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PubMed2003

Leucine-rich repeat-mediated intramolecular interactions in nematode recognition and cell death signaling by the tomato resistance protein Mi.

The root-knot nematode resistance gene Mi from tomato encodes a nucleotide-binding/leucine-rich repeat (NB/LRR) protein with a novel amino-terminal domain compared to related disease-resistance genes. The closely linked paralog Mi-1.1, which does not confer nematode resistance, encodes a protein 91% identical to the functional copy, Mi-1.2. The chimeric construct Mi-DS3, which encodes the 161 amino-terminal residues from Mi-1.1 fused to the remainder of Mi-1.2, induces localized necrosis when transiently expressed in Nicotiana benthamiana leaves. We produced mutant constructs that exchanged sequences encoding each of the 40 amino acid differences from the Mi-1.1 LRR region into Mi-DS3 and into Mi-1.2. For 23 of the substitutions, necrosis was lost upon transient expression of the mutated Mi-DS3 in N. benthamiana, and nematode resistance was lost when the altered Mi-1.2 was expressed in the tomato roots. One substitution, R961D, failed to give Mi-DS3-induced necrosis, but produced a dominant lethal phenotype when introduced into Mi-1.2. This gain-of-function phenotype was suppressed by co-expression with the amino-terminal region of Mi-1.1, suggesting that residue 961 is critical for negative regulation by the corresponding N-terminal region. Substitutions of Mi-1.1 residues 984-986 retained the ability to cause necrosis in Mi-DS3, but resulted in loss-of-nematode resistance in Mi-1.2, suggesting that these residues are essential for nematode recognition. None of the loss-of-function mutations in Mi-1.2 had a dominant negative phenotype. These results indicate that the Mi-1.2 LRR is involved in regulation of the transmission of the resistance response as well as in recognition of the nematode.

Amino Acid Motifs↗
PubMed1996

Interferon regulatory factor-2 directs transcription from the gp91phox promoter.

Repressor elements in the gp91(phox) promoter are necessary to restrict tissue-specific transcription to mature phagocytes. Deletion of these elements leads to significant promoter activity in cell lines such as HEL and K562 that do not normally express gp91(phox). The -100 to +12 base pair gp91(phox) promoter region is sufficient to direct maximal de-repressed transcription in these cells. However, promoter activity is dramatically decreased following a 16-base pair truncation that deletes an interferon-stimulated response element. This element interacts with IRF-1 and IRF-2, members of the interferon regulatory factor family of transcription factors. In addition, this promoter region is bound by a factor with properties similar to BID, a DNA-binding protein that also interacts with three upstream sites within the gp91(phox) promoter. Transient transfection studies using mutated promoters indicate that both the IRF and BID binding sites are required for maximal gp91(phox) promoter activity. Overexpression of IRF-1 or IRF-2 in K562 cells leads to transactivation of gp91(phox) promoter constructs, which is dependent on the presence of an intact IRF binding site. IRF-2 predominates in macrophages that express the gp91(phox) gene as well as in HEL and K562 cells. We conclude that IRF-2 and BID activate gp91(phox) promoter activity in the absence of transcriptional repression.

Binding Sites↗
PubMed1998

UGA codon position affects the efficiency of selenocysteine incorporation into glutathione peroxidase-1.

A UGA codon and a selenocysteine insertion sequence in the 3'-untranslated region are the only established mRNA elements necessary for selenocysteine (Sec or U) incorporation during translation. These two elements, however, do not universally confer efficient Sec incorporation. The objective of this study was to systematically examine the effect of UGA codon position on efficiency of Sec insertion. In a glutathione peroxidase-1 (F-GPX1) expression vector, the UGA at the native position (U47) was mutated to a cysteine codon, and codons for Ser-7, Ser-12, Ser-18, Ser-29, Ser-45, Ser-93, Cys-154, Val-172, Ser-178, and Ser-195 were individually mutated to UGA and transiently expressed in COS-7 cells. 75Se incorporation at the 11 positions was 31, 72, 54, 105, 90, 100, 146, 135, 13, 11, and 43%, respectively, of 75Se incorporation at U47, suggesting that Sec is more efficiently incorporated at UGA codons positioned in the middle of the coding region rather than close to the 5' or 3' ends. Ribonuclease protection showed that these differences were not due to differences in mRNA level. When the green fluorescence protein (GFP) coding region was placed in-frame at the 5' or 3' ends of the coding region in F-GPX1 to produce chimeric 50-51-kDa GFP/GPX1 proteins, Sec incorporation at UGA codons, formerly close to the 5' or 3' ends, was increased to levels comparable to the UGA at U47. Insertion of GFP after the UAA-stop was just as effective in increasing Sec insertion efficiency as GFP inserted before the stop. These studies used a recombinant expression model that incorporated Sec at non-native UGA codons at rates equal to those of endogenous glutathione peroxidase-1 and showed that the efficiency of Sec incorporation can be modulated by UGA position; Sec incorporation at high efficiency appears to require that the UGA be >21 nucleotides from the AUG-start and >204 nucleotides from the selenocysteine insertion sequence element.

