Composition-based prediction and rational manipulation of prion-like domain recruitment to stress granules.

Mutations in a number of stress granule-associated proteins have been linked to various neurodegenerative diseases. Several of these mutations are found in aggregation-prone prion-like domains (PrLDs) within these proteins. In this work, we examine the sequence features governing PrLD localization to stress granules upon stress. We demonstrate that many yeast PrLDs are sufficient for stress-induced assembly into microscopically visible foci that colocalize with stress granule markers. Additionally, compositional biases exist among PrLDs that assemble upon stress, and these biases are consistent across different stressors. Using these biases, we have developed a composition-based prediction method that accurately predicts PrLD assembly into foci upon heat shock. We show that compositional changes alter PrLD assembly behavior in a predictable manner, while scrambling primary sequence has little effect on PrLD assembly and recruitment to stress granules. Furthermore, we were able to design synthetic PrLDs that were efficiently recruited to stress granules, and found that aromatic amino acids, which have previously been linked to PrLD phase separation, were dispensable for this recruitment. These results highlight the flexible sequence requirements for stress granule recruitment and suggest that PrLD localization to stress granules is driven primarily by amino acid composition, rather than primary sequence.

A Hybrid DNA-Templated Gold Nanocluster For Enhanced Enzymatic Reduction of Oxygen.

We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC) of ∼1 nm in diameter and possessing ∼7 Au atoms. When integrated with bilirubin oxidase (BOD) and single walled carbon nanotubes (SWNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the overpotential of electrocatalytic oxygen reduction reaction (ORR) by ∼15 mV as compared to the enzyme alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is specific to nanoclusters and not to plasmonic gold particles. Rotating ring disk electrode (RRDE) measurements confirm 4e(-) reduction of O2 to H2O with minimal production of H2O2, suggesting that the presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication between the electrode surface and the enzyme active site. Finally, the AuNC displays phosphorescence with large Stokes shift and microsecond lifetime.

Structural and Biochemical Characterization of Staphylococcus aureus Cysteine Desulfurase Complex SufSU.

In this work, we provide the first in vitro characterization of two essential proteins from Staphylococcus aureus (S. aureus) involved in iron-sulfur (Fe-S) cluster biogenesis: the cysteine desulfurase SufS and the sulfurtransferase SufU. Together, these proteins form the transient SufSU complex and execute the first stage of Fe-S cluster biogenesis in the SUF-like pathway in Gram-positive bacteria. The proteins involved in the SUF-like pathway, such as SufS and SufU, are essential in Gram-positive bacteria since these bacteria tend to lack redundant Fe-S cluster biogenesis pathways. Most previous work characterizing the SUF-like pathway has focused on Bacillus subtilis (B. subtilis). We focus on the SUF-like pathway in S. aureus because of its potential to serve as a therapeutic target to treat S. aureus infections. Herein, we characterize S. aureus SufS (SaSufS) by X-ray crystallography and UV-vis spectroscopy, and we characterize S. aureus SufU (SaSufU) by a zinc binding fluorescence assay and small-angle X-ray scattering. We show that SaSufS is a type II cysteine desulfurase and that SaSufU is a Zn2+-containing sulfurtransferase. Additionally, we evaluated the cysteine desulfurase activity of the SaSufSU complex and compared its activity to that of B. subtilis SufSU. Subsequent cross-species activity analysis reveals a surprising result: SaSufS is significantly less stimulated by SufU than BsSufS. Our results set a basis for further characterization of SaSufSU as well as the development of new therapeutic strategies for treating infections caused by S. aureus by inhibiting the SUF-like pathway.

Effects of Mutations on the Aggregation Propensity of the Human Prion-Like Protein hnRNPA2B1.

Hundreds of human proteins contain prion-like domains, which are a subset of low-complexity domains with high amino acid compositional similarity to yeast prion domains. A recently characterized mutation in the prion-like domain of the human heterogeneous nuclear ribonucleoprotein hnRNPA2B1 increases the aggregation propensity of the protein and causes multisystem proteinopathy. The mutant protein forms cytoplasmic inclusions when expressed in Drosophila, the mutation accelerates aggregation in vitro, and the mutant prion-like domain can substitute for a portion of a yeast prion domain in supporting prion activity. To examine the relationship between amino acid sequence and aggregation propensity, we made a diverse set of point mutations in the hnRNPA2B1 prion-like domain. We found that the effects on prion formation in Saccharomyces cerevisiae and aggregation in vitro could be predicted entirely based on amino acid composition. However, composition was an imperfect predictor of inclusion formation in Drosophila; while most mutations showed similar behaviors in yeast, in vitro, and in Drosophila, a few showed anomalous behavior. Collectively, these results demonstrate the significant progress that has been made in predicting the effects of mutations on intrinsic aggregation propensity while also highlighting the challenges of predicting the effects of mutations in more complex organisms.