The N-terminal Helix Controls the Transition between the Soluble and Amyloid States of an FF Domain

Background

Protein aggregation is linked to the onset of an increasing number of human nonneuropathic (either localized or systemic) and neurodegenerative disorders. In particular, misfolding of native α-helical structures and their self-assembly into nonnative intermolecular β-sheets has been proposed to trigger amyloid fibril formation in Alzheimer’s and Parkinson’s diseases.

Methods

Here, we use a battery of biophysical techniques to elucidate the conformational conversion of native α-helices into amyloid fibrils using an all-α FF domain as a model system.

Results

We show that under mild denaturing conditions at low pH this FF domain self-assembles into amyloid fibrils. Theoretical and experimental dissection of the secondary structure elements in this domain indicates that the helix 1 at the N-terminus has both the highest α-helical and amyloid propensities, controlling the transition between soluble and aggregated states of the protein.

Conclusions

The data illustrates the overlap between the propensity to form native α-helices and amyloid structures in protein segments.

Significance

The results presented contribute to explain why proteins cannot avoid the presence of aggregation-prone regions and indeed use stable α-helices as a strategy to neutralize such potentially deleterious stretches.     

Structural and Phylogenetic Studies with MjTX-I Reveal a Multi-Oligomeric Toxin – a Novel Feature in Lys49-PLA2s Protein Class

The mortality caused by snakebites is more damaging than many tropical diseases, such as dengue haemorrhagic fever, cholera, leishmaniasis, schistosomiasis and Chagas disease. For this reason, snakebite envenoming adversely affects health services of tropical and subtropical countries and is recognized as a neglected disease by the World Health Organization. One of the main components of snake venoms is the Lys49-phospholipases A₂, which is catalytically inactive but possesses other toxic and pharmacological activities. Preliminary studies with MjTX-I from Bothrops moojeni snake venom revealed intriguing new structural and functional characteristics compared to other bothropic Lys49-PLA₂s. We present in this article a comprehensive study with MjTX-I using several techniques, including crystallography, small angle X-ray scattering, analytical size-exclusion chromatography, dynamic light scattering, myographic studies, bioinformatics and molecular phylogenetic analyses.Based in all these experiments we demonstrated that MjTX-I is probably a unique Lys49-PLA₂, which may adopt different oligomeric forms depending on the physical-chemical environment. Furthermore, we showed that its myotoxic activity is dramatically low compared to other Lys49-PLA₂s, probably due to the novel oligomeric conformations and important mutations in the C-terminal region of the protein. The phylogenetic analysis also showed that this toxin is clearly distinct from other bothropic Lys49-PLA₂s, in conformity with the peculiar oligomeric characteristics of MjTX-I and possible emergence of new functionalities inresponse to environmental changes and adaptation to new preys.     

Natural history collections and the future legacy of ecological research

Oecologia - Natural history collections are now being championed as key to broad ecological studies, especially those involving human impacts in the Anthropocene. However, collections are going...     

The extrapituitary prolactin promoter polymorphism is associated with rheumatoid arthritis and anti-CCP antibodies in Mexican population

Prolactin (PRL) is a hormone–cytokine that has been involved in autoimmunity due to its immunoregulatory and lymphoproliferative effects. It is produced by various extrapituitary sites including immune cells, under control of a superdistal promoter that contains a single nucleotide polymorphism − 1149 G/T previously associated with rheumatoid arthritis (RA) susceptibility in European population. The aim of this study was to investigate the association of the extrapituitary PRL − 1149 G/T promoter polymorphism with clinical parameters, clinical activity and disability indices in RA patients from Western Mexico and to analyze the PRL mRNA expression according to the PRL − 1149 G/T promoter polymorphism in total leucocytes from RA patients and controls. We conducted a case–control study that included 258 RA patients and 333 control subjects (CS). The DNA samples were genotyped using the PCR–RFLP method and the PRL mRNA expression was determined by quantitative real time PCR. PRL serum levels and antibodies to cyclic citrullinated peptides (anti-CCP) were measured with ELISA. We found significant differences in the genotype (p = 0.022) and allelic (p = 0.046) distribution of the polymorphism between RA patients and control subjects. According to the dominant genetic model, there is an association between the T allele (GT + TT genotypes) and decreased RA susceptibility in comparison to the G allele carriers (GG genotype) (OR 0.64, 95% CI 0.45–0.92; p = 0.011). The T allele carriers (GT + TT genotypes) had lower titers of anti-CCP antibodies in comparison to the G allele carriers (GG genotype) (median, 66 U/mL vs. 125 U/mL; p = 0.03). Furthermore, the GG homozygotes had higher PRL mRNA expression in comparison to the GT heterozygotes, and this latter with respect to the TT homozygotes, in both groups (RA: 1 > 0.72 > 0.19; CS: 1 > 0.54 > 0.28). However, PRL serum levels were similar in both groups. Our results suggest that the PRL − 1149 T allele is a genetic marker for decreased RA susceptibility and is associated with lower titers of anti-CCP antibodies in Mexican population. We also suggest influence of genotype upon PRL mRNA expression.     

