Biology subjects

Miller, S. J.

Publications and source records attributed to Miller, S. J..

7 recordsLinked to original sources

bioRxiv2025

A painless nerve growth factor variant uncouples nociceptive and neurotrophic TrkA signaling

Nerve growth factor (NGF) binding to the receptor tyrosine kinase, TrkA, drives neurotrophic signaling essential for neuronal development and survival. This interaction simultaneously drives peripheral pain, making this pathway an attractive but complicated therapeutic target for chronic pain. By integrating single-molecule microscopy, structural and electrophysiology analyses, with a human NGF variant, NGFpainless, which retains neurotrophic effects but abolishes pain, we delineate the molecular mechanisms that bias TrkA signaling towards neurotrophic functions without triggering nociception. We show that, unlike wild-type NGF, NGFpainless fails to sensitize TRPV1 channels to capsaicin, thus disengaging TrkA from the nociceptive pathway. We further show that this selective loss of nociceptive TrkA signaling by NGFpainless results from its reduced ability to activate PLC{gamma}1 and trigger calcium release compared to NGF, while still preserving the ERK and AKT signaling essential for neurotrophic functions. This biased signaling arises from reduced electrostatic complementarity at the TrkA:NGFpainless complex interface, which shortens the lifetime of this functional complex on native membranes. Mutations in TrkA that restore the electrostatic complementarity at the TrkA:NGFpainless interface eliminate biased signaling. This mechanistic understanding of TrkA binding by NGFpainless, and how it differs from NGF, will spur the development of two therapeutic classes of molecules - one that selectively suppresses nociceptive signaling while preserving neurotrophic functions in chronic pain, and another that enhances neurotrophic activity without evoking peripheral pain in neurodegenerative conditions.

neuroscience↗

bioRxiv2025

A cross model spatial and single-cell atlas reveals the conserved involvement of osteopontin in polycystic kidney disease

Polycystic kidney disease (PKD) arises from mutations in cilia-associated genes, such as PKD1 and PKD2, expressed in renal epithelial cells, leading to progressive kidney dysfunction and end- stage kidney disease (ESKD). PKD patients exhibit significant heterogeneity in disease progression, largely due to genetic and environmental modifiers. Like patients, mouse models of PKD also exhibit significant heterogeneity with regards to the gene mutated, age of disease onset, and rate of disease progression. To elucidate the cellular and molecular consequences of these variables, we constructed an integrated single-cell and spatial transcriptomics atlas across mouse models of PKD, mapping changes in cell type composition, gene expression, and intercellular signaling networks across the whole atlas and within individual models. Consistently across models, single cell RNA sequencing (scRNAseq) data revealed increased Spp1 (osteopontin) expression and signaling from PKD-enriched clusters to Ly6clo monocytes. Global deletion of Spp1 in Pkd1RC/RC mice resulted in reduced cyst severity, improved kidney function, and reduced Ly6clo monocyte numbers, suggesting that SPP1 signaling to Ly6clo monocytes promotes PKD progression. We also created a freely available, searchable website (https://bmblx.bmi.osumc.edu/scPKD/) that can be used to identify cross- and intra-model specific changes in gene expression, guiding researchers to new therapeutic targets for treating PKD.

bioinformatics↗

bioRxiv2024

β-amino acids reduce ternary complex stability and alter the translation elongation mechanism

Templated synthesis of proteins containing non-natural amino acids (nnAAs) promises to vastly expand the chemical space available to biological therapeutics and materials. Existing technologies limit the identity and number of nnAAs than can be incorporated into a given protein. Addressing these bottlenecks requires deeper understanding of the mechanism of messenger RNA (mRNA) templated protein synthesis and how this mechanism is perturbed by nnAAs. Here we examine the impact of both monomer backbone and side chain on formation and ribosome-utilization of the central protein synthesis substate: the ternary complex of native, aminoacylated transfer RNA (aa-tRNA), thermally unstable elongation factor (EF-Tu), and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer (FRET) measurements, we reveal the dramatic effect of monomer backbone on ternary complex formation and protein synthesis. Both the (R) and (S)-{beta}2 isomers of Phe disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-{beta}3-Phe reduce ternary complex stability by approximately one order of magnitude. Consistent with these findings, (R)- and (S)-{beta}2-Phe-charged tRNAs were not utilized by the ribosome, while (R)- and (S)-{beta}3-Phe stereoisomers were utilized inefficiently. The reduced affinities of both species for EF-Tu ostensibly bypassed the proofreading stage of mRNA decoding. (R)-{beta}3-Phe but not (S)-{beta}3-Phe also exhibited order of magnitude defects in the rate of substrate translocation after mRNA decoding, in line with defects in peptide bond formation that have been observed for D--Phe. We conclude from these findings that non-natural amino acids can negatively impact the translation mechanism on multiple fronts and that the bottlenecks for improvement must include consideration of the efficiency and stability of ternary complex formation.

biochemistry↗

bioRxiv2023

Backbone extension acyl rearrangements enable cellular synthesis of proteins with internal β2-peptide linkages