3' Untranslated Regions↗
PubMed2000

Interaction of nectin with afadin is necessary for its clustering at cell-cell contact sites but not for its cis dimerization or trans interaction.

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, a homophilic cell adhesion molecule, and afadin, an actin filament-binding protein, which connects nectin to the actin cytoskeleton. Here we studied a mechanism of cell-cell adhesion of the nectin-afadin system by use of a cadherin-deficient L cell line stably expressing the intact form of mouse nectin-2alpha, a truncated form of nectin-2alpha incapable of interacting with afadin (nectin-2alpha-DeltaC), or a point-mutated form of nectin-2alpha capable of interacting with afadin and a cadherin-expressing EL cell line, which transiently expressed the point-mutated form of nectin-2alpha. We found that the interaction of nectin-2alpha with afadin was necessary for their clustering at cell-cell contact sites. However, nectin-2alpha-DeltaC showed cis dimerization and trans interaction, both of which did not require the interaction of nectin-2alpha with afadin. We have previously shown in EL cells that the interaction of nectin-1 with afadin is necessary for its recruitment to adherens junctions. We found that the trans interaction of nectin-2alpha was furthermore necessary for this recruitment. On the basis of these observations, we propose a model for the mechanism of cell-cell adhesion of nectin and roles of afadin in this mechanism.

Animals↗
PubMed2000

NF-IL6 (C/EBPbeta ) vigorously activates il1b gene expression via a Spi-1 (PU.1) protein-protein tether.

Two classes of transcription factors, ETS and bZIP, stand out as key mediators of monocyte commitment and differentiation. The ETS domain factor Spi-1 (also called PU.1) and the bZIP factor NF-IL6 (also called C/EBPbeta) have been shown to be involved in the transcriptional regulation of interleukin-1beta gene (il1b) and other monocyte-specific genes. We now show that these two factors strongly cooperate on the il1b core promoter (-59/+12) in the absence of direct NF-IL6 binding to DNA. Transient transfection assays, using mutated il1b core promoters, showed that the Spi-1, but not the NF-IL6, binding site is absolutely required for functional cooperativity. Furthermore, the NF-IL6 transactivation domain (TAD) is functionally indispensable and more critical than that of Spi-1. Additionally, TAD-deficient NF-IL6 functions as a dominant negative for Spi-1-mediated activation, suggesting the involvement of the bZIP DNA binding domain. This is supported by the demonstration of in vitro interaction between the NF-IL6 bZIP and Spi-1 winged helix-turn-helix (wHTH) DNA binding domains, arguing that NF-IL6 vigorously activates the il1b core promoter via protein-tethered transactivation mediated by Spi-1.

Base Sequence↗
PubMed2001

Glycosylation signals that separate the trimerization from the mhc class II-binding domain control intracellular degradation of invariant chain.

