Structure-specific effects of protein topology on cross-beta assembly: studies of insulin fibrillation

Systemic amyloidoses, an important class of protein misfolding diseases, are often due to fibrillation of disulfide-cross-linked globular proteins otherwise unrelated in sequence or structure. Although cross-beta assembly is regarded as a universal property of polypeptides, it is not understood how such amyloids accommodate diverse disulfide connectivities. Does amyloidogenicity depend on protein topology? A model is provided by insulin, a two-chain protein containing three disulfide bridges. The importance of chain topology is demonstrated by mini-proinsulin (MP), a single-chain analogue in which the C-terminus of the B chain (residue B30) is tethered to the N-terminus of the A chain (A1). The B30-A1 tether impedes the fiber-specific alpha --> beta transition, leading to slow formation of a structurally nonuniform amorphous precipitate. Conversely, fibrillation is robust to interchange of disulfide bridges. Whereas native insulin exhibits pairings [A6-A11, A7-B7, and A20-B19], metastable isomers with alternative pairings [A6-B7, A7-A11, A20-B19] or [A6-A7, A11-B7, A20-B1] readily undergo fibrillation with essentially identical alpha --> beta transitions. Respective pairing schemes are in each case retained. Isomeric fibrils and the amorphous MP precipitate are each able to seed the fibrillation of wild-type insulin, suggesting a structural correspondence between respective nuclei or modes of assembly. Together, our results demonstrate that effects of polypeptide topology on amyloidogenicity depend on structural context. Although the native structures and stabilities of single-chain insulin analogues are similar to those of wild-type insulin, the interchain tether constrains the extent of conformational distortion at elevated temperature, retards initial non-native aggregation, and is apparently incompatible with the mature structure of an insulin protofilament. We speculate that the general danger of fibrillation has imposed a constraint in protein evolution, selecting for topologies unfavorable to amyloid formation.     

Distance from Home to Study Clinic and Risk of Follow-Up Interruption in a Cohort of HIV-1-Discordant Couples in Nairobi, Kenya

Background

Longitudinal studies of HIV-1-infected individuals or those at risk of infection are subject to missed study visits that may have negative consequences on the care of participants and can jeopardize study validity due to bias and loss of statistical power. Distance between participant residence and study clinic, as well as other socioeconomic and demographic factors, may contribute to interruptions in patient follow-up.

Methods

HIV-1-serodiscordant couples were enrolled between May 2007 and October 2009 and followed for two years in Nairobi, Kenya. At baseline, demographic and home location information was collected and linear distance from each participant’s home to the study clinic was determined. Participants were asked to return to the study clinic for quarterly visits, with follow-up interruptions (FUI) defined as missing two consecutive visits. Cox proportional hazards regression was used to assess crude and adjusted associations between FUI and home-to-clinic distance, and other baseline characteristics.

Results

Of 469 enrolled couples, 64% had a female HIV-1-infected partner. Overall incidence of FUI was 13.4 per 100 person-years (PY), with lower incidence of FUI in HIV-1-infected (10.8 per 100 PY) versus -uninfected individuals (16.1 per 100 PY) (hazard ratio [HR] = 0.66; 95% confidence interval [CI]: 0.50, 0.88). Among HIV-1-infected participants, those living between 5 and 10 kilometers (km) from the study clinic had a two-fold increased rate of FUI compared to those living <5 km away (HR = 2.17; 95% CI: 1.09, 4.34). Other factors associated with FUI included paying higher rent (HR = 1.67; 95% CI: 1.05, 2.65), having at least primary school education (HR = 1.96; 95% CI: 1.02, 3.70), and increased HIV-1 viral load (HR = 1.23 per log₁₀ increase; 95% CI: 1.01, 1.51).

Conclusions

Home-to-clinic distance, indicators of socioeconomic status, and markers of disease progression may affect compliance with study follow-up schedules. Retention strategies should focus on participants at greatest risk of FUI to ensure study validity.     

