Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Circular plasmids containing telomeric TG1-3 arrays or the HMR E silencer segregate efficiently between dividing cells of the yeast Saccharomyces cerevisiae. Subtelomeric X repeats augment the TG1-3 partitioning activity by a process that requires the SIR2, SIR3, and SIR4 genes, which are also required for silencer-based partitioning. Here we show that targeting Sir4p to DNA directly via fusion to the bacterial repressor LexA confers efficient mitotic segregation to otherwise unstable plasmids. The Sir4p partitioning activity resides within a 300-amino-acid region (residues 950 to 1262) which precedes the coiled-coil dimerization motif at the extreme carboxy end of the protein. Using a topology-based assay, we demonstrate that the partitioning domain also retards the axial rotation of LexA operators in vivo. The anchoring and partitioning properties of LexA-Sir4p chimeras persist despite the loss of the endogenous SIR genes, indicating that these functions are intrinsic to Sir4p and not to a complex of Sir factors. In contrast, inactivation of the Sir4p-interacting protein Rap1p reduces partitioning by a LexA-Sir4p fusion. The data are consistent with a model in which the partitioning and anchoring domain of Sir4p (PAD4 domain) attaches to a nuclear component that divides symmetrically between cells at mitosis; DNA linked to Sir4p by LexA serves as a reporter of protein movement in these experiments. We infer that the segregation behavior of telomere- and silencer-based plasmids is, in part, a consequence of these Sir4p-mediated interactions. The assays presented herein illustrate two novel approaches to monitor the intracellular dynamics of nuclear proteins.
Mol Cell Biol 1997 Dec
PMID:The yeast silent information regulator Sir4p anchors and partitions plasmids. 937 37

Many retrotransposons and retroviruses are thought to select integration sites through interactions with specific chromosomal proteins. In yeast, the Ty5 retrotransposon integrates preferentially with regions bound by silent chromatin, namely the telomeres and the HMR and HML mating loci. A Ty5 mutant (M3) was identified with an approximately 20-fold decrease in targeted integration as measured by a plasmid-based targeting assay. Often chromosomal insertions generated by M3, none were located at the telomeres or silent mating loci. A single amino acid change at the boundary of integrase and reverse transcriptase is responsible for the mutant phenotype. We predict that this mutation lies within a targeting domain that mediates Ty5 target choice by interacting with a component of silent chromatin.
Mol Cell 1998 Jun
PMID:A single amino acid change in the yeast retrotransposon Ty5 abolishes targeting to silent chromatin. 965 88

Silent information regulator 3 is an essential component of the Saccharomyces cerevisiae silencing complex that functions at telomeres and the silent mating-type loci, HMR and HML. We show that expression of the N- and C-terminal-encoding halves of SIR3 in trans partially complements the mating defect of the sir3 null allele, suggesting that the two domains have distinct functions. We present here a functional characterization of these domains. The N-terminal domain (Sir3N) increases both the frequency and extent of telomere-proximal silencing when expressed ectopically in SIR+ yeast strains, although we are unable to detect interaction between this domain and any known components of the silencing machinery. In contrast to its effect at telomeres, Sir3N overexpression derepresses transcription of reporter genes inserted in the ribosomal DNA (rDNA) array. Immunolocalization of Sir3N-GFP and Sir2p suggests that Sir3N directly antagonizes nucleolar Sir2p, releasing an rDNA-bound population of Sir2p so that it can enhance repression at telomeres. Overexpression of the C-terminal domain of either Sir3p or Sir4p has a dominant-negative effect on telomeric silencing. In strains overexpressing the C-terminal domain of Sir4p, elevated expression of either full-length Sir3p or Sir3N restores repression and the punctate pattern of Sir3p and Rap1p immunostaining. The similarity of Sir3N and Sir3p overexpression phenotypes suggests that Sir3N acts as an allosteric effector of Sir3p, either enhancing its interactions with other silencing components or liberating the full-length protein from nonfunctional complexes.
Mol Cell Biol 1998 Oct
PMID:Functional characterization of the N terminus of Sir3p. 974 28

