The RING ubiquitin E3 RNF114 interacts with A20 and modulates NF-κB activity and T-cell activation.

Accurate regulation of nuclear factor-κB (NF-κB) activity is crucial to prevent a variety of disorders including immune and inflammatory diseases. Active NF-κB promotes IκBα and A20 expression, important negative regulatory molecules that control the NF-κB response. In this study, using two-hybrid screening we identify the RING-type zinc-finger protein 114 (RNF114) as an A20-interacting factor. RNF114 interacts with A20 in T cells and modulates A20 ubiquitylation. RNF114 acts as negative regulator of NF-κB-dependent transcription, not only by stabilizing the A20 protein but also IκBα. Importantly, we demonstrate that in T cells, the effect of RNF114 is linked to the modulation of T-cell activation and apoptosis but is independent of cell cycle regulation. Altogether, our data indicate that RNF114 is a new partner of A2O involved in the regulation of NF-κB activity that contributes to the control of signaling pathways modulating T cell-mediated immune response.

Self-assembly of phosphate amphiphiles in mixtures of prebiotically plausible surfactants.

The spontaneous formation of closed bilayer structures from prebiotically plausible amphiphiles is an essential requirement for the emergence of early cells on prebiotic Earth. The sources of amphiphiles could have been both endo- and exogenous (accretion of meteorite carbonaceous material or interstellar dust particles). Among all prebiotic possible amphiphile candidates, those containing phosphate are the least investigated species because their self-assembly occurs in a seemingly too narrow range of conditions. The self-assembly of simple phosphate amphiphiles should, however, be of great interest, as contemporary membranes predominantly contain phospholipids. In contrast to common expectations, we show that these amphiphiles can be easily synthesized under prebiotically plausible environmental conditions and can efficiently form bilayer structures in the presence of various co-surfactants across a large range of pH values. Vesiculation was even observed in crude reaction mixtures that contained 1-decanol as the amphiphile precursor. The two best co-surfactants promoted vesicle formation over the entire pH range in aqueous solutions. Expanding the pH range where bilayer membranes self-assemble and remain intact is a prerequisite for the emergence of early cell-like compartments and their preservation under fluctuating environmental conditions. These mixed bilayers also retained small charged solutes, such as dyes. These results demonstrate that alkyl phosphate amphiphiles might have played a significant role as early compartment building blocks.

Mutational analysis of the kinetics and thermodynamics of transcription factor NF-kappaB homodimerisation.

Dimeric transcription factors of the NF-kappaB/Rel family are sequence-specific DNA-binding proteins that mediate the inducible expression of immunologically important eukaryotic genes by competing for kappaB sites. The kinetic and thermodynamic components of these interactions were probed by mutation of the subunit interface of the p50 homodimer, a paradigm for other family members. Guided by the crystal structure, we selected the side chains of five key residues (R255, Y270, L272, A311 and V313) for individual and combinatorial truncation, with the aim of generating a mutant panel. Homodimerisation was assessed indirectly by measurement of DNA binding with an optical biosensor in order to unmask the relative contributions of each residue. Surface plasmon resonance revealed that a unanimous bias for a palindromic kappaB site over an asymmetric one was mainly the result of a slower dissociation rate for the DNA/homodimer complex in the case of the palindromic kappaB site. Y270 and L272 were individually the most critical residues in homodimerisation. DNA binding was abolished when all five residues were substituted, which reinforces the notion that only a subset of residues contributes crucial dimer-forming contacts. The role of Y270 was unique, since its mutation to glycine dramatically slowed both the association and dissociation rates for DNA binding. Surprisingly, R255 was shown to be of little importance in the stability of the p50 homodimer, despite its apparent participation in a salt bridge at the dimer interface. Our results suggest that binding modes inferred from structural data should be treated cautiously.

A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins.

BACKGROUND: Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor kappa B (NF-kappa B) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. RESULTS: A range of model fusion proteins to either the amino- or carboxy-terminus of NF-kappa B p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-kappa B p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:10(8) and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. CONCLUSIONS: A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

Analysis of the NF-kappaB p50 dimer interface by diversity screening.

