

Background  

Localized network patterns are assumed to represent an optimal design principle in different biological networks. A widely used method for identifying functional components in biological networks is looking for network motifs – over-represented network patterns. A number of recent studies have undermined the claim that these over-represented patterns are indicative of optimal design principles and question whether localized network patterns are indeed of functional significance. This paper examines the functional significance of regulatory network patterns via their biological annotation and evolutionary conservation.     

Y chromosome lineage- and village-specific genes on chromosomes 1p22 and 6q27 control visceral leishmaniasis in Sudan

Familial clustering and ethnic differences suggest that visceral leishmaniasis caused by Leishmania donovani is under genetic control. A recent genome scan provided evidence for a major susceptibility gene on Chromosome 22q12 in the Aringa ethnic group in Sudan. We now report a genome-wide scan using 69 families with 173 affected relatives from two villages occupied by the related Masalit ethnic group. A primary ten-centimorgan scan followed by refined mapping provided evidence for major loci at 1p22 (LOD score 5.65; nominal p = 1.72 × 10⁻⁷; empirical p < 1 × 10⁻⁵; λ_S = 5.1) and 6q27 (LOD score 3.74; nominal p = 1.68 × 10⁻⁵; empirical p < 1 × 10⁻⁴; λ_S = 2.3) that were Y chromosome–lineage and village-specific. Neither village supported a visceral leishmaniasis susceptibility gene on 22q12. The results suggest strong lineage-specific genes due to founder effect and consanguinity in these recently immigrant populations. These chance events in ethnically uniform African populations provide a powerful resource in the search for genes and mechanisms that regulate this complex disease.     

Fc glycans terminated with N-acetylglucosamine residues increase antibody resistance to papain

Glycosylation in the CH2 domain of Fc is required for immunoglobulins G (IgGs) to exhibit immune effector functions including complement-dependent cytotoxicity (CDC) and antibody-dependent (Ab-dependent) cellular cytotoxicity (ADCC). We recently established that glycosylated Abs are more resistant to papain digestion than non-glycosylated IgGs (Biochem. Biophys. Res. Commun. 2006, 341, 797-803). To test whether specific Fc glycan structures affect Ab resistance to papain, we used in vitro glycoengineering methods to prepare homogeneous Ab glycoforms terminated with either sialic acid (G2S2), beta-galactose (G2), or N-acetylglucosamine (G0) and subjected them to papain digestions. Analyses of aliquots taken at different times during the digestions by matrix-assisted laser desorption-time-of-flight-mass spectroscopy (MALDI-TOF-MS) and high-performance liquid chromatography (HPLC) methods showed that the G0 glycoform was at least two times more resistant to papain digestion than the G2 and G2S2 glycoforms. The increased resistance of the G0 glycoform over the G2 and G2S2 glycoforms was independent of the specific Ab analyzed. A mouse/human chimeric version of Ab1, a fully human version of Ab2, and a humanized version of Ab3 exhibited a similar pattern of glycoform-dependent resistance. These data suggest that terminal sugars of Fc glycans may play important roles in Ab stability and affect resistance to proteases in addition to impacting Ab effector functions.     

Niacin deficiency modulates genes involved in cancer: Are smokers at higher risk?

The role of niacin’s metabolite, nicotinamide adenine dinucleotide (NAD), in DNA repair via base-excision repair pathway is well documented. We evaluated if niacin deficiency results in genetic instability in normal human fetal lung fibroblasts (MRC-5), and further, does it leads to enhanced accumulation of cigarette smoke–induced genetic damage? MRC-5 cells were grown discretely in niacin-proficient/deficient media, and exposed to nicotine-derived nitrosamine ketone (NNK, a cigarette smoke carcinogen). Niacin deficiency abated the NAD polymerization, augmented the spontaneous induction of micronuclei (MN) and chromosomal aberrations (CA) and raised the expression of 10 genes and suppressed 12 genes involved in different biological functions. NNK exposure resulted in genetic damage as measured by the induction of MN and CA in cells grown in niacin-proficient medium, but the damage became practically marked when niacin-deficient cells were exposed to NNK. NNK exposure raised the expression of 16 genes and suppressed the expression of 56 genes in cells grown in niacin-proficient medium. NNK exposure to niacin-deficient cells raised the expression of eight genes including genes crucial in promoting cancer such as FGFR3 and DUSP1 and suppressed the expression of 33 genes, including genes crucial in preventing the onset and progression of cancer like RASSF2, JUP, and IL24, in comparison with the cells grown in niacin-proficient medium. Overall, niacin deficiency interferes with the DNA damage repair process induced by chemical carcinogens like NNK, and niacin-deficient population are at the higher risk of genetic instability caused by cigarette smoke carcinogen NNK.     

Glycoengineering of therapeutic glycoproteins: in vitro galactosylation and sialylation of glycoproteins with terminal N-acetylglucosamine and galactose residues

Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.     

