Biology subjects

Stewart, L. J.

Publications and source records attributed to Stewart, L. J..

5 recordsLinked to original sources

bioRxiv2025

A Multivalent Pan-Ebolavirus Nanoparticle Vaccine Provides Protection in Rodents from Lethal Infection by Adapted Zaire and Sudan Viruses

Both Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) are members of the family Filoviridae, first discovered in 1976 during outbreaks of hemorrhagic fever in northern Zaire and southern Sudan. Ebola virus disease outbreaks are major public health events because of their potential for human-to-human transmission with high case fatality rates. Filoviral surface glycoproteins (GPs) are known to be the primary targets of neutralizing antibodies for protection from disease, and are the relevant immunogens in the two approved EBOV vaccines. Here we describe the design, electron microscopy-based structural characterization, and efficacy testing of a series of icosahedral I53-50 nanoparticles displaying prefusion trimeric EBOV and SUDV GP antigens. Mice and guinea pigs vaccinated with either a cocktail of EBOV-GP-I53-50 plus SUDV-GP-I53-50 or mosaic EBOV / SUDV-GP-I53-50 nanoparticles were protected from death or severe clinical signs of disease and weight loss, respectively, when challenged with either mouse-adapted EBOV or guinea pig-adapted SUDV.

immunology↗

bioRxiv2023

De novo design of buttressed loops for sculpting protein functions

In natural proteins, structured loops play central roles in molecular recognition, signal transduction and enzyme catalysis. However, because of the intrinsic flexibility and irregularity of loop regions, organizing multiple structured loops at protein functional sites has been very difficult to achieve by de novo protein design. Here we describe a solution to this problem that generates structured loops buttressed by extensive hydrogen bonding interactions with two neighboring loops and with secondary structure elements. We use this approach to design tandem repeat proteins with buttressed loops ranging from 9 to 14 residues in length. Experimental characterization shows the designs are folded and monodisperse, highly soluble, and thermally stable. Crystal structures are in close agreement with the computational design models, with the loops structured and buttressed by their neighbors as designed. We demonstrate the functionality afforded by loop buttressing by designing and characterizing binders for extended peptides in which the loops form one side of an extended binding pocket. The ability to design multiple structured loops should contribute quite generally to efforts to design new protein functions.

biochemistry↗

bioRxiv2022

Copper stress perturbs zinc and manganese homeostasis in Streptococcus pyogenes.

All bacteria possess homeostastic mechanisms that control the availability of micronutrient metals within the cell. Cross-talks between different metal homeostasis pathways within the same bacterial organism have been reported widely. In addition, there have been previous suggestions that some metal uptake transporters can promote adventitious uptake of the wrong metal. This work describes the cross-talk between Cu and the Zn and Mn homeostasis pathways in Group A Streptococcus (GAS). Using a {Delta}copA mutant strain that lacks the primary Cu efflux pump and thus traps excess Cu in the cytoplasm, we show that growth in the presence of supplemental Cu promotes downregulation of genes that contribute to Zn or Mn uptake. This effect is not associated with changes in cellular Zn or Mn levels. Co-supplementation of the culture medium with Zn or, to a lesser extent, Mn alleviates key Cu stress phenotypes, namely bacterial growth and secretion of the fermentation end-product lactate. However, neither co-supplemental Zn nor Mn influences cellular Cu levels or Cu availability in Cu-stressed cells. In addition, we provide evidence that the Zn or Mn uptake transporters in GAS do not promote Cu uptake. Together, the results from this study strengthen and extend our previous proposal that mis-regulation of Zn and Mn homeostasis is a key phenotype of Cu stress in GAS. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/509676v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@b33b23org.highwire.dtl.DTLVardef@efaa7borg.highwire.dtl.DTLVardef@a18b5dorg.highwire.dtl.DTLVardef@1c4913f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Large-scale design and refinement of stable proteins using sequence-only models

Engineered proteins generally must possess a stable structure in order to achieve their designed function. Stable designs, however, are astronomically rare within the space of all possible amino acid sequences. As a consequence, many designs must be tested computationally and experimentally in order to find stable ones, which is expensive in terms of time and resources. Here we report a neural network model that predicts protein stability based only on sequences of amino acids, and demonstrate its performance by evaluating the stability of almost 200,000 novel proteins. These include a wide range of sequence perturbations, providing a baseline for future work in the field. We also report a second neural network model that is able to generate novel stable proteins. Finally, we show that the predictive model can be used to substantially increase the stability of both expert-designed and model-generated proteins.

biophysics↗

bioRxiv2020

A role for glutathione in buffering excess intracellular copper in Streptococcus pyogenes

Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally-responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (Group A Streptococcus, GAS), this system is encoded by the copYAZ operon. In this study, we demonstrate that the site of GAS infection in vivo represents a Cu-rich environment but inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde 3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of non-cognate metal-binding sites. Surprisingly, the onset of these effects is delayed by [~]4 h even though expression of copZ is upregulated immediately upon exposure to Cu. We further show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. Our results indicate that GSH contributes to buffering of excess intracellular Cu when the transcriptionally-responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance that allows bacteria to maintain metabolism even in the presence of an excess of this metal ion. This study advances fundamental understanding of Cu handling in the bacterial cytoplasm. IMPORTANCEThe control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it is established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and is long assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this role of glutathione beyond measurement of the effect of Cu on growth of glutathione-deficient mutants. Our work with Group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.

microbiology↗