Cholesterol gallstone dissolution in bile. Dissolution kinetics of crystalline cholesterol monohydrate by conjugated chenodeoxycholate-lecithin and conjugated ursodeoxycholate-lecithin mixtures: dissimilar phase equilibria and dissolution mechanisms

Using compressed discs and microcrystals of cholesterol monohydrate, we evaluated the mechanisms and kinetics of dissolution in conjugated bile salt-lecithin solutions. In stirred conjugated ursodeoxycholate-lecithin and cheno-deoxycholate-lecithin solutions, dissolution of 10,000-psi discs was micellar and linear with time for 10 hours. The dissolution rate constants (k) decreased in proportion to the lecithin content and dissolution rates and k values were appreciably smaller in conjugated ursodeoxycholate-lecithin solutions. After dissolution for 5 to 10 days the discs incubated with ursodeoxycholate-lecithin systems became progressively transformed into macroscopic liquid crystals. Unstirred dissolution of 3,000-psi discs in "simulated" human bile containing physiological lecithin concentrations gave apparent k values that decreased in the following order: ursodeoxycholate-rich >/= chenodeoxycholate-rich > normal. In most cases the discs incubated with ursodeoxycholate-rich bile became covered with a microscopic liquid-crystalline layer. With 20-25 moles % lecithin, these layers eventually dispersed into the bulk solution as microscopic vesicles. During dissolution of microcrystalline cholesterol in conjugated ursodeoxycholate-lecithin systems, a bulk liquid-crystalline phase formed rapidly (within 12 hours) and the final cholesterol solubilities were greater than those in conjugated chenodeoxycholate-lecithin micellar systems. Prolonged incubation of cholesterol microcrystals with pure lecithin or lecithin plus bile salt liposomes did not reproduce these effects. Condensed ternary phase diagrams of conjugated ursodeoxycholate-lecithin-cholesterol systems established that cholesterol-rich liquid crystals constituted an equilibrium precipitate phase that coexisted with cholesterol monohydrate crystals and saturated micelles under physiological conditions. Similar phase dissolution-relationships were observed at physiological lecithin-bile salt ratios for a number of other hydrophilic bile salts (e.g., conjugated ursocholate, hyocholate, and hyodeoxycholate). In contrast, liquid crystals were not observed in conjugated chenodeoxycholate-lecithin-cholesterol systems except at high (nonphysiological) lecithin contents. Based on these and other results we present a molecular hypothesis for cholesterol monohydrate dissolution by any bile salt-lecithin system and postulate that enrichment of bile with highly hydrophilic bile salts will induce crystalline cholesterol dissolution by a combination of micellar and liquid crystalline mechanisms. Since bile salt polarity can be measured and on this basis the ternary phase diagram deduced, we believe that the molecular mechanisms of cholesterol monohydrate dissolution as well as the in vivo cholelitholytic potential of uncommon bile salts can be predicted.-Salvioli, G., H. Igimi, and M. C. Carey. Cholesterol gallstone dissolution in bile. Dissolution kinetics of crystalline cholesterol monohydrate by conjugated chenodeoxycholate-lecithin and conjugated ursodeoxycholate-lecithin mixtures: dissimilar phase equilibria and dissolution mechanisms.     

Influence of dilution on the physical state of model bile systems: NMR and quasi-elastic light-scattering investigations

Multinuclear (1H and 31P) nuclear magnetic resonance (NMR) spectroscopy and quasi-elastic light scattering have been used to characterize molecular aggregates formed in dilute sodium taurocholate--egg lecithin solutions. When mixed micelles (1.25 g/dL) are diluted with 150 mM aqueous sodium chloride, light-scattering measurements suggest a transformation from mixed micelles to unilamellar vesicle species. Decreased 1H NMR line widths for bile salt resonances are consistent with predominance of a monomer form. The concurrent appearance of a second phospholipid choline methyl resonance indicates two types of phospholipid environment in slow chemical exchange: this behavior is consistent with small unilamellar vesicles. The appearance of bilayer vesicles in dilute model bile solutions is confirmed by addition of a lanthanide shift reagent (Pr3+), which splits the 1H or 31P head-group peak into two components with distinct chemical shift sensitivities. These mixed micelle and vesicle aggregates are also distinguished by their susceptibility to the lipolytic enzyme phospholipase A2 from cobra venom.     

