Mechanism of Fine-tuning pH Sensors in Proprotein Convertases: IDENTIFICATION OF A pH-SENSING HISTIDINE PAIR IN THE PROPEPTIDE OF PROPROTEIN CONVERTASE 1/3*

The propeptides of proprotein convertases (PCs) regulate activation of cognate protease domains by sensing pH of their organellar compartments as they transit the secretory pathway. Earlier experimental work identified a conserved histidine-encoded pH sensor within the propeptide of the canonical PC, furin. To date, whether protonation of this conserved histidine is solely responsible for PC activation has remained unclear because of the observation that various PC paralogues are activated at different organellar pH values. To ascertain additional determinants of PC activation, we analyzed PC1/3, a paralogue of furin that is activated at a pH of ∼5.4. Using biophysical, biochemical, and cell-based methods, we mimicked the protonation status of various histidines within the propeptide of PC1/3 and examined how such alterations can modulate pH-dependent protease activation. Our results indicate that whereas the conserved histidine plays a crucial role in pH sensing and activation of this protease an additional histidine acts as a “gatekeeper” that fine-tunes the sensitivity of the PC1/3 propeptide to facilitate the release inhibition at higher proton concentrations when compared with furin. Coupled with earlier analyses that highlighted the enrichment of the amino acid histidine within propeptides of secreted eukaryotic proteases, our work elucidates how secreted proteases have evolved to exploit the pH of the secretory pathway by altering the spatial juxtaposition of titratable groups to regulate their activity in a spatiotemporal fashion.     

Naphthoquinone-mediated Inhibition of Lysine Acetyltransferase KAT3B/p300, Basis for Non-toxic Inhibitor Synthesis

Hydroxynaphthoquinone-based inhibitors of the lysine acetyltransferase KAT3B (p300), such as plumbagin, are relatively toxic. Here, we report that free thiol reactivity and redox cycling properties greatly contribute to the toxicity of plumbagin. A reactive 3rd position in the naphthoquinone derivatives is essential for thiol reactivity and enhances redox cycling. Using this clue, we synthesized PTK1, harboring a methyl substitution at the 3rd position of plumbagin. This molecule loses its thiol reactivity completely and its redox cycling ability to a lesser extent. Mechanistically, non-competitive, reversible binding of the inhibitor to the lysine acetyltransferase (KAT) domain of p300 is largely responsible for the acetyltransferase inhibition. Remarkably, the modified inhibitor PTK1 was a nearly non-toxic inhibitor of p300. The present report elucidates the mechanism of acetyltransferase activity inhibition by 1,4-naphthoquinones, which involves redox cycling and nucleophilic adduct formation, and it suggests possible routes of synthesis of the non-toxic inhibitor.     

Functional domains of the 50S subunit mature late in the assembly process

Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages of assembly of the 50S subunit using Bacillus subtilis cells depleted of RbgA, a highly conserved GTPase. We found that RbgA-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins L16, L27, L28, L33a, L35 and L36. Using a novel pulse labeling/quantitative mass spectrometry technique, we show that this particle is physiologically relevant and is capable of maturing into a complete 50S particle. Cryo-electron microscopy and chemical probing revealed that the central protuberance, the GTPase associating region and tRNA-binding sites in this intermediate are unstructured. These findings demonstrate that key functional sites of the 50S subunit remain unstructured until late stages of maturation, preventing the incomplete subunit from prematurely engaging in translation. Finally, structural and biochemical analysis of a ribosome particle depleted of L16 indicate that L16 binding is necessary for the stimulation of RbgA GTPase activity and, in turn, release of this co-factor, and for conversion of the intermediate to a complete 50S subunit.     

The Macroecology of Airborne Pollen in Australian and New Zealand Urban Areas

The composition and relative abundance of airborne pollen in urban areas of Australia and New Zealand are strongly influenced by geographical location, climate and land use. There is mounting evidence that the diversity and quality of airborne pollen is substantially modified by climate change and land-use yet there are insufficient data to project the future nature of these changes. Our study highlights the need for long-term aerobiological monitoring in Australian and New Zealand urban areas in a systematic, standardised, and sustained way, and provides a framework for targeting the most clinically significant taxa in terms of abundance, allergenic effects and public health burden.     

