Biology subjects

White, W. L.

Publications and source records attributed to White, W. L..

4 recordsLinked to original sources

bioRxiv2025

Design of solubly expressed miniaturized SMART MHCs

The precise recognition of specific peptide-MHC (pMHC) complexes by T-cell receptors (TCRs) plays a key role in infectious disease, cancer and autoimmunity. A critical step in many immunobiological studies is the identification of T-cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in Escherichia coli (E. coli) and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing {beta}2-microglobulin ({beta}2m) and the heavy chain 3 domain, and enable soluble expression of both H-2Db and A*02:01 in E. coli. We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity, and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T-cell repertoire in approaches ranging from yeast display to T-cell staining. SignificanceDespite the critical role that TCR/pMHC interactions play in human health, it has remained difficult to produce reagents necessary to study them. Requirements for refolding or sequence optimization limit immunologists and biochemists ability to characterize diverse pMHC/TCR interactions. Here, we develop a de-novo designed protein domain that stabilizes the H-2Db and A*02:01 class I MHC allomorphs, allowing soluble expression in E. coli without the need for a stabilizing peptide, and improving display on the yeast surface, while maintaining peptide and TCR binding interactions. These features facilitate a wide range of experiments to more fully understand the nature of pMHC/TCR interactions, and pave the way for the development of stabilizing domains for all MHC allomorphs.

biochemistry↗

bioRxiv2024

Proofreading and single-molecule sensitivity in T-cellreceptor signaling by condensate nucleation

T-cells display the remarkable ability to detect single foreign peptides displayed on target cells, while ignoring highly abundant self peptides. This selectivity has been explained by kinetic proofreading in the T-cell receptor (TCR) signaling pathway, which prevents responses to short-lived binding events regardless of their abundance. However, the biochemical mechanisms that drive kinetic proofreading have remained unclear. Here, using computational modeling, we show that these key signaling properties of the TCR pathway can emerge from the dynamics of LAT phosphorylation, diffusion, and condensation following TCR-pMHC binding. In this model, time delays in LAT condensate nucleation underlie kinetic proofreading, enabling selective signaling responses to high-affinity pMHC ligands. The cooperativity in the nucleation and growth of LAT condensates also provides a mechanism to amplify weak signals from single foreign peptides and for condensates to grow with increasing antigen numbers. In contrast to other models, condensate-nucleation proofreading predicts a dependence of signal strength on pMHC spacing at fixed number, a prediction we validated experimentally using a protein scaffold to present pMHCs at defined intervals. Our results suggest that nucleation-condensation proofreading underlies the remarkable antigen detection capabilities of the TCR signaling pathway. SignificanceTo fight infections and cancer, T-cells must selectively recognize low levels of foreign peptides from pathogens or cancer cells, but the mechanisms that enable these properties have remained unclear.Using mathematical modeling and experiments, we find that T-cells can selectively detect single foreign peptides through a clustering process where a key protein downstream of the T-cell receptor forms condensates containing hundreds of signaling proteins. This condensate nucleation process can explain experimentally observed features of T-cell signaling, including our finding that the size of signaling clusters depends on the distance between foreign peptides. Our work reveals a key role for the condensation of signaling molecules in setting the spatial and temporal thresholds that control the sensitivity and selectivity of T-cells.

biophysics↗

bioRxiv2023

Bacteroidia and Clostridia perform a progressive cascade of polysaccharide degradation along the hindgut of the herbivorous fish Kyphosus sydneyanus

The gut microbiota of the marine herbivorous fish Kyphosus sydneyanus are thought to play an important role in host nutrition by supplying short-chain fatty acids (SCFA) through fermentation of dietary macroalgae. Here, we assembled 645 metagenome-assembled genomes (MAGs) from wild fish to determine the capacity of different bacterial taxa to degrade seaweed carbohydrates along the gut. Most bacteria (99%) were unclassified at the species level, highlighting taxonomic novelty dominated by Bacteroidia and Clostridia within the gut community. The presence of genes encoding endo-acting CAZymes in both phyla suggest they have a role in initiating glycan depolymerization. Bacteroidia also contributed the most to CAZyme-related gene expression in the distal hindgut, and encoded the highest densities of CAZymes within the community. In particular, the enrichment of CAZyme gene clusters (CGCs) within the Bacteroidia genus Alistipes (n = 73 versus just 59 distributed across all other taxa) points to an enhanced capacity for macroalgal polysaccharide utilization (e.g., alginate, laminarin and sulfated polysaccharides). Pairwise correlations of MAG relative abundances and encoded CAZyme compositions provide evidence of potential inter-species collaborations, whereby co-abundant MAGs exhibited complementary degradative capacities for specific substrates. Results indicated flexibility across these co-abundant groups in their capacity to source carbon (e.g., glucose or galactose-rich glycans), which possibly facilitates coexistence via niche partitioning. Our results indicate the potential for collaborative microbial carbohydrate metabolism in the gut of K. sydneyanus by Bacteroidia and Clostridia, and suggest that members of the genus Alistipes are a metabolically and taxonomically diverse group of specialized macroalgae biomass degraders.

microbiology↗

bioRxiv2023

Antigen perception in T cells by long-term Erk and NFAT signaling dynamics.

Immune system threat detection hinges on T cells ability to perceive varying peptide major-histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs, but diverge only over longer (9+ hrs) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception, and establish a framework for understanding T cell responses under diverse contexts. SIGNIFICANCE STATEMENTTo counter diverse pathogens, T cells mount distinct responses to varying peptide-major histocompatibility complex ligands (pMHCs). They perceive the affinity of pMHCs for the T cell receptor (TCR), which reflects its foreignness, as well as pMHC abundance. By tracking signaling responses in single living cells to different pMHCs, we find that T cells can independently perceive pMHC affinity vs dose, and encode this information through the dynamics of Erk and NFAT signaling pathways downstream of the TCR. These dynamics are jointly decoded by gene regulatory mechanisms to produce pMHC-specific activation responses. Our work reveals how T cells can elicit tailored functional responses to diverse threats and how dysregulation of these responses may lead to immune pathologies.

immunology↗