Proteins and polypeptides containing extended backbone monomers embody highly desirable structures and functions, but they cannot yet be biosynthesized in cells. There are two challenges at work. First is the ribosome, whose ability to promote rapid bond-forming reactions to and from anything other than an -amino acid or -hydroxy acid is unknown. The second challenge is the absence of orthogonal enzymes that acylate tRNA with extended backbone monomers. Here we describe a general approach to the programmed cellular synthesis of proteins containing extended backbone monomers that circumvents both of these challenges. Rather than relying on direct and uncharacterized reactions of non--amino acid monomers within the ribosomal PTC, we develop a proximity-guided intramolecular rearrangement that effectively edits the protein backbone post-translationally. The method relies on the ability of PylRS-like aminoacyl-tRNA synthetase enzymes to accept diverse -hydroxy acid monomers, including those whose side chains contain masked nucleophiles. Introduction of such an -hydroxy acid monomer into a protein translated in vivo, followed by nucleophile unmasking, sets up a thermodynamically favored and quantitative intramolecular Backbone Extension Acyl Rearrangement (BEAR) reaction that edits the protein backbone to install an extended backbone monomer. In the examples described here, the intramolecular rearrangement converts an -peptide backbone directly into a {beta}-backbone. As far as we know, this report represents the first example in which a much-desired expanded backbone {beta}-amino acid linkage has been introduced site-selectively into a protein in a cell.

synthetic biology↗

bioRxiv2023

Addition of adjuvant to DTaP modulates vaccine-induced immunological responses but is insufficient to improve protection in CD-1 mice

3.1Pertussis is a vaccine-preventable respiratory disease caused by the Gram-negative bacterium Bordetella pertussis. While vaccination rates remain high in developed countries, incidence of pertussis has increased following the transition from wP vaccines to aP vaccines. The reemergence of pertussis is attributed, in part, to waning immunity induced by aP vaccination. Therefore, the objective of this work was to determine if addition of adjuvant to DTaP can modulate the immune response and improve protection compared to DTaP alone. In this study we immunized outbred, female CD-1 mice with 1/320th the human dose of vehicle control, DTaP, and DTaP supplemented with adjuvant. Markers of early vaccine-induced memory were measured using a chemokine assay or by flow cytometry. Protection was assessed by measuring serological responses and quantifying bacterial burden in the respiratory tract at day 3 post-challenge. From this work we identified a partially protective aP vaccine dose to use for vaccination and challenge studies. We observed that MPLA and SWE promote robust anti-B. pertussis antibody responses and stimulate significant increases in early markers of vaccine-induced memory such as CXCL13, FDCs, and TFH cells. Quil-A induced Th1 responses compared to DTaP alone, but none of the adjuvants improved protection against challenge with B. pertussis. Overall, the data suggests that addition of adjuvant modulates the protective immune responses induced by aPs. Further studies are needed to evaluate the B cell compartment and longevity of protection.

immunology↗

bioRxiv2022

Atomistic simulations of the E. coli ribosome provide selection criteria for translationally active substrates

As genetic code expansion advances beyond L--amino acids to backbone modifications and new polymerization chemistries, the field faces an increasingly broad challenge to discover what the ribosome can accommodate. Although the E. coli ribosome tolerates non-L--amino acids in vitro, few structural insights are available, and the boundary conditions for efficient bond formation are unknown. We describe a 2.1 [A] cryo-EM structure of the E. coli ribosome containing well-resolved -amino acid monomers coupled with a computational approach for which energy surface minima produced by metadynamics trend in agreement with established incorporation efficiencies. Reactive monomers across diverse structural classes favor a conformational space characterized by an A-site nucleophile to P-site carbonyl distance of < 4 [A] and a Burgi-Dunitz angle of 90-110{degrees}. Monomers whose free energy minima fall outside these regions do not react. Application of this model should accelerate the in vivo and in vitro ribosomal synthesis and application of sequence-defined, non-peptide heterooligomers.

synthetic biology↗

bioRxiv2020

Temperature variability alters the stability and thresholds for collapse of interacting species

Temperature variability and extremes can have profound impacts on populations and ecological communities. Predicting impacts of thermal variability poses a challenge because it has both direct physiological effects and indirect effects through species interactions. In addition, differences in thermal performance between predators and prey and non-linear averaging of temperature-dependent performance can result in complex and counterintuitive population dynamics in response to climate change. Yet the combined consequences of these effects remain underexplored. Here, modeling temperature-dependent predator-prey dynamics, we study how changes in temperature variability affect population size, collapse, and stable coexistence of both predator and prey, relative to under constant environments or warming alone. We find that the effects of temperature variation on interacting species can lead to a diversity of outcomes, from predator collapse to stable coexistence, depending on interaction strengths and differences in species thermal performance. Temperature variability also alters predictions about population collapse - in some cases allowing predators to persist for longer than predicted when considering warming alone, and in others accelerating collapse. To inform management responses that are robust to future climates with increasing temperature variability and extremes, we need to incorporate the consequences of temperature variation in complex ecosystems.

ecology↗