An in vivo screen has been devised for NF-kappaB p50 activity in Escherichia coli exploiting the ability of the mammalian transcription factor to emulate a prokaryotic repressor. Active intracellular p50 was shown to repress the expression of a green fluorescent protein reporter gene allowing for visual screening of colonies expressing active p50 on agar plates. A library of mutants was constructed in which the residues Y267, L269, A308 and V310 of the dimer interface were simultaneously randomised and twenty-five novel functional interfaces were selected which repressed the reporter gene to similar levels as the wild-type protein. The leucine-269 alanine-308 core was repeatedly, but not exclusively, selected from the library whilst a diversity of predominantly non-polar residues were selected at positions 267 and 310. These results indicate that L269 and A308 may form a hot spot of interaction and allow an insight into the processes of dimer selectivity and evolution within this family of transcription factors.

Profilin I is essential for cell survival and cell division in early mouse development.

Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1(ko)) allele in mice. Homozygous pfn1(ko/ko) mice are not viable. Pfn1(ko/ko) embryos died as early as the two-cell stage, and no pfn1(ko/ko) blastocysts were detectable. Adult pfn1(ko/wt) mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1(ko/wt) embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.

Comparative value of 99mTc-sestamibi scintimammography and sonography in the diagnostic workup of breast masses.

OBJECTIVE: This study was conducted to assess the relative roles of 99mTc-sestamibi scintimammography and sonography in the evaluation of breast lesions that are indeterminate or suspicious on mammography or clinical examination. SUBJECTS AND METHODS: Twenty-five patients with 33 biopsy-proven breast lesions underwent both scintimammography and sonography. Lesions were categorized as benign or requiring biopsy on the basis of the absence or presence of a focus of increased activity on scintimammography and the shape, orientation, and echogenicity of the lesion on sonography. RESULTS: Sensitivity and specificity in detecting breast cancer were 92% and 95%, respectively, for scintimammography and 100% and 48%, respectively, for sonography. The higher specificity of scintimammography was statistically significant (p < 0.01). CONCLUSION: Although the overall accuracy of 99mTc-sestamibi scintimammography in the diagnosis of breast cancer was high, it has several disadvantages in comparison with sonography. Scintimammography has a slightly higher false-negative rate for breast cancer, is unable to reveal cysts, is more expensive, takes longer to perform, and involves ionizing radiation. For these reasons, scintimammography with 99mTc-sestamibi is unlikely to either replace sonography or be frequently used in addition to sonography.

Evolutionary optimisation of enzymes.

Aside from the demonstration that individual molecular traits of enzymes can be evolutionarily optimised, the discovery that several traits can be simultaneously optimised is a major advance. The first observations of the effects of evolutionary optimisation at the structural level, through X-ray crystallography, reinforce the view that enzymes are best optimised by evolution and not by design.

Molecular genetic approaches to understanding the actin cytoskeleton.

New tools in molecular genetics, such as genetic interaction screens and conditional gene targeting, have advanced the study of actin dynamics in a number of model systems. Yeast, Dictyostelium, Caenorhabditis elegans, Drosophila, and mice have contributed much in recent years to a better understanding of both the numerous functions and modes of regulation of the actin cytoskeleton.

The salt dependence of DNA recognition by NF-kappaB p50: a detailed kinetic analysis of the effects on affinityand specificity.

The binding kinetics of NF-kappaB p50 to the Ig-kappaB site and to a DNA duplex with no specific binding site were determined under varying conditions of potassium chloride concentration using a surface plasmonresonance biosensor. Association and dissociation rate constants were measured enabling calculation of the dissociation constants. Under previously established high affinity buffer conditions, the k a for both sequences was in the order of 10(7) M-1s-1whilst the k d values varied 600-fold in a sequence-dependent manner between 10(-1) and 10(-4 )s-1, suggesting that the selectivity of p50 for different sequences is mediated primarily through sequence-dependent dissociation rates. The calculated K D value for the Ig-kappaB sequence was 16 pM, whilst the K D for the non-specific sequence was 9.9 nM. As the ionic strength increased to levels which are closer to that of the cellular environment, the binding of p50 to the non-specific sequence was abolished whilst the specific affinity dropped to nanomolar levels. From these results, a mechanism is proposed in which p50 binds specific sequences with high affinity whilst binding non-specific sequences weakly enough to allow efficient searching of the DNA.