A gene-driven ENU-based approach to generating an allelic series in any gene

N-ethyl-N-nitrosourea (ENU) introduces mutations throughout the mouse genome at relatively high efficiency. Successful high-throughput phenotype screens have been reported and alternative screens using sequence-based approaches have been proposed. For the purpose of generating an allelic series in selected genes by a sequence-based approach, we have constructed an archive of over 4000 DNA samples from individual F₁ ENU-mutagenized mice paralleled by frozen sperm samples. Together with our previously reported archive, the total size now exceeds 6000 individuals. A gene-based screen of 27.4 Mbp of DNA, carried out using denaturing high-performance liquid chromatography (DHPLC), found a mutation rate of 1 in 1.01 Mbp of which 1 in 1.82 Mbp were potentially functional. Screening of whole or selected regions of genes on subsets of the archive has allowed us to identify 15 new alleles from 9 genes out of 15 tested. This is a powerful adjunct to conventional mutagenesis strategies and has the advantage of generating a variety of alleles with potentially different phenotypic outcomes that facilitate the investigation of gene function. It is now available to academic collaborators as a community resource.     

Medfly populations differ in diel and age patterns of sexual signalling

Insect populations may differ in several life history traits, including behavioural ones such as sexual signalling. We tested whether male Mediterranean fruit fly (medlfy), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), populations obtained from geographically isolated areas exhibit differences in quantitative and qualitative aspects of male sexual signalling. Male sexual signalling was studied in four medfly populations (originating from Brazil, Portugal, Kenya, and Greece) under identical laboratory conditions (25 °C, 60% r.h., and L14:D10). The four populations had been reared for one generation in the laboratory. Sexual signalling was studied in the F₁ progeny that were fed one of two diets (yeast hydrolysate plus sugar or sugar only). On both diets, the four populations differed significantly in the progress of maturity (indicated by the average number of males exhibiting sexual signalling) and in the quantity of signalling after attaining maturity. Yeast availability significantly increased sexual signalling; however, it had a different impact on the quantity of signalling in the different populations. A bi-modal pattern of sexual signalling, with one peak at approximately 08:00–09:00 hours and the second at approximately 13:00–14:00 hours, was recorded for all populations and diets. However, quantitative differences among the populations within the 'sexually active' period of the day resulted in significant differences in the daily pattern of sexual signalling. The significance of these findings for understanding adaptations of geographically isolated medfly populations to different ecosystems, as well as its practical importance for the application of the sterile insect technique against C. capitata , is discussed.     

Photosynthetic and Phylogenetic Primers for Detection of Anoxygenic Phototrophs in Natural Environments

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.     

A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase

Prostate cancer progresses from a hormone-sensitive, androgen-dependent stage to a hormone-refractory, androgen-independent tumor. The androgen receptor pathway functions in these androgen-independent tumors despite anti-androgen therapy. In our LAPC-4 prostate cancer model, androgen-independent sublines expressed higher levels of the HER-2/neu receptor tyrosine kinase than their androgen-dependent counterparts. Forced overexpression of HER-2/neu in androgen-dependent prostate cancer cells allowed ligand-independent growth. HER-2/neu activated the androgen receptor pathway in the absence of ligand and synergized with low levels of androgen to 'superactivate' the pathway. By modulating the response to low doses of androgen, a tyrosine kinase receptor can restore androgen receptor function to prostate cancer cells, a finding directly related to the clinical progression of prostate cancer.     

Mutually antagonistic effects of androgen and activin in the regulation of prostate cancer cell growth

Activin, a member of the TGFbeta superfamily, is expressed in the prostate and inhibits growth. We demonstrate that the effects of activin and androgen on regulation of prostate cancer cell growth are mutually antagonistic. In the absence of androgen, activin induced apoptosis in the androgen-dependent human prostate cancer cell line LNCaP, an effect suppressed by androgen administration. Although activin by itself did not alter the cell cycle distribution, it potently suppressed androgen- induced progression of cells into S-phase of the cell cycle and thus inhibited androgen-stimulated growth of LNCaP cells. Expression changes in cell cycle regulatory proteins such as Rb, E2F-1, and p27 demonstrated a strong correlation with the mutually antagonistic growth regulatory effects of activin and androgen. The inhibitory effect of activin on growth was independent of serine, serine, valine, serine motif phosphorylation of Smad3. Despite their antagonistic effect on growth, activin and androgen costimulated the expression of prostate-specific antigen through a Smad3-mediated mechanism. These observations indicate the existence of a complex cross talk between activin and androgen signaling in regulation of gene expression and growth of the prostate.