Selective roles for tumor necrosis factor alpha-converting enzyme/ADAM17 in the shedding of the epidermal growth factor receptor ligand family: the juxtamembrane stalk determines cleavage efficiency

Epidermal growth factor (EGF) family ligands are derived by proteolytic cleavage of the ectodomains of integral membrane precursors. Previously, we established that tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) is a physiologic transforming growth factor-alpha (TGF-alpha) sheddase, and we also demonstrated enhanced shedding of amphiregulin (AR) and heparin-binding (HB)-EGF upon restoration of TACE activity in TACE-deficient EC-2 fibroblasts. Here we extended these results by showing that purified soluble TACE cleaved single sites in the juxtamembrane stalks of mouse pro-HB-EGF and pro-AR ectodomains in vitro. For pro-HB-EGF, this site matched the C terminus of the purified human growth factor, and we speculate that the AR cleavage site is also physiologically relevant. In contrast, ADAM9 and -10, both implicated in HB-EGF shedding, failed to cleave the ectodomain or cleaved at a nonphysiologic site, respectively. Cotransfection of TACE in EC-2 cells enhanced phorbol myristate acetate-induced but not constitutive shedding of epiregulin and had no effect on betacellulin (BTC) processing. Additionally, soluble TACE did not cleave the juxtamembrane stalks of either pro-BTC or pro-epiregulin ectodomains in vitro. Substitution of the shorter pro-BTC juxtamembrane stalk or truncation of the pro-TGF-alpha stalk to match the pro-BTC length reduced TGF-alpha shedding from transfected cells to background levels, whereas substitution of the pro-BTC P2-P2' sequence reduced TGF-alpha shedding less dramatically. Conversely, substitution of the pro-TGF-alpha stalk or lengthening of the pro-BTC stalk, especially when combined with substitution of the pro-TGF-alpha P2-P2' sequence, markedly increased BTC shedding. These results indicate that efficient TACE cleavage is determined by a combination of stalk length and scissile bond sequence.     

Compound 800, a natural product isolated from genetically engineered Pseudomonas: proposed structure, reactivity, and putative relation to heme d1

Genetically engineered strains of Escherichia coli and Pseudomonas aeruginosa were prepared harboring the gene cluster nirFDLGH from Pseudomonas stutzeri substrain ZoBell on a high copy plasmid. These genes have been previously implicated as being essential for the biosynthesis of heme d(1), the prosthetic group of dissimilatory nitrite reductases in anaerobic, denitryfying bacteria. Tetrapyrroles detectable at steady-state levels were identified from both organisms, and cell-free extracts from each were also used to transform uroporphyrinogen in vitro. E. coli does not naturally produce d(1), and the engineered strain failed to produce d(1) or any tetrapyrrole foreign to E. coli. Therefore, while nirFDLGHmay be necessary for d(1) biosynthesis, it is not sufficient. In the denitrifier P. aeruginosa, the results were more positive. The presence of the plasmid led to increased levels of d(1). In addition, a previously unidentified tetrapyrrole was detected. This compound was characterized by visible absorption spectroscopy, infrared spectroscopy, X-ray photoelectron spectroscopy, mass spectrometry, and NMR, and a tentative structure was proposed for this compound. The tetrapyrrole has structural features similar to sirohydrochlorin (as precorrin-2 or sirotetrahydrochlorin, a known intermediate of d(1)) and d(1) itself. The most unusual substituents are epoxide and sulfoxide moieties. When this tetrapyrrole was treated with strong mineral acid and heat, it was converted into natural d(1).     

Sensitivity of Bayes estimators to hyper-parameters with an application to maximum yield from fisheries

Priors are seldom unequivocal and an important component of Bayesian modeling is assessment of the sensitivity of the posterior to the specified prior distribution. This is especially true in fisheries science where the Bayesian approach has been promoted as a rigorous method for including existing information from previous surveys and from related stocks or species. These informative priors may be highly contested by various interest groups. Here, formulae for the first and second derivatives of Bayes estimators with respect to hyper-parameters of the joint prior density are given. The formula for the second derivative provides a correction to a previously published result. The formulae are shown to reduce to very convenient and easily implemented forms when the hyper-parameters are for exponential family marginal priors. For model parameters with such priors it is shown that the ratio of posterior variance to prior variance can be interpreted as the sensitivity of the posterior mean to the prior mean. This methodology is applied to a nonlinear state-space model for the biomass of South Atlantic albacore tuna and sensitivity of the maximum sustainable yield to the prior specification is examined.     