Drosophila hormone receptor 38 functions in metamorphosis: a role in adult cuticle formation.

DHR38 is a member of the steroid receptor superfamily in Drosophila homologous to the vertebrate NGFI-B-type orphan receptors. In addition to binding to specific response elements as a monomer, DHR38 interacts with the USP component of the ecdysone receptor complex in vitro, in yeast and in a cell line, suggesting that DHR38 might modulate ecdysone-triggered signals in the fly. We characterized the molecular structure and expression of the Dhr38 gene and initiated an in vivo analysis of its function(s) in development. The Dhr38 transcription unit spans more than 40 kb in length, includes four introns, and produces at least four mRNA isoforms differentially expressed in development; two of these are greatly enriched in the pupal stage and encode nested polypeptides. We characterized four alleles of Dhr38: a P-element enchancer trap line, l(2)02306, which shows exclusively epidermal staining in the late larval, pre-pupal and pupal stages, and three EMS-induced alleles. Dhr38 alleles cause localized fragility and rupturing of the adult cuticle, demonstrating that Dhr38 plays an important role in late stages of epidermal metamorphosis.

Analysis of the conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate by active-site mutants of Aspergillus nidulans isopenicillin N synthase.

BACKGROUND: Penicillins and cephalosporins constitute a major class of clinically useful antibiotics. A key step in their biosynthesis involves the oxidative cyclisation of delta-(Lalpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N by isopenicillin N synthase (IPNS). This chemically remarkable transformation has been extensively studied using substrate analogues. The conversion of an analogue in which the valine is replaced by alpha-aminobutyrate results in three products, two epimeric penams and a cepham. The ratio of these products in reactions catalysed by four different IPNS isozymes has been used previously to probe the thermicity of the chemical mechanism. But how IPNS restricts the products from the natural substrate to a single penam (isopenicillin N) has remained unknown. RESULTS: A key active-site residue, Leu223, identified according to a model of enzyme-substrate binding, has been altered to sterically less demanding residues. As the steric constraints on the upper part of the active site are reduced, the ratio of the beta-methyl penam to the cepham increases when the alpha-aminobutyrate-containing substrate analogue is used. These results suggest a mechanism for processing of the natural substrate in which IPNS uses steric control to restrict the conformational freedom of an intermediate such that the only product is the penam. CONCLUSIONS: Using steric pressure to control conformation, and hence to disfavour reactions leading to alternate products, is probably the result of evolutionary selection for a biologically active product at the expense of biologically inactive byproducts. It is likely that this sort of enzymatic catalysis is used in situations where substrate conversion is highly exothermic and a variety of products are possible.

In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly.

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho-associated coiled-coil kinase, the Rac-associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co-localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.

Killing two birds with one stone: a chemically plausible scheme for linked nucleic acid replication and coded peptide synthesis.

To understand how life began, we must explain the origins of nucleic acid replication and genetically coded peptide synthesis. Neither of these is easy to explain individually; here, we propose a chemically plausible scheme for the evolution of a process that simultaneously produced both polymers. Later, two separate machineries could have evolved from the linked process.

The transcriptional factor CF2 is a mediator of EGF-R-activated dorsoventral patterning in Drosophila oogenesis.

Establishment of dorsoventral polarity during Drosophila oogenesis requires localized intercellular communication between the follicular cells and the oocyte. This is initiated by the transmission of a "dorsal signal" from the oocyte to the anterior dorsal follicle cells by the EGF receptor (EGF-R) pathway and is followed by transmission of a second signal from the ventral follicle cells back to the embryo. We show that the zinc finger transcription factor CF2 participates in these processes. CF2 is suppressed by EGF-R signaling in the anterior dorsal follicle cells. Altered expression patterns of CF2 result in specific dorsoventral patterning defects in egg chambers and in embryos, as demonstrated phenotypically and with molecular markers. CF2 appears to act as a repressor of dorsal follicle cell fates and specifically as a repressor of the rhomboid gene transcription.