Role of Central Metabolism in the Osmoadaptation of the Halophilic Bacterium Chromohalobacter salexigens

Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 m NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-¹³C]-, [2-¹³C]-, [6-¹³C]-, and [U-¹³C₆]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria.     

A population-based study of first and second-line drug-resistant tuberculosis in a high-burden area of the Mexico/United States border

The resistance of 139 Mycobacterium tuberculosis (MTB) isolates from the city of Monterrey, Northeast Mexico, to first and second-line anti-TB drugs was analysed. A total of 73 isolates were susceptible and 66 were resistant to anti-TB drugs. Monoresistance to streptomycin, isoniazid (INH) and ethambutol was observed in 29 cases. Resistance to INH was found in 52 cases and in 29 cases INH resistance was combined with resistance to two or three drugs. A total of 24 isolates were multidrug-resistant (MDR) resistant to at least INH and rifampicin and 11 MDR cases were resistant to five drugs. The proportion of MDR-TB among new TB cases in our target population was 0.72% (1/139 cases). The proportion of MDR-TB among previously treated cases was 25.18% (35/139 cases). The 13 polyresistant and 24 MDR isolates were assayed against the following seven second-line drugs: amikacin (AMK), kanamycin (KAN), capreomycin (CAP), clofazimine (CLF), ethionamide (ETH), ofloxacin (OFL) and cycloserine (CLS). Resistance to CLF, OFL or CLS was not observed. Resistance was detected to ETH (10.80%) and to AMK (2.70%), KAN (2.70%) and CAP (2.70%). One isolate of MDR with primary resistance was also resistant to three second-line drugs. Monterrey has a high prevalence of MDR-TB among previously treated cases and extensively drug-resistant-MTB strains may soon appear.     

Roles of Wnt8a during formation and patterning of the mouse inner ear

Fgf and Wnt signalling have been shown to be required for formation of the otic placode in vertebrates. Whereas several Fgfs including Fgf3, Fgf8 and Fgf10 have been shown to participate during early placode induction, Wnt signalling is required for specification and maintenance of the otic placode, and dorsal patterning of the otic vesicle. However, the requirement for specific members of the Wnt gene family for otic placode and vesicle formation and their potential interaction with Fgf signalling has been poorly defined. Due to its spatiotemporal expression during placode formation in the hindbrain Wnt8a has been postulated as a potential candidate for its specification. Here we have examined the role of Wnt8a during formation of the otic placode and vesicle in mouse embryos. Wnt8a expression depends on the presence of Fgf3 indicating a serial regulation between Fgf and Wnt signalling during otic placode induction and specification. Wnt8a by itself however is neither essential for placode specification nor redundantly required together with Fgfs for otic placode and vesicle formation. Interestingly however, Wnt8a and Fgf3 are redundantly required for expression of Fgf15 in the hindbrain indicating additional reciprocal interactions between Fgf and Wnt signalling. Further reduction of Wnt signalling by the inactivation of Wnt1 in a Wnt8a mutant background revealed a redundant requirement for both genes during morphogenesis of the dorsal portion of the otic vesicle.     

The role of very small fragments in conserving genetic diversity of a common tree in a hyper fragmented Brazilian Atlantic forest landscape

In hyper fragmented biomes, conservation of extant biota relies on preservation and proper management of remnants. The maintenance of genetic diversity and functional connectivity in a landscape context is probably key to long-term conservation of remnant populations. We measured the genetic diversity in seedlings and adults of tree Copaifera langsdorffii and evaluated whether edge and density-dependent effects drive natural regeneration in a set of very small and degraded Brazilian Atlantic forest fragments. We evaluated the role of small remnants in the conservation of genetic diversity in a hyper fragmented landscape and discuss the challenge of long-term population sustainability of such altered habitats. High genetic diversity in adults indicated these fragments are valuable targets for C. langsdorffii in situ conservation, but both genetic diversity and divergence among patches decreased in seedlings. In our landscape, regeneration increased as it neared edges and adults; suggesting this population is resilient to fragmentation. However, at a broader scale, current levels of gene flow have not been sufficient to prevent the loss of genetic diversity across generations. Restoration plans, even at a small scale, are necessary to promote fragment connectivity and spatially expand opportunities for the fairly restricted gene flow observed in this severely fragmented Brazilian Atlantic forest region.