The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments

The [URE3] nonchromosomal genetic element is a prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. Ure2p^1–65 is the prion domain of Ure2p, sufficient to propagate [URE3] in vivo. We show that full length Ure2p–green fluorescent protein (GFP) or a Ure2p^1–65-GFP fusion protein is aggregated in cells carrying [URE3] but is evenly distributed in cells lacking the [URE3] prion. This indicates that [URE3] involves a self-propagating aggregation of Ure2p. Overexpression of Ure2p^1–65 induces the de novo appearance of [URE3] by 1,000-fold in a strain initially [ure-o], but cures [URE3] from a strain initially carrying the [URE3] prion. Overexpression of several other fragments of Ure2p or Ure2-GFP fusion proteins also efficiently cures the prion. We suggest that incorporation of fragments or fusion proteins into a putative [URE3] “crystal” of Ure2p poisons its propagation.     

[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis

The role of the high potential [3Fe-4S]^1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]^2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.     

Functional expression of a novel human neurokinin-3 receptor homolog that binds [3H]senktide and [125I-MePhe7]neurokinin B, and is responsive to tachykinin peptide agonists

In 1992, Xie et al. identified a cDNA sequence in the expression cloning search for the κ opioid receptor. When the cDNA was expressed in Cos-7 cells, binding of opioid compounds was observed to be of low affinity and without κ, μ, or δ selectivity [Xie, G.-X., Miyajima, A. and Goldstein, A. (1992) Proc. Natl. Acad. Sci. USA 89, 4124–4128]. This cDNA was highly homologous to the human neurokinin-3 (NK-3) receptor sequence, and displayed lower homology to NK-1 and NK-2 sequences. This sequence was stably expressed in Chinese hamster ovary cells, which do not express neurokinin receptors naturally, and ligand binding and second messenger characteristics were compared with a human NK-3 receptor. The NK-3 receptor homolog bound [³H]senktide with a K_d of 39 nM, similar to that of the NK-3 receptor. The rank order of tachykinin peptides competing for [³H]senktide binding at the NK-3 receptor homolog was [MePhe⁷]neurokinin B > senktide > substance P = neurokinin A > neurokinin B. This cell line also bound [¹²⁵I-MePhe⁷]neurokinin B; however, neurokinin B was an effective competitor. Tachykinin peptides stimulated both inositol phospholipid hydrolysis and arachidonic acid release at NK-3 and NK-3 receptor homolog cell lines, with similar rank orders of potency of [MePhe⁷]neurokinin B = neurokinin B = senktide > NKA = substance P. These results indicate that expression of the NK-3 receptor homolog cDNA in the Chinese hamster ovary cell system induces the expression of a receptor site with many similarities but certain key differences from that of the human NK-3 receptor. The results are discussed with reference to the existence of a novel human tachykinin receptor.     

Autoradiographic characterization of [3H]-5-HT-moduline binding sites in rodent brain and their relationship to 5-HT1B receptors

5-HT-moduline is an endogenous tetrapeptide [Leu-Ser-Ala-Leu (LSAL)] that was first isolated from bovine brain tissue. To understand the physiological role of this tetrapeptide, we studied the localization of 5-HT-moduline binding sites in rat and mouse brains. Quantitative data obtained with a gaseous detector of β-particles (β-imager) indicated that [³H]-5-HT-moduline bound specifically to rat brain sections with high affinity (K_d = 0.77 nM and B_max = 0.26 dpm/mm²). Using film autoradiography in parallel, we found that 5-HT-moduline binding sites were expressed in a variety of rat and mouse brain structures. In 5-HT_1B receptor knock-out mice, the specific binding of [³H]-5-HT-moduline was not different from background labeling, indicating that 5-HT-moduline targets are exclusively located on the 5-HT_1B receptors. Although the distribution of 5-HT-moduline binding sites was similar to that of 5-HT_1B receptors, they did not overlap totally. Differences in distribution patterns were found in regions containing either high levels of 5-HT_1B receptors such as globus pallidus and subiculum that were poorly labeled or in other regions such as dentate gyrus of hippocampus and cortex where the relative density of 5-HT-moduline binding sites was higher than that of 5-HT_1B receptors. In conclusion, our data, based on autoradiographic localization, indicate that 5-HT-moduline targets are located on 5-HT_1B receptors present both on 5-HT afferents and postsynaptic neurons. By interacting specifically with 5-HT_1B receptors, this tetrapeptide may play a pivotal role in pathological states such as stress that involves the dysfunction of 5-HT neurotransmission.     