Technetium-99m-MAG3 renal studies in spinal cord injury patients: normal range, reproducibility, and change as a function of duration and level of injury.

UNLABELLED: The normal range, reproducibility, and change as a function of duration and level of injury for Tc-99m-MAG3 renal studies were quantitated in spinal cord injury (SCI) patients. METHODS: Five SCI patients without evidence of renal disease in each of four groups: less than 2 months, 2-12 months, 1-2 years, and greater than 2 years from time of injury, were each studied twice. There were at least two patients with paraplegia and two with tetraplegia in each group. Renal clearance (camera based method), percent function in each kidney, time of peak renal parenchymal activity, and half time of parenchymal activity following the peak were evaluated. The peak and half times were determined with regions of interest (ROIs) over the entire kidney and over just the cortex. All results were compared to normal ranges previously established in normal subjects of the same age range using the same methodology. RESULTS: Renal clearance in the less than 2 month SCI patients was not significantly different from normal subjects in either paraplegic or in tetraplegic individuals. However, clearance in tetraplegics was increased by 28.5% at 2-12 month, increased by 50.6% at 1-2 years, and decreased by 25.9% at greater than 2 years compared to normal subjects (all P < 0.02). Clearance in those with paraplegia showed a similar, but less marked, trend (P = NS). The time of peak parenchymal activity when measured with cortical ROIs did not vary among patient groups or level of injury, but was increased compared to normal subjects (P < 0.05). The percent function in each kidney and half time following the peak were symmetrical, did not differ among patient groups or with level of injury, and did not differ from normal subjects. The parenchymal peak time was significantly shorter with cortical rather than renal ROIs in all patient groups (P < 0.05). In serial studies in the same patient the percent standard deviation in total renal clearance was less than between single studies in different patients, but the decrease was significant for only the right kidney (P < 0.03), and the decrease was not as great as in normal subjects. In addition, the percent standard deviation for percent function in each kidney was significantly less than the percent standard deviations in single studies (P < 0.02). There were no significant differences between intra- and interpatient studies for any other parameter. CONCLUSION: We conclude that: (1) renal clearance measured with Tc-99m-MAG3 in tetraplegic patients increases significantly during the first 2 years following injury and decreases significantly thereafter; there is a similar, but much less marked, trend in paraplegics, (2) parenchymal peak times with cortical ROIs occur later for SCI patients than for normal subjects, and (3) there is more intrapatient variation in total renal clearance and percent renal clearance on a side in SCI patients than in normal subjects suggesting that it may be harder to study SCI patients reproducibly. These findings should be taken into account when performing and interpreting Tc-99m-MAG3 renal studies in SCI patients.

Amino-acid substitutions in the cleavage site of acyl-coenzyme A:isopenicillin N acyltransferase from Penicillium chrysogenum: effect on proenzyme cleavage and activity.

Site-directed mutagenesis of the penDE gene and expression in Escherichia coli has produced recombinant acylcoenzyme A:isopenicillin N acyltransferase (re-AT) containing amino-acid substitutions in the proenzyme cleavage site (decreases) region (Asp-Gly102 decreases Cys103-Thr-Thr). The effect of these substitutions on proenzyme cleavage and AT activity has been investigated. The re-AT with substitutions at Cys103 (Cys103-->Ser, Cys103-->Ala and Cys103-->Trp) were uncleaved and inactive. Substitutions at Asp101 and Gly102 (Asp101-->Gly, Gly102-->Ala, Gly102-->Val, Gly102-->Met, Gly102-->Val and Asp101Gly102-->GlyPhe) did not prevent proenzyme cleavage or abolish AT activity. Thr105-->Ser and Thr105-->Ala substitutions did not prevent proenzyme cleavage or AT activity; however, AT containing Thr105-->Val resulted in a significant inhibition of proenzyme cleavage.

Drosophila hormone receptor 38: a second partner for Drosophila USP suggests an unexpected role for nuclear receptors of the nerve growth factor-induced protein B type.

