Engineered Pathways for Correct Disulfide Bond Oxidation

Correct formation of disulfide bonds is critical for protein folding. We find that cells lacking protein disulfide isomerases (PDIs) can use alternative mechanisms for correct disulfide bond formation. By linking correct disulfide bond formation to antibiotic resistance, we selected mutants that catalyze correct disulfide formation in the absence of DsbC, Escherichia coli's PDI. Most of our mutants massively overproduce the disulfide oxidase DsbA and change its redox status. They enhance DsbA's ability to directly form the correct disulfides by increasing the level of mixed disulfides between DsbA and substrate proteins. One mutant operates via a different mechanism; it contains mutations in DsbB and CpxR that alter the redox environment of the periplasm and increases the level of the chaperone/protease DegP, allowing DsbA to gain disulfide isomerase ability in vivo. Thus, given the proper expression level, redox status, and chaperone assistance, the oxidase DsbA can readily function in vivo to catalyze the folding of proteins with complex disulfide bond connectivities. Our selection reveals versatile strategies for correct disulfide formation in vivo. Remarkably, our evolution of new pathways for correct disulfide bond formation in E. coli mimics eukaryotic PDI, a highly abundant partially reduced protein with chaperone activity. Antioxid. Redox Signal. 14, 2399-2412.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90429/1/ars-2E2010-2E3782.pd

From Biology to Biotechnology: Disulfide Bond Formation in <i>Escherichia coli</i>

Disulfide bonds formed between a pair of oxidized cysteines are important to the structural integrity and proper folding of many proteins. Accordingly, Nature has evolved several systems for the genesis and maintenance of such bonds. Beginning with the discovery of protein disulfide isomerase, which provided the first evidence for enzyme-catalyzed disulfide-bond formation, many years of research have resulted in the explication of the complex network of electron transport pathways needed for this process. Herein, we take a historical approach in describing the elucidation of disulfide-bond formation in E. coli. We frame this topic in the context of genome sequencing eras. The first section describes the discovery of eukaryotic protein disulfide isomerase and the subsequent research that followed from the early 1960s to the early 1990s, a time period we have named the pre-genomic sequencing era. The second section details the renaissance in research on disulfide-bond formation in the periplasm of prokaryotes, fueled by bacterial genetic screens and the development of genomic sequencing technology. Accordingly, we have named this section the genomic sequencing era, which ranges from the early 1990s to approximately 2010. The final section outlines the use of bacterial genetic screens to select for new oxidoreductase enzymes and their potential uses in biotechnological and pharmaceutical applications. This era we have dubbed the post-genomic sequencing era, and we envision it to represent the future of research on oxidative folding

Surplus or Deficit? Spatiotemporal Variations of the Supply, Demand, and Budget of Landscape Services and Landscape Multifunctionality in Suburban Shanghai, China

Landscape services are inevitably interlinked with human wellbeing. It is essential to assess landscape services and multifunctionality from both supply and demand points of view toward sustainable landscape management. This study focused on the spatiotemporal variations of the supply, demand, and budget of landscape services in suburban Shanghai, China, including crop production, nutrient regulation, air-quality regulation, soil-erosion regulation, water purification, and recreation and aesthetical value. A new index landscape multifunctionality budget (BMFI) was developed, integrating the budget status of surplus and deficit with landscape management. Spatial autocorrelation analysis and regression analysis were conducted to identify spatial agglomeration and influencing factors of BMFI. Pronounced spatiotemporal heterogeneity of landscape services was observed. BMFI was in surplus status in 2005 and 2010, but turned to deficit in 2015. Landscape service budgets generally followed the spatial pattern of positive in the west and negative in the east. Budget deficits covered half of the villages in 2015, which were mainly situated near central Shanghai with high population density, high average income, and a fragmented and less diverse landscape pattern. Rapid urban sprawl and the following land-cover changes are the main drivers for the spatiotemporal variations. Landscape function zoning with effective economic development and ecological conservation policies can comprehensively improve the competitiveness achieving sustainable future.National Natural Science Foundation of ChinaPeer Reviewe

KCAT: A Knowledge-Constraint Typing Annotation Tool

Fine-grained Entity Typing is a tough task which suffers from noise samples extracted from distant supervision. Thousands of manually annotated samples can achieve greater performance than millions of samples generated by the previous distant supervision method. Whereas, it's hard for human beings to differentiate and memorize thousands of types, thus making large-scale human labeling hardly possible. In this paper, we introduce a Knowledge-Constraint Typing Annotation Tool (KCAT), which is efficient for fine-grained entity typing annotation. KCAT reduces the size of candidate types to an acceptable range for human beings through entity linking and provides a Multi-step Typing scheme to revise the entity linking result. Moreover, KCAT provides an efficient Annotator Client to accelerate the annotation process and a comprehensive Manager Module to analyse crowdsourcing annotations. Experiment shows that KCAT can significantly improve annotation efficiency, the time consumption increases slowly as the size of type set expands.Comment: 6 pages, acl2019 demo pape

Utilizing redox-sensitive GFP fusions to detect in vivo redox changes in a genetically engineered prokaryote

Understanding the in vivo redox biology of cells is a complex albeit important biological problem. Studying redox processes within living cells without physical disruption or chemical modifications is essential in determining the native redox states of cells. In this study, the previously characterized reduction-oxidation sensitive green fluorescent protein (roGFP2) was used to elucidate the redox changes of the genetically engineered Escherichia coli strain, SHuffle. SHuffle cells were demonstrated to be under constitutive oxidative stress and responding transcriptionally in an OxyR-dependent manner. Using roGFP2 fused to either glutathione (GSH)- or hydrogen peroxide (H2O2)- sensitive proteins (glutaredoxin 1 or Orp1), the cytosolic redox state of both wild type and SHuffle cells based on GSH/GSSG and H2O2 pools was measured. These probes open the path to in vivo studies of redox changes and genetic selections in prokaryotic hosts

Genetic Selection for Enhanced Folding In Vivo Targets the Cys14-Cys38 Disulfide Bond in Bovine Pancreatic Trypsin Inhibitor

The periplasm provides a strongly oxidizing environment; however, periplasmic expression of proteins with disulfide bonds is often inefficient. Here, we used two different tripartite fusion systems to perform in vivo selections for mutants of the model protein bovine pancreatic trypsin inhibitor (BPTI) with the aim of enhancing its expression in Escherichia coli. This trypsin inhibitor contains three disulfides that contribute to its extreme stability and protease resistance. The mutants we isolated for increased expression appear to act by eliminating or destabilizing the Cys14-Cys38 disulfide in BPTI. In doing so, they are expected to reduce or eliminate kinetic traps that exist within the well characterized in vitro folding pathway of BPTI. These results suggest that elimination or destabilization of a disulfide bond whose formation is problematic in vitro can enhance in vivo protein folding. The use of these in vivo selections may prove a valuable way to identify and eliminate disulfides and other rate-limiting steps in the folding of proteins, including those proteins whose in vitro folding pathways are unknown. Antioxid. Redox Signal. 14, 973-984.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90494/1/ars-2E2010-2E3712.pd