Overexpression of the SUP45 gene encoding a Sup35p-binding protein inhibits the induction of the de novo appearance of the [PSI+] prion

[PSI⁺], a non-Mendelian element found in some strains of Saccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p). Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p). As predicted by the prion model, overproduction of Sup35p induces the de novo appearance of [PSI⁺]. However, another non-Mendelian determinant, [PIN⁺], is required for this induction. We now show that SUP45 overexpression inhibits the induction of [PSI⁺] by Sup35p overproduction in [PIN⁺] strains, but has no effect on the propagation of [PSI⁺] or on the [PIN] status of the cells. We also show that SUP45 overexpression counteracts the growth inhibition usually associated with overexpression of SUP35 in [PSI⁺] strains. We argue that excess Sup45p inhibits [PSI⁺] seed formation. Because Sup45p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation. This in vivo yeast result is reminiscent of the in vitro finding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid.     

Widespread accumulation of [3H]testosterone in the spinal cord of a wild bird with an elaborate courtship display

Elaborate courtship displays are relatively common features of the masculine reproductive behavior in birds. However, little is known about their neural and hormonal control. One bird that performs such a display is the golden-collared manakin (Manacus vitellinus) of Panamanian forests. Adult males, but not females, perform a physically intense display requiring substantial neuromuscular control of the wings and legs. We tested the hypothesis that steroid sensitivity is a property of neurons in the manakin spinal cord. Males and females were captured from active courtship leks, treated with drugs to block steroidogenesis, injected with (3)H-labeled testosterone, and the spinal cords were removed and processed for autoradiography. Sex steroid-accumulating cells were widely distributed in the spinal cords in each of six males and in one of five females. Cells, including presumptive motoneurons, reached their highest density in the ventral horns of the cervical and lumbosacral enlargements, regions associated with motor control of the wings and legs. These results suggest that neurons in the adult manakin spinal cord can express sex-steroid receptors, but do so less in females than in males. This evidence for androgen sensitivity and sexual dimorphism in the adult avian spinal cord suggests that sex steroids may control diverse behaviors in male birds in part by acting directly on the spinal neural circuits.     

Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1α, a potent ligand for the HIV-1 “fusin” coreceptor

Stromal cell-derived factor-1α (SDF-1α ) is a member of the chemokine superfamily and functions as a growth factor and chemoattractant through activation of CXCR4/LESTR/Fusin, a G protein-coupled receptor. This receptor also functions as a coreceptor for T-tropic syncytium-inducing strains of HIV-1. SDF-1α antagonizes infectivity of these strains by competing with gp120 for binding to the receptor. The crystal structure of a variant SDF-1α ([N33A]SDF-1α ) prepared by total chemical synthesis has been refined to 2.2-Å resolution. Although SDF-1α adopts a typical chemokine β-β-β-α topology, the packing of the α-helix against the β-sheet is strikingly different. Comparison of SDF-1α with other chemokine structures confirms the hypothesis that SDF-1α may be either an ancestral protein from which all other chemokines evolved or the chemokine that is the least divergent from a primordial chemokine. The structure of SDF-1α reveals a positively charged surface ideal for binding to the negatively charged extracellular loops of the CXCR4 HIV-1 coreceptor. This ionic complementarity is likely to promote the interaction of the mobile N-terminal segment of SDF-1α with interhelical sites of the receptor, resulting in a biological response.     

Crystal structure of desheptapeptide(B24–B30)insulin at 1.6 Å resolution: Implications for receptor binding

The crystal structure of desheptapeptide (B24–B30) insulin (DHPI), a virtually inactive analog of insulin, was determined at 1.6 Å resolution. In the refined structure model, DHPI retains three α-helices (A1–A8, A12–A18, and B9–B19) as its structural framework, while great conformational changes occur in the N and C termini of B-chain. The β-turn, which lies in B20–B30 in insulin and insulin analogs with high potency, no longer exists in DHPI. Relative motion is observed among the three α-helices, each as a rigid functional group. In contrast, a region covering B5–B6 and A6–A11 exhibits a relatively stable conformation. We interpret our results as identifying: (i) the importance of β-turn in determining the receptor-binding potency of insulin and (ii) a leading role of Phe^B24 in maintaining the β-turn structure.     

Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation

The proteasome is responsible for degradation of substrates of the ubiquitin pathway. 20S proteasomes are cylindrical particles with subunits arranged in a stack of four heptameric rings. The outer rings are composed of α subunits, and the inner rings are composed of β subunits. A well-characterized archaeal proteasome has a single type of each subunit, and the N-terminal threonine of the β subunit is the active-site nucleophile. Yeast proteasomes have seven different β subunits and exhibit several distinct peptidase activities, which were proposed to derive from disparate active sites. We show that mutating the N-terminal threonine in the yeast Pup1 β subunit eliminates cleavage after basic residues in peptide substrates, and mutating the corresponding threonine of Pre3 prevents cleavage after acidic residues. Surprisingly, neither mutation has a strong effect on cell growth, and they have at most minor effects on ubiquitin-dependent proteolysis. We show that Pup1 interacts with Pup3 in each β subunit ring. Our data reveal that different proteasome active sites contribute very differently to protein breakdown in vivo, that contacts between particular subunits in each β subunit ring are critical for active-site formation, and that active sites in archaea and different eukaryotes are highly similar.     

A protein required for prion generation: [URE3] induction requires the Ras-regulated Mks1 protein

Infectious proteins (prions) can arise de novo as well as by transmission from another individual. De novo prion generation is believed responsible for most cases of Creutzfeldt-Jakob disease and for initiating the mad cow disease epidemic. However, the cellular components needed for prion generation have not been identified in any system. The [URE3] prion of Saccharomyces cerevisiae is an infectious form of Ure2p, apparently a self-propagating amyloid. We now demonstrate a protein required for de novo prion generation. Mks1p negatively regulates Ure2p and is itself negatively regulated by the presence of ammonia and by the Ras-cAMP pathway. We find that in mks1Delta strains, de novo generation of the [URE3] prion is blocked, although [URE3] introduced from another strain is expressed and propagates stably. Ras2(Val19) increases cAMP production and also blocks [URE3] generation. These results emphasize the distinction between prion generation and propagation, and they show that cellular regulatory mechanisms can critically affect prion generation.     

The 3D NOESY-[1H,15N,1H]-ZQ-TROSY NMR experiment with diagonal peak suppression

In our 3D NOESY-[(1)H,(15)N,(1)H]-ZQ-TROSY experiment, the TROSY principle (transverse relaxation-optimized spectroscopy) is used in three-dimensional (3D) (15)N-resolved nuclear Overhauser enhancement spectroscopy (NOESY), which enables resonance assignments by sequential nuclear Overhauser effects and the collection of structural constraints in large (15)N- or (2)H,(15)N-labeled proteins. Our experiment affords optimization of the transverse relaxation in all three frequency dimensions, provides suppression of the strong diagonal autorelaxation peaks, which otherwise tend to interfere with the analysis of nearby informative crosspeaks, and yields improved resolution for the entire spectrum when compared with conventional 3D (15)N-resolved-[(1)H,(1)H]-NOESY, because of the narrower lineshapes along both proton dimensions. The key element of this experiment is an approach for correlating the (15)N and (1)H chemical shifts with two-dimensional ZQ-[(15)N,(1)H]-TROSY, where zero-quantum (ZQ) coherence is generated and the remote cross-correlation between the (1)H and (15)N chemical shift anisotropy interactions is used to reduce transverse relaxation during (15)N evolution. Practical applications are illustrated with spectra of a protein with a molecular mass of 110,000 Da.     

The atomic model of the human protective protein/cathepsin A suggests a structural basis for galactosialidosis