Invariant chain (Ii) serves as a chaperone for folding and intracellular transport of major histocompatibility complex class II (MHCII) molecules. Early in biosynthesis, Ii associates with MHCII molecules and directs their intracellular transport to endocytic compartments where vesicular proteinases sequentially release Ii from the MHCII heterodimer. The detachment of Ii makes the MHCII groove susceptible for binding of antigenic peptides. We investigated the role of N-linked glycosylation in the controlled intracellular degradation of Ii. Motifs for asparagine-linked glycosylation were altered, and mutated Ii (IiNmut) was transiently expressed in COS cells. The half-life of IiNmut was strongly reduced compared with wild-type Ii although the sensitivity of the N glycan-free polypeptide to in vitro proteinase digestion was not substantially increased. Inhibition of vesicular proteinases revealed endosomal degradation of IiNmut. Intracellular proteolysis of IiNmut is substantially impaired by serine proteinase inhibitors. Thus, a considerable amount of IiNmut is degraded in nonacidic intracellular compartments. The data suggest that N-linked glycosylation of Ii hinders premature proteolysis in nonacidic vesicles resulting in Ii degradation in acidic MHC class II-processing compartments.

Amino Acid Sequence↗
PubMed2003

Farnesoid X receptor regulates bile acid-amino acid conjugation.

The farnesoid X receptor (FXR; NR1H4) regulates bile acid and lipid homeostasis by acting as an intracellular bile acid-sensing transcription factor. Several identified FXR target genes serve critical roles in the synthesis and transport of bile acids as well as in lipid metabolism. Here we used Affymetrix micro-array and Northern analysis to demonstrate that two enzymes involved in conjugation of bile acids to taurine and glycine, namely bile acid-CoA synthetase (BACS) and bile acid-CoA: amino acid N-acetyltransferase (BAT) are induced by FXR in rat liver. Analysis of the human BACS and BAT genes revealed the presence of functional response elements in the proximal promoter of BACS and in the intronic region between exons 1 and 2 of the BAT gene. The response elements resemble the consensus FXR binding site consisting of two nuclear receptor half-sites organized as an inverted repeat and separated by a single nucleotide (IR-1). These response elements directly bind FXR/retinoid X receptor (RXR) heterodimers and confer the activity of FXR ligands in transient transfection experiments. Further mutational analysis confirms that the IR-1 sequence of the BACS and BAT genes mediate transactivation by FXR/RXR heterodimers. Finally, Fisher rats treated with the synthetic FXR ligand GW4064 clearly show increased transcript levels of both the BACS and BAT mRNA. These studies demonstrate a mechanism by which FXR regulates bile acid amidation, a critical component of the enterohepatic circulation of bile acids.

Acyltransferases↗
PubMed2006

Estrogen receptor isoform-specific regulation of the retinoblastoma-binding protein 1 (RBBP1) gene: roles of AF1 and enhancer elements.

Estrogen (E2) is involved in mediating many important functions relevant to osteoblast biology through the actions of the estrogen receptors (ER) alpha and beta. To further understand the mechanisms of ER-specific regulation, we used microarray and reverse transcription-PCR analyses of E2-treated U2OS-ERalpha or -ERbeta cells and identified retinoblastoma-binding protein 1 (RBBP1) as a major E2-regulated gene. RBBP1 is a retinoblastoma cofactor involved in the control of osteoblastic proliferation. Although RBBP1 mRNA levels rapidly increased after 2 h of E2 treatment in both U2OS-ER-expressing lines, a sustained induction was only observed in U2OS-ERalpha cells. Examination of the RBBP1 genomic sequence revealed an ER response element and a Sp1 site located within the first intron. Chromatin immunoprecipitation analyses demonstrated that E2-dependent ERalpha binding to the intron 1 enhancer region was constitutive, whereas ERbeta binding was transient, consistent with the mRNA time course. Interestingly, transient transfection and receptor mutational studies revealed that RBBP1 induction by ERalpha only requires the Sp1 site, whereas ERbeta utilizes both the Sp1 and estrogen response elements binding sites for maximal E2-dependent activation. Stable U2OS transfectants containing a deletion of the ERalpha activation function 1 (AF1) resulted in a temporal mRNA induction profile similar to that of wild type ERbeta. Further, overexpression and chromatin immunoprecipitation analyses also demonstrated that E2-dependent RBBP1 induction is SRC2-dependent for both ER isoforms. These results describe an E2-dependent, ER isoform-specific transcriptional activation of the RBBP1 gene, which in part, is explained by the differential activity of ER AF1 and enhancer element binding.

Base Sequence↗
PubMed2005

Polymorphism of the PEMT gene and susceptibility to nonalcoholic fatty liver disease (NAFLD).