Analysis of unassisted translesion replication by the DNA polymerase III holoenzyme

DNA damage-induced mutations are formed when damaged nucleotides present in single-stranded DNA are replicated. We have developed a new method for the preparation of gapped plasmids containing site-specific damaged nucleotides, as model DNA substrates for translesion replication. Using these substrates, we show that the DNA polymerase III holoenzyme from Escherichia coli can bypass a synthetic abasic site analogue with high efficiency (30% bypass in 16 min), unassisted by other proteins. The theta and tau subunits of the polymerase were not essential for bypass. No bypass was observed when the enzyme was assayed on a synthetic 60-mer oligonucleotide carrying the same lesion, and bypass on a linear gapped plasmid was 3-4-fold slower than on a circular gapped plasmid. There was no difference in the bypass when standing-start and running-start replication were compared. A comparison of translesion replication by DNA polymerase I, DNA polymerase II, the DNA polymerase III core, and the DNA polymerase III holoenzyme clearly showed that the DNA polymerase III holoenzyme was by far the most effective in performing translesion replication. This was not only due to the high processivity of the pol III holoenzyme, because increasing the processivity of pol II by adding the gamma complex and beta subunit, did not increase bypass. These results support the model that SOS regulation was imposed on a fundamentally constitutive translesion replication reaction to achieve tight control of mutagenesis.     

Yeast and mammalian alpha-glucosidase inhibitory constituents from Himalayan rhubarb Rheum emodi Wall.ex Meisson

The methanolic extract of rhizome of Himalayan rhubarb Rheum emodi displayed mild yeast as well as mammalian intestinal alpha-glucosidase inhibitory activity. However, further fractionation of active extract led to the isolation of several potent molecules in excellent yields, displaying varying degrees of inhibition on two test models of alpha-glucosidase. Rhapontigenin, desoxyrhapontigenin, chrysophanol-8-O-beta-d-glucopyranoside, torachrysone-8-O-beta-d-glucopyranoside displayed potent yeast alpha-glucosidase inhibition. However chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside displayed potent to moderate mammalian alpha-glucosidase inhibitory activity. Other compounds displayed mild activity on both the tests. Except desoxyrhapontigenin and rhapontigenin that increased Vmax, other compounds including crude extract decreased the Vmax significantly (p<0.02) in yeast alpha-glucosidase test. Further kinetic analysis on mammalian alpha-glucosidase inhibition showed that chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside may be classified as mixed-noncompetitive inhibitors. However, desoxyrhapontigenin and rhapontigenin may be classified as modulators of enzyme activity. Presence and position of glycoside moiety in compounds appear important for better inhibition of mammalian alpha-glucosidase. This is the first report assigning particularly, mammalian intestinal alpha-glucosidase inhibitory activity to these compounds. Chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin, desoxyrhapontigenin and rhapontigenin have been isolated in substantial yields from R. emodi for the first time. Therefore, these compounds may have value in the treatment and prevention of hyperglycemia associated diabetes mellitus.     

DNA binding by yeast Mlh1 and Pms1: implications for DNA mismatch repair

The yeast Mlh1–Pms1 heterodimer required for mismatch repair (MMR) binds to DNA. Here we map DNA binding to N-terminal fragments of Mlh1 and Pms1. We demonstrate that Mlh1 and Pms1 N-terminal domains (NTDs) independently bind to double-stranded and single-stranded DNA, in the absence of dimerization and with different affinities. Full-length Mlh1p alone, which can homodimerize, also binds to DNA. Substituting conserved positively charged amino acids in Mlh1 produces mutator phenotypes in a haploid yeast strain characteristic of reduced MMR. These substitutions strongly reduce DNA binding by the Mlh1 NTD and, to a lesser extent, they also reduce DNA binding by full-length Mlh1 and the Mlh1–Pms1 heterodimer. Replacement of a homologous Pms1 residue has a much smaller effect on mutation rate and does not reduce DNA binding. The results demonstrate that NTDs of yeast Mlh1 and Pms1 contain independent DNA binding sites and they suggest that the C-terminal region of Mlh1p may also contribute to DNA binding. The differential mutator effects and binding properties observed here further suggest that Mlh1 and Pms1 differ in their interactions with DNA. Finally, the results are consistent with the hypothesis that DNA binding by Mlh1 is important for MMR.     

SANE,a novel LEM domain protein,regulates bone morphogenetic protein signaling through interaction with Smad1

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that play important roles in bone formation, embryonic patterning, and epidermal-neural cell fate decisions. BMPs signal through pathway specific mediators such as Smads1 and 5, but the upstream regulation of BMP-specific Smads has not been fully characterized. Here we report the identification of SANE (Smad1 Antagonistic Effector), a novel protein with significant sequence similarity to nuclear envelop proteins such as MAN1. SANE binds to Smad1/5 and to BMP type I receptors and regulates BMP signaling. SANE specifically blocks BMP-dependent signaling in Xenopus embryos and in a mammalian model of bone formation but does not inhibit the TGF-beta/Smad2 pathway. Inhibition of BMP signaling by SANE requires interaction between SANE and Smad1, because a SANE mutant that does not bind Smad1 does not inhibit BMP signaling. Furthermore, inhibition appears to be mediated by inhibition of BMP-induced Smad1 phosphorylation, blocking ligand-dependent nuclear translocation of Smad1. These studies define a new mode of regulation for intracellular BMP/Smad1 signaling.