Village-Randomized Clinical Trial of Home Distribution of Zinc for Treatment of Childhood Diarrhea in Rural Western Kenya

Background

Zinc treatment shortens diarrhea episodes and can prevent future episodes. In rural Africa, most children with diarrhea are not brought to health facilities. In a village-randomized trial in rural Kenya, we assessed if zinc treatment might have a community-level preventive effect on diarrhea incidence if available at home versus only at health facilities.

Methods

We randomized 16 Kenyan villages (1,903 eligible children) to receive a 10-day course of zinc and two oral rehydration solution (ORS) sachets every two months at home and 17 villages (2,241 eligible children) to receive ORS at home, but zinc at the health–facility only. Children’s caretakers were educated in zinc/ORS use by village workers, both unblinded to intervention arm. We evaluated whether incidence of diarrhea and acute lower respiratory illness (ALRI) reported at biweekly home visits and presenting to clinic were lower in zinc villages, using poisson regression adjusting for baseline disease rates, distance to clinic, and children’s age.

Results

There were no differences between village groups in diarrhea incidence either reported at the home or presenting to clinic. In zinc villages (1,440 children analyzed), 61.2% of diarrheal episodes were treated with zinc, compared to 5.4% in comparison villages (1,584 children analyzed, p<0.0001). There were no differences in ORS use between zinc (59.6%) and comparison villages (58.8%). Among children with fever or cough without diarrhea, zinc use was low (<0.5%). There was a lower incidence of reported ALRI in zinc villages (adjusted RR 0.68, 95% CI 0.46–0.99), but not presenting at clinic.

Conclusions

In this study, home zinc use to treat diarrhea did not decrease disease rates in the community. However, with proper training, availability of zinc at home could lead to more episodes of pediatric diarrhea being treated with zinc in parts of rural Africa where healthcare utilization is low.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00530829","term_id":"NCT00530829"}}NCT00530829     

Using Microwave and Macroscopic Samples of Dielectric Solids to Study the Photonic Properties of Disordered Photonic Bandgap Materials

Recently, disordered photonic materials have been suggested as an alternative to periodic crystals for the formation of a complete photonic bandgap (PBG). In this article we will describe the methods for constructing and characterizing macroscopic disordered photonic structures using microwaves. The microwave regime offers the most convenient experimental sample size to build and test PBG media. Easily manipulated dielectric lattice components extend flexibility in building various 2D structures on top of pre-printed plastic templates. Once built, the structures could be quickly modified with point and line defects to make freeform waveguides and filters. Testing is done using a widely available Vector Network Analyzer and pairs of microwave horn antennas. Due to the scale invariance property of electromagnetic fields, the results we obtained in the microwave region can be directly applied to infrared and optical regions. Our approach is simple but delivers exciting new insight into the nature of light and disordered matter interaction.Our representative results include the first experimental demonstration of the existence of a complete and isotropic PBG in a two-dimensional (2D) hyperuniform disordered dielectric structure. Additionally we demonstrate experimentally the ability of this novel photonic structure to guide electromagnetic waves (EM) through freeform waveguides of arbitrary shape.     

High quality draft genome sequence of Streptomyces sp. strain AW19M42 isolated from a sea squirt in Northern Norway

Here we report the 8 Mb high quality draft genome of Streptomyces sp. strain AW19M42, together with specific properties of the organism and the generation, annotation and analysis of its genome sequence. The genome encodes 7,727 putative open reading frames, of which 6,400 could be assigned with COG categories. Also, 62 tRNA genes and 8 rRNA operons were identified. The genome harbors several gene clusters involved in the production of secondary metabolites. Functional screening of the isolate was positive for several enzymatic activities, and some candidate genes coding for those activities are listed in this report. We find that this isolate shows biotechnological potential and is an interesting target for bioprospecting.