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes phosphatidylcholine synthesis. PEMT knockout mice have fatty livers, and it is possible that, in humans, nonalcoholic fatty liver disease (NAFLD) might be associated with PEMT gene polymorphisms. DNA samples from 59 humans without fatty liver and from 28 humans with NAFLD were genotyped for a single nucleotide polymorphism in exon 8 of PEMT, which leads to a V175M substitution. V175M is a loss of function mutation, as determined by transiently transfecting McArdle-RH7777 cells with constructs of wild-type PEMT open reading frame or the V175M mutant. Met/Met at residue 175 (loss of function SNP) occurred in 67.9% of the NAFLD subjects and in only 40.7% of control subjects (P<0.03). For the first time we report that a polymorphism of the human PEMT gene (V175M) is associated with diminished activity and may confer susceptibility to NAFLD.

Adolescent↗
PubMed2004

Infected cell protein 0 encoded by bovine herpesvirus 1 can activate caspase 3 when overexpressed in transfected cells.

Infection of cattle or bovine cells with bovine herpesvirus 1 (BHV-1) leads to increased apoptosis. Previous studies indicated that BHV-1 infected cell protein 0 (bICP0), the major transcriptional regulatory protein of BHV-1, is toxic in transiently transfected cells. Point mutations within the zinc RING finger of bICP0 reduced toxicity and eliminated the ability of bICP0 to activate viral gene expression. In mouse neuroblastoma cells (neuro-2A) and bovine turbinate cells, bICP0 activated caspase 3, a key regulatory protein in the apoptotic pathway. A pro-apoptotic gene (Bax), but not bICP0, induced caspase 3 cleavage and activation by 8 h after transfection of neuro-2A cells. Conversely, bICP0 or the N-terminal 356 aa of bICP0 did not induce caspase 3 cleavage in neuro-2A cells until 30 h after transfection, suggesting that bICP0 stimulates caspase 3 cleavage by an indirect mechanism. These studies indicate that the toxic functions of bICP0 correlate with caspase 3 cleavage and activation.

Amino Acid Sequence↗
PubMed2004

Roles of the two Drosophila CRYPTOCHROME structural domains in circadian photoreception.

CRYPTOCHROME (CRY) is the primary circadian photoreceptor in Drosophila. We show that CRY binding to TIMELESS (TIM) is light-dependent in flies and irreversibly commits TIM to proteasomal degradation. In contrast, CRY degradation is dependent on continuous light exposure, indicating that the CRY-TIM interaction is transient. A novel cry mutation (cry(m)) reveals that CRY's photolyase homology domain is sufficient for light detection and phototransduction, whereas the carboxyl-terminal domain regulates CRY stability, CRY-TIM interaction, and circadian photosensitivity. This contrasts with the function of Arabidopsis CRY domains and demonstrates that insect and plant cryptochromes use different mechanisms.

Animals↗
PubMed2000

Delavirdine in combination with zidovudine in treatment of human immunodeficiency virus type 1-infected patients: evaluation of efficacy and emergence of viral resistance in a randomized, comparative phase III trial. The M/3331/0013B Study Group.

We compared the activity of delavirdine (DLV) plus zidovudine (AZT) (n = 300) with that of AZT (n = 297) against human immunodeficiency virus type 1 in a randomized, double-blind, placebo-controlled trial. DLV exerted a transient antiviral effect, and mutations for resistance to DLV were found in more than 90% of subjects at week 12. The K103N mutation, which confers nonnucleoside reverse transcriptase inhibitor cross-resistance, was found in 85% of the patients.

Anti-HIV Agents↗
PubMed1989

Biosynthesis of hepatitis B virus e antigen: directed mutagenesis of the putative aspartyl protease site.

The C gene products of all mammalian hepadnaviruses contain a region with sequence similarities to the catalytic center of the aspartyl proteases. This region could have the capacity to cleave precore proteins, leading to the synthesis of e antigen. By site-directed mutagenesis on a plasmid containing the hepatitis B virus C gene, we have replaced either the Asp residue of the putative aspartyl protease catalytic center or an Asp residue located 3 amino acids upstream. Transient expression of the mutated hepatitis B virus C gene in human and mouse cells showed that none of these mutations prevented the secretion of an accurately processed HBe antigen. Thus, we demonstrated that the aspartyl protease responsible for e antigen precursor processing is not C gene encoded but is more likely to be a cellular enzyme. From these results, we suggest a model for the mechanism of e antigen synthesis.