Characterization of a small metal binding protein from Nitrosomonas europaea

A small metal-binding protein (SmbP) with no known similarity to other proteins in current databases was isolated and characterized from the periplasm of Nitrosomonas europaea. The primary structure of this small (9.9 kDa) monomeric protein is characterized by a series of 10 repeats of a seven amino acid motif and an unusually high number of histidine residues. The protein was isolated from N. europaea with Cu(II) bound but was found to be capable of binding multiple equivalents of a variety of divalent and trivalent metals. The protein was overexpressed in Escherichia coli and used for the study of its metal-binding properties by UV/vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy and equilibrium dialysis and isothermal titration calorimetry. The protein was found to bind up to six Cu(II) atoms with dissociation constants of approximately 0.1 microM for the first two metal ions and approximately 10 microM for the next four. Binding of Cu(II) resulted in spectroscopic features illustrating two distinctive geometries, as determined by EPR spectroscopy. The levels of SmbP in the periplasm were found to increase by increasing the levels of copper in the growth media. This protein is proposed to have a role in cellular copper management in the ammonia-oxidizing bacterium N. europaea.     

Kinetic mechanism and quaternary structure of Aminobacter aminovorans NADH:flavin oxidoreductase: an unusual flavin reductase with bound flavin

The homodimeric NADH:flavin oxidoreductase from Aminobacter aminovorans is an NADH-specific flavin reductase herein designated FRD(Aa). FRD(Aa) was characterized with respect to purification yields, thermal stability, isoelectric point, molar absorption coefficient, and effects of phosphate buffer strength and pH on activity. Evidence from this work favors the classification of FRD(Aa) as a flavin cofactor-utilizing class I flavin reductase. The isolated native FRD(Aa) contained about 0.5 bound riboflavin-5'-phosphate (FMN) per enzyme monomer, but one bound flavin cofactor per monomer was obtainable in the presence of excess FMN or riboflavin. In addition, FRD(Aa) holoenzyme also utilized FMN, riboflavin, or FAD as a substrate. Steady-state kinetic results of substrate titrations, dead-end inhibition by AMP and lumichrome, and product inhibition by NAD(+) indicated an ordered sequential mechanism with NADH as the first binding substrate and reduced FMN as the first leaving product. This is contrary to the ping-pong mechanism shown by other class I flavin reductases. The FMN bound to the native FRD(Aa) can be fully reduced by NADH and subsequently reoxidized by oxygen. No NADH binding was detected using 90 microM FRD(Aa) apoenzyme and 300 microM NADH. All results favor the interpretation that the bound FMN was a cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanism would require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRD(Aa) exhibited a monomer-dimer equilibrium with a K(d) of 2.7 microM. Similarities and differences between FRD(Aa) and certain flavin reductases are discussed.     

Aminobacter aminovorans NADH:flavin oxidoreductase His140: a highly conserved residue critical for NADH binding and utilization

Homodimeric FRD(Aa) Class I is an NADH:flavin oxidoreductase from Aminobacter aminovorans. It is unusual because it contains an FMN cofactor but utilizes a sequential-ordered kinetic mechanism. Because little is known about NADH-specific flavin reductases in general and FRD(Aa) in particular, this study aimed to further explore FRD(Aa) by identifying the functionalities of a key residue. A sequence alignment of FRD(Aa) with several known and hypothetical flavoproteins in the same subfamily reveals within the flavin reductase active-site domain a conserved GDH motif, which is believed to be responsible for the enzyme and NADH interaction. Mutation of the His140 in this GDH motif to alanine reduced FRD(Aa) activity to <3%. An ultrafiltration assay and fluorescence quenching demonstrated that H140A FRD(Aa) binds FMN in the same 1:1 stoichiometric ratio as the wild-type enzyme, but with slightly weakened affinity (K(d) = 0.9 microM). Anaerobic stopped-flow studies were carried out using both the native and mutated FRD(Aa). Similar to the native enzyme, H140A FRD(Aa) was also able to reduce the FMN cofactor by NADH although much less efficiently. Kinetic analysis of anaerobic reduction measurements indicated that the His140 residue of FRD(Aa) was essential to NADH binding, as well as important for the reduction of the FMN cofactor. For the native enzyme, the cofactor reduction was followed by at least one slower step in the catalytic pathway.     

Conserved repeat motifs and glucan binding by glucansucrases of oral streptococci and Leuconostoc mesenteroides

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.     

