Biology subjects

McCarty, J.

Publications and source records attributed to McCarty, J..

9 recordsLinked to original sources

bioRxiv2026

Undergraduate Biophysical Chemistry Series: Teaching through a Combination of a Purpose-built Textbook, Research-derived Biomolecular Samples and Computer Labs

Biophysics is a rapidly advancing field with an incredible breadth of topics. Thus, undergraduate biophysics instructors have to strategize and decide what topics they will cover in their courses. Educational institutions utilize a variety of biophysics textbooks. A common deficiency of each of the existing texts is that it serves well a given set of topics (theory, illustrations, practice problems) and leaves out other areas. A typical example includes good theory and problems for thermodynamics and kinetics while presenting molecular dynamics and various spectroscopic methods in a lacking or outdated way. The authors of this manuscript teach a capstone Biophysical Chemistry three-quarter series (Western Washington University/WWU, Bellingham, WA) which ideally should resonate with the general and major-specific courses the students take within their major at WWU. To achieve this goal and to enrich the traditional lecture-based delivery, the instructors have developed and brought together key pedagogical elements: purpose-built online textbook with a uniform structure of the academic content and practice problems, a study sample (oligopeptide) of biophysical significance with a growing set of experimental and computational data and student-centric in-class activities including computer labs. Our Biophysical series emphasizes concepts and methods of computational structural biology (Molecular Dynamics) and spectroscopic approaches (IR, UV and NMR). Here we describe the details of our integrative approach, summarize key outcomes and chart ways to advance the biophysical chemistry series further. Our textbook can be found through LibreText.

scientific communication and education↗

bioRxiv2025

GlialCAM Cytoplasmic Signaling in Oligodendrocytes and Astrocytes is Essential for White Matter Homeostasis in the Brain

Glial cell adhesion molecule (GlialCAM) is an astrocyte- and oligodendrocyte-expressed transmembrane protein with two extracellular IgG-like domains and a cytoplasmic tail with putative signaling functions. While numerous studies have explored functions for the GlialCAM IgG-like domains in brain development and physiology, functions for its cytoplasmic signaling tail remain largely unknown. Therefore, we developed a mutant mouse model that expresses a truncated GlialCAM construct (GlialCAM {Delta}CT) that contains intact extracellular and transmembrane domains but lacks the cytoplasmic tail. Deletion of the