Amino Acid Sequence↗
PubMed1995

Mutational analysis of the 18-base-pair inverted repeat element at the bovine papillomavirus origin of replication: identification of critical sequences for E1 binding and in vivo replication.

Replication of bovine papillomavirus requires two viral proteins, E1 and E2-TA. Previously we demonstrated that sequences within an imperfect 18-bp inverted repeat (IR) element were sufficient to confer specific binding of the E1 protein to the origin region (S. E. Holt, G. Schuller, and V. G. Wilson, J. Virol. 68:1094-1102, 1994). To identify critical nucleotides for E1 binding and origin function, a series of individual point mutations was constructed at each nucleotide position in the 18-bp IR. Binding of E1 to these point mutations established that both the position of the mutation and the specific nucleotide change were important for the E1-DNA interaction. Equivalent mutations from each half of the IR exhibited similar binding, suggesting that the halves were functionally symmetric for E1 interactions. Each of these mutations was evaluated also for origin function in vivo by a transient-replication assay. No single point mutation eliminated replication capacity completely, though many mutants were severely impaired, demonstrating an important functional contribution for the E1 binding site. Furthermore, E1 binding was not sufficient for replication, as several origin mutants bound E1 well in vitro but replicated poorly in vivo. This suggests that certain nucleotides within the 18-bp IR may be involved in postbinding events necessary for replication initiation. The results with the point mutations suggest that E1-E1 interactions are important for stable complex formation and also indicate that there is some flexibility with regard to formation of a functional E1 replication complex at the origin.

Adenovirus E1A Proteins↗
PubMed1996

E2 represses the late gene promoter of human papillomavirus type 8 at high concentrations by interfering with cellular factors.

The late gene promoter P7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is regulated by the viral E2 protein. Transfection experiments performed with the human skin keratinocyte cell line RTS3b and P7535 reporter plasmids revealed transactivation at low amounts and a repression of basal promoter activity at high amounts of E2 expression vector. This repression was promoter specific and correlated with the amount of transiently expressed E2 protein. Mutational analyses revealed that the negative regulation of P7535 activity is mediated by the low-affinity E2 binding site P2, which is separated by one nucleotide from the P7535 TATA box. Biochemical and genetic analyses suggested that repression is due to a displacement of the TATA-box binding protein by E2 and an interference of E2 with promoter-activating cellular factors that specifically recognize the P2 sequence. The high conservation of the P2 sequence among several papillomaviruses (epidermodysplasia verruciformis-associated HPVs, HPV1, cottontail rabbit papillomavirus, and bovine papillomavirus type 1) in the vicinity of the late gene promoter cap site suggests that an interplay of E2 and cellular factors at this sequence element is important for the expression of structural proteins.

Animals↗
PubMed1991

Sequences and factors required for the F9 embryonal carcinoma stem cell E1a-like activity.

F9 embryonal carcinoma (EC) stem cells contain an E1a-like activity that is absent from differentiated derivatives. We have previously characterized proteins present in F9 EC cell extracts that bind to the E1a-dependent E2A promoter and have shown that two of them, TF68 and DRTF1, are required for efficient transcription in vitro (N. B. La Thangue, B. Thimmapaya, and P. W. J. Rigby, Nucleic Acids Res. 18:2929-2938, 1990). We now show that the E1a-like activity is detectable in transient transfection assays. Deletion mutations show that a distal sequence element, which includes the ATF/CREB consensus, is required for expression in both cell types, although it does not mediate the down-regulation of promoter activity that accompanies differentiation. A series of point mutations generated by in vitro mutagenesis confirm this and show that sequences around -60 are necessary for efficient expression in stem cells but not in differentiated derivatives. These sequences bind DRTF1, the activity of which is strongly down-regulated during differentiation. Surprisingly, mutations in a previously uncharacterized region of the promoter restore activity to a promoter carrying the -60 mutation and lead to the formation of a new DNA-protein complex.

Adenovirus Early Proteins↗