Human protective protein/cathepsin A (PPCA), a serine carboxypeptidase, forms a multienzyme complex with β-galactosidase and neuraminidase and is required for the intralysosomal activity and stability of these two glycosidases. Genetic lesions in PPCA lead to a deficiency of β-galactosidase and neuraminidase that is manifest as the autosomal recessive lysosomal storage disorder galactosialidosis. Eleven amino acid substitutions identified in mutant PPCAs from clinically different galactosialidosis patients have now been modeled in the three-dimensional structure of the wild-type enzyme. Of these substitutions, 9 are located in positions likely to alter drastically the folding and stability of the variant protein. In contrast, the other 2 mutations that are associated with a more moderate clinical outcome and are characterized by residual mature protein appeared to have a milder effect on protein structure. Remarkably, none of the mutations occurred in the active site or at the protein surface, which would have disrupted the catalytic activity or protective function. Instead, analysis of the 11 mutations revealed a substantive correlation between the effect of the amino acid substitution on the integrity of protein structure and the general severity of the clinical phenotype. The high incidence of PPCA folding mutants in galactosialidosis reflects the fact that a single point mutation is unlikely to affect both the β-galactosidase and the neuraminidase binding sites of PPCA at the same time to produce the double glycosidase deficiency. Mutations in PPCA that result in defective folding, however, disrupt every function of PPCA simultaneously.     

Reciprocal regulation of Gsα by palmitate and the βγ subunit

Hormonal activation of G_s, the stimulatory regulator of adenylyl cyclase, promotes dissociation of α_s from Gβγ, accelerates removal of covalently attached palmitate from the Gα subunit, and triggers release of a fraction of α_s from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant α_s prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated α_s (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound βγ with 5-fold higher affinity. βγ protected GDP-bound α_s, but not α_s· GTP[γS], from depalmitoylation by a recombinant esterase. We conclude that βγ binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of α_s and that dissociation of α_s·GTP from βγ is likely to mediate receptor-induced α_s depalmitoylation and translocation of the protein to cytosol in intact cells.     

Enzymatic reaction with unnatural substrates: DNA photolyase (Escherichia coli) recognizes and reverses thymine [2+2] dimers in the DNA strand of a DNA/PNA hybrid duplex

Peptide nucleic acids (PNA) are mimics with normal bases connected to a pseudopeptide chain that obey Watson–Crick rules to form stable duplexes with itself and natural nucleic acids. This has focused attention on PNA as therapeutic or diagnostic reagents. Duplexes formed with PNA mirror some but not all properties of DNA. One fascinating aspect of PNA biochemistry is their reaction with enzymes. Here we show an enzyme reaction that operates effectively on a PNA/DNA hybrid duplex. A DNA oligonucleotide containing a cis, syn-thymine [2+2] dimer forms a stable duplex with PNA. The hybrid duplex is recognized by photolyase, and irradiation of the complex leads to the repair of the thymine dimer. This finding provides insight into the enzyme mechanism and provides a means for the selective repair of thymine photodimers.     

Formation of stable adducts and absence of depurinating DNA adducts in cells and DNA treated with the potent carcinogen dibenzo[a,l]pyrene or its diol epoxides

Polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants, and some are potent carcinogens in rodents. Carcinogenic PAH are activated in cells to metabolites that react with DNA to form stable covalent DNA adducts. It has been proposed [Cavalieri, E. L. & Roger, E. G. (1995) Xenobiotica 25, 677–688] that unstable DNA adducts are also formed and that apurinic sites in the DNA resulting from unstable PAH adducts play a key role in the initiation of cancer. The potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) is activated in cells to (+)-syn- and (−)-anti-DB[a,l]P-11,12-diol-13,14-epoxide (DB[a,l]PDE), which have been shown to form stable adducts with DNA. To evaluate the importance of unstable PAH adducts, we compared stable adduct formation to apurinic site formation. Stable DB[a,l]PDE adducts were determined by ³³P-postlabeling and HPLC. To measure apurinic sites they were converted to strand breaks, and these were monitored by examining the integrity of a particular restriction fragment of the dihydrofolate reductase gene. The method easily detected apurinic sites resulting from methylation by treatment of cells or DNA with dimethyl sulfate or from reaction of DNA with DB[a,l]P in the presence of horseradish peroxidase. We estimate the method could detect 0.1 apurinic site in the 14-kb fragment examined. However, apurinic sites were below our limit of detection in DNA treated directly with (+)-syn- or (−)-anti-DB[a,l]PDE or in DNA from Chinese hamster ovary B11 cells so treated, although in these samples the frequency of stable adducts ranged from 3 to 10 per 14 kb. We also treated the human mammary carcinoma cell line MCF-7 with DB[a,l]P and again could not detect significant amounts of unstable adducts. These results indicate that the proportion of stable adducts formed by DB[a,l]P activated in cells and its diol epoxides is greater than 99% and suggest a predominant role for stable DNA adducts in the carcinogenic activity of DB[a,l]P.