Extracellular superoxide dismutase (EC-SOD) binds to type i collagen and protects against oxidative fragmentation

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is mainly found in the extracellular matrix of tissues. EC-SOD participates in the detoxification of reactive oxygen species by catalyzing the dismutation of superoxide radicals. The tissue distribution of the enzyme is particularly important because of the reactive nature of its substrate, and it is likely essential that EC-SOD is positioned at the site of superoxide production to prevent adventitious oxidation. EC-SOD contains a C-terminal heparin-binding region thought to be important for modulating its distribution in the extracellular matrix. This paper demonstrates that, in addition to binding heparin, EC-SOD specifically binds to type I collagen with a dissociation constant (K(d)) of 200 nm. The heparin-binding region was found to mediate the interaction with collagen. Notably, the bound EC-SOD significantly protects type I collagen from oxidative fragmentation. This expands the known repertoire of EC-SOD binding partners and may play an important physiological role in preventing oxidative fragmentation of collagen during oxidative stress.     

In vitro identification and characterization of an early complex linking HIV-1 genomic RNA recognition and Pr55Gag multimerization

The minimal protein requirements that drive virus-like particle formation of human immunodeficiency virus type 1 (HIV-1) have been established. The C-terminal domain of capsid (CTD-CA) and nucleocapsid (NC) are the most important domains in a so-called minimal Gag protein (mGag). The CTD is essential for Gag oligomerization. NC is known to bind and encapsidate HIV-1 genomic RNA. The spacer peptide, SP1, located between CA and NC is important for the multimerization process, viral maturation and recognition of HIV-1 genomic RNA by NC. In this study, we show that NC in the context of an mGag protein binds HIV-1 genomic RNA with almost 10-fold higher affinity. The protein region encompassing the 11th alpha-helix of CA and the proposed alpha-helix in the CA/SP1 boundary region play important roles in this increased binding capacity. Furthermore, sequences downstream from stem loop 4 of the HIV-1 genomic RNA are also important for this RNA-protein interaction. In gel shift assays using purified mGag and a model RNA spanning the region from +223 to +506 of HIV-1 genomic RNA, we have identified an early complex (EC) formation between 2 proteins and 1 RNA molecule. This EC was not present in experiments performed with a mutant mGag protein, which contains a CTD dimerization mutation (M318A). These data suggest that the dimerization interface of the CTD plays an important role in EC formation, and, as a consequence, in RNA-protein association and multimerization. We propose a model for the RNA-protein interaction, based on previous results and those presented in this study.     

Association analysis of the plasminogen activator inhibitor-1 4G/5G polymorphism in Hispanics and African Americans: the IRAS family study

OBJECTIVE: Plasminogen activator inhibitor type-1 (PAI-1) plays a central role in fibrolysis and has recently been hypothesized to influence components of the insulin resistance syndrome. We consider whether the 4G/5G polymorphism influences components of insulin resistance and obesity solely through PAI-1 protein levels or also though a secondary pathway. In addition, we explore whether transforming growth factor (TGF-beta1), a key regulator of PAI-1 expression, modifies the influence of the PAI-1 4G/5G polymorphism on these traits. METHODS AND RESULTS: The Insulin Resistance and Atherosclerosis (IRAS) Family Study genotyped 287 African American (18 pedigrees) and 811 Hispanic American (45 pedigrees) individuals for the 4G/5G PAI-1 and two TGF-beta1 polymorphisms (R25P, C-509T). Individuals were recruited from three clinical centers located in San Antonio (urban Hispanic), San Luis Valley (rural Hispanic) and Los Angeles (African American). The presence of the 4G PAI-1 allele was positively associated with PAI-1 protein level (combined sample p < 0.0001). Hispanic Americans average 65% higher PAI-1 protein levels than African Americans (p < 0.0001). Consistently across ethnic groups, increased PAI-1 protein levels were associated with increased insulin resistance and overall and central obesity (p value < 0.0001, combined sample). Adjusting for PAI-1 protein levels, there was evidence of an association of PAI-1 genotype (4G) with insulin sensitivity (p < 0.002) and subcutaneous fat (p < 0.01). These associations were not influenced by TGF-beta1 genotypes. CONCLUSIONS: PAI-1 protein is a strong correlate of insulin resistance (IR) and obesity in Hispanics and African Americans. However, PAI-1 4G/5G polymorphism appears to influence insulin resistance and obesity beyond its direct influence on serum PAI-1 protein levels.