In Drosophila the response to the hormone ecdysone is mediated in part by Ultraspiracle (USP) and ecdysone receptor (EcR), which are members of the nuclear receptor superfamily. Heterodimers of these proteins bind to ecdysone response elements (EcREs) and ecdysone to modulate transcription. Herein we describe Drosophila hormone receptor 38 (DHR38) and Bombyx hormone receptor 38 (BHR38), two insect homologues of rat nerve growth factor-induced protein B (NGFI-B). Although members of the NGFI-B family are thought to function exclusively as monomers, we show that DHR38 and BHR38 in fact interact strongly with USP and that this interaction is evolutionarily conserved. DHR38 can compete in vitro against EcR for dimerization with USP and consequently disrupt EcR-USP binding to an EcRE. Moreover, transfection experiments in Schneider cells show that DHR38 can affect ecdysone-dependent transcription. This suggests that DHR38 plays a role in the ecdysone response and that more generally NGFI-B type receptors may be able to function as heterodimers with retinoid X receptor type receptors in regulating transcription.

A heuristic approach to the analysis of enzymic catalysis: reaction of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate and delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-allylglycine catalyzed by isopenicillin N synthase isozymes.

Isopenicillin N synthase (IPNS) catalyzes the oxidative cyclization of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N. It is proposed that the multiple products produced from certain substrate analogues result from pathway branching after formation of a ferryl oxene intermediate. We have been interested in ascertaining the reasons for multiple product formation. One possibility is that the products are predisposed toward formation once the beta-lactam ring and the ferryl oxene are produced. Alternately, the products may be persuaded into being by the enzyme restricting conformations such that otherwise less favorable chemistry can take place. With the existing description of the IPNS catalytic cycle, this fundamental question has not been answerable. We describe here the application of a heuristic method to resolve this key issue. It was reasoned that by comparing the ratios of products formed by a set of perturbed IPNS variants it might be possible to generate qualitative information about the relative magnitude of certain activation parameters. If certain product ratios are affected but others are not, then it should be possible to say which steps in the reaction are dictated merely by chemical fundamentals and which steps are directly effected by the enzyme. In this paper we report the high-level expression, purification, and characterization of four IPNS isozymes. Comparison of the product ratios obtained on incubation of unnatural substrate analogues with four IPNS isozymes corresponding to perturbed active site variants shows substantial variation in some cases and little in others. Interpretation of the results obtained with delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alpha-aminobutyrate (ACAB) allows conclusions to be drawn regarding the role of the enzyme in restricting available conformations of the natural substrate to disfavor certain otherwise chemically favorable pathways and hence products. The results obtained with delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-allylglycine, while rather more complex, substantiate the conclusions drawn from the ACAB data. A major conclusion is that, in the oxidation of ACV, IPNS is a negative catalyst of cepham formation but a positive catalyst of penam formation.

Acyl-coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum: effect of amino acid substitutions at Ser227, Ser230 and Ser309 on proenzyme cleavage and activity.

Using a high level Escherichia coli expression system for the Penicillium chrysogenum penDE gene, we have produced acyl-coenzyme A: isopenicillin N acyltransferase (AT) enzymes containing amino acid substitutions at three conserved Ser residues. Chosen for study based on amino acid sequence homologies to other proteins, Ser227, Ser230 and Ser309 were changed to Cys or Ala to assess amino acid side chain involvement in proenzyme cleavage and AT enzyme mechanism. Substitutions at Ser230 had no effect on proenzyme cleavage, acyl-coenzyme A: IPN acyltransferase (IAT) or acyl-coenzyme A:6-aminopenicillanic acid acyltransferase (AAT) activities. While Ser227-->Cys had no effect, Ser227-->Ala produced uncleaved proenzyme lacking both AAT and IAT activities, suggesting that the presence of a nucleophilic side chain at this residue is required for proenzyme cleavage and AT activity. Substitution of Ser309-->Cys did not appreciably prevent proenzyme cleavage, IAT or AAT activity. Recombinant AT (recAT) proenzyme containing Ser309-->Ala was cleaved; however, IAT and AAT activities were not observed. This separation of proenzyme cleavage from IAT and AAT activities has not been previously observed, and suggests that Ser309 is involved in substrate acylation.