Transcriptional silencing in Saccharomyces cerevisiae occurs at several genetic loci, including the ribosomal DNA (rDNA). Silencing at telomeres (telomere position effect [TPE]) and the cryptic mating-type loci (HML and HMR) depends on the silent information regulator genes, SIR1, SIR2, SIR3, and SIR4. However, silencing of polymerase II-transcribed reporter genes integrated within the rDNA locus (rDNA silencing) requires only SIR2. The mechanism of rDNA silencing is therefore distinct from TPE and HM silencing. Few genes other than SIR2 have so far been linked to the rDNA silencing process. To identify additional non-Sir factors that affect rDNA silencing, we performed a genetic screen designed to isolate mutations which alter the expression of reporter genes integrated within the rDNA. We isolated two classes of mutants: those with a loss of rDNA silencing (lrs) phenotype and those with an increased rDNA silencing (irs) phenotype. Using transposon mutagenesis, lrs mutants were found in 11 different genes, and irs mutants were found in 22 different genes. Surprisingly, we did not isolate any genes involved in rRNA transcription. Instead, multiple genes associated with DNA replication and modulation of chromatin structure were isolated. We describe these two gene classes, and two previously uncharacterized genes, LRS4 and IRS4. Further characterization of the lrs and irs mutants revealed that many had alterations in rDNA chromatin structure. Several lrs mutants, including those in the cdc17 and rfc1 genes, caused lengthened telomeres, consistent with the hypothesis that telomere length modulates rDNA silencing. Mutations in the HDB (RPD3) histone deacetylase complex paradoxically increased rDNA silencing by a SIR2-dependent, SIR3-independent mechanism. Mutations in rpd3 also restored mating competence selectively to sir3Delta MATalpha strains, suggesting restoration of silencing at HMR in a sir3 mutant background.
Mol Cell Biol 1999 Apr
PMID:A genetic screen for ribosomal DNA silencing defects identifies multiple DNA replication and chromatin-modulating factors. 1008 85

The macrolide antibiotic erythromycin and its 6-O-methyl derivative (clarithromycin) bind to bacterial ribosomes primarily through interactions with nucleotides in domains II and V of 23S rRNA. The domain II interaction occurs between nucleotide A752 and the macrolide 3-cladinose moiety. Removal of the cladinose, and substitution of a 3-keto group (forming the ketolide RU 56006), results in loss of the A752 interaction and an approximately 100-fold drop in drug binding affinity. Within domain V, the key determinant of drug binding is nucleotide A2058 and substitution of G at this position is the major cause of drug resistance in some clinical pathogens. The 2058G mutation disrupts the drug-domain V contact and leads to a further > 25 000-fold decrease in the binding of RU 56006. Drug binding to resistant ribosomes can be improved over 3000-fold by forming an alternative and more effective contact to A752 via alkyl-aryl groups linked to a carbamate at the drug 11/12 position (in the ketolide antibiotics HMR 3647 and HMR 3004). The data indicate that simultaneous drug interactions with domains II and V strengthen binding and that the domain II contact is of particular importance to achieve binding to the ribosomes of resistant pathogens in which the domain V interaction is perturbed.
Mol Microbiol 2000 Apr
PMID:Macrolide-ketolide inhibition of MLS-resistant ribosomes is improved by alternative drug interaction with domain II of 23S rRNA. 1076 Jan 75

Silencing at HMR requires silencers, and one of the roles of the silencer is to recruit Sir proteins. This work focuses on the function of Sir1p once it is recruited to the silencer. We have generated mutants of Sir1p that are recruited to the silencer but are unable to silence, and we have utilized these mutants to identify four proteins, Sir3p, Sir4p, Esc2p, and Htz1p, that when overexpressed, restored silencing. The isolation of Sir3p and Sir4p validated this screen. Molecular analysis suggested that Esc2p contributed to silencing in a manner similar to Sir1p and probably helped recruit or stabilize the other Sir proteins, while Htz1p present at HMR assembled a specialized chromatin structure necessary for silencing.
Mol Cell 2000 Oct
PMID:A histone variant, Htz1p, and a Sir1p-like protein, Esc2p, mediate silencing at HMR. 1109 Jun 16

Many mammalian cells have two distinct types of ATP-sensitive potassium (K(ATP)) channels: the classic ones in the surface membrane (sK(ATP)) and others in the mitochondrial inner membrane (mitoK(ATP)). Cardiac mitoK(ATP) channels play a pivotal role in ischemic preconditioning, and thus represent interesting drug targets. Unfortunately, the molecular structure of mitoK(ATP) channels is unknown, in contrast to sK(ATP) channels, which are composed of a pore-forming subunit (Kir6.1 or Kir6.2) and a sulfonylurea receptor (SUR1, SUR2A, or SUR2B). As a means of probing the molecular makeup of mitoK(ATP) channels, we compared the pharmacology of native cardiac mitoK(ATP) channels with that of molecularly defined sK(ATP) channels expressed heterologously in human embryonic kidney 293 cells. Using mitochondrial oxidation to index mitoK(ATP) channel activity in rabbit ventricular myocytes, we found that pinacidil and diazoxide open mitoK(ATP) channels, but P-1075 does not. On the other hand, 5-hydroxydecanoic acid (5HD), but not HMR-1098, blocks mitoK(ATP) channels. Although pinacidil is a nonselective activator of expressed sK(ATP) channels, diazoxide did not open channels formed by Kir6.1/SUR2A, Kir6.2/SUR2A (known components of cardiac sK(ATP) channels) or Kir6.2/SUR2B. P-1075 activated all the K(ATP) channels, except Kir6.1/SUR1 channels. Glybenclamide potently blocked all sK(ATP) channels, but 5HD only blocked channels formed by SUR1/Kir6.1 or Kir6.2 (IC(50)s of 66 and 81 microM, respectively). This potency is similar to that for block of mitoK(ATP) channels (IC(50) = 95 microM). In addition, HMR-1098 potently blocked Kir6.2/SUR2A channels (IC(50) = 1.5 microM), but was 67 times less potent in blocking Kir6.1/SUR1 channels (IC(50) = 100 microM). Our results demonstrate that mitoK(ATP) channels closely resemble Kir6.1/SUR1 sK(ATP) channels in their pharmacological profiles.
Mol Pharmacol 2001 Feb
PMID:Pharmacological comparison of native mitochondrial K(ATP) channels with molecularly defined surface K(ATP) channels. 1116 Aug 57

The ability to form selective cell-cell adhesions is an essential property of metazoan cells. Members of the cadherin superfamily are important regulators of this process in both vertebrates and invertebrates. With the advent of genome sequencing projects, determination of the full repertoire of cadherins available to an organism is possible and here we present the identification and analysis of the cadherin repertoires in the genomes of Caenorhabditis elegans and Drosophila melanogaster. Hidden Markov models of cadherin domains were matched to the protein sequences obtained from the translation of the predicted gene sequences. Matches were made to 21 C. elegans and 18 D. melanogaster sequences. Experimental and theoretical work on C. elegans sequences, and data from ESTs, show that three pairs of genes, and two triplets, should be merged to form five single genes. It also produced sequence changes at one or both of the 5' and 3' termini of half the sequences. In D. melanogaster it is probable that two of the cadherin genes should also be merged together and that three cadherin genes should be merged with other neighbouring genes. Of the 15 cadherin proteins found in C. elegans, 13 have the features of cell surface proteins, signal sequences and transmembrane helices; the other two have only signal sequences. Of the 17 in D. melanogaster, 11 at present have both features and another five have transmembrane helices. The evidence currently available suggests about one-third of the cadherins in the two organisms can be grouped into subfamilies in which all, or parts of, the molecules are conserved. Each organism also has a approximately 980 residue protein (CDH-11 and CG11059) with two cadherin domains and whose sequences match well over their entire length two proteins from human brain. Two proteins in C. elegans, HMR-1A and HMR-1B, and three in D. melanogaster, CadN, Shg and CG7527, have cytoplasmic domains homologous to those of the classical cadherin genes of chordates but their extracellular regions have different domain structures. Other common subclasses include the seven-helix membrane cadherins, Fat-like protocadherins and the Ret-like cadherins. At present, the remaining cadherins have no obvious similarities in their extracellular domain architecture or homologies to their cytoplasmic domains and may, therefore, represent species-specific or phylum-specific molecules.
J Mol Biol 2001 Feb 02
PMID:Cadherin superfamily proteins in Caenorhabditis elegans and Drosophila melanogaster. 1116 10

In the yeast Saccharomyces cerevisiae, a and alpha mating-type information is stored in transcriptionally silenced cassettes called HML and HMR. Silencing of these loci, maintained by the formation of a specialized type of heterochromatin, requires trans-acting proteins and cis-acting elements. Proteins required for silencing include the Sir2 NAD(+)-dependent deacetylase, Sir3, and Sir4. Factors that bind to the cis elements at HMR and HML and that are important for silencing include the origin recognition complex (ORC). Mutations of any of these Sir proteins or combinations of cis elements result in loss of silencing. SUM1-1 was previously identified as a dominant mutation that restores silencing to HMR in the absence of either the Sir proteins or some of the cis elements. We have investigated the novel mechanism whereby Sum1-1 causes Sir-independent silencing at HMR and present the following findings: Sum1-1 requires the Sir2 homolog, Hst1, for silencing and most probably requires the NAD(+)-dependent deacetylase activity of this protein. Sum1-1 interacts strongly with ORC, and this strong interaction is dependent on HMR DNA. Furthermore, ORC is required for Sum1-1-mediated silencing at HMR. These observations lead to a model for Sum1-1 silencing of HMR in which Sum1-1 is recruited to HMR by binding to ORC. Sum1-1, in turn, recruits Hst1. Hst1 then deacetylates histones or other chromatin-associated proteins to cause chromatin condensation and transcriptional silencing.
Mol Cell Biol 2001 May
PMID:A novel form of transcriptional silencing by Sum1-1 requires Hst1 and the origin recognition complex. 1131 77

X. Kong, J. S. Tweddell, G. J. Gross and J. E. Baker. Sarcolemmal and Mitochondrial K(ATP)Channels Mediate Cardioprotection in Chronically Hypoxic Hearts. Journal of Molecular and Cellular Cardiology (2001) 33, 1041-1045. Hypoxia from birth increases the resistance of the isolated neonatal heart to ischemia. We determined if increased resistance to ischemia was due to activation of sarcolemmal or mitochondrial K(ATP)channels. Rabbits (n=8/group) were raised from birth in a normoxic (F(I)O(2)=0.21) or hypoxic (F(I)O(2)=0.12) environment for 8-10 days and the heart perfused with Krebs-Henseleit bicarbonate buffer. A mitochondrial-selective K(ATP)channel blocker 5-hydroxydecanoate (5-HD) (300 micromol/l) or a sarcolemmal-selective K(ATP)channel blocker HMR 1098 (30 micromol/l) were added alone or in combination for 20 min prior to a global ischemic period of 30 min, followed by 35 min reperfusion. Recovery of ventricular developed pressure was higher in chronically hypoxic than normoxic hearts. 5-HD and HMR 1098 partially reduced the cardioprotective effect of chronic hypoxia, but had no effect in normoxic hearts. The combination of 5-HD and HMR 1098 abolished the cardioprotective effect of chronic hypoxia. We conclude that both sarcolemmal and mitochondrial K(ATP)channels contribute to cardioprotection in the chronically hypoxic heart.
J Mol Cell Cardiol 2001 May
PMID:Sarcolemmal and mitochondrial K(atp)channels mediate cardioprotection in chronically hypoxic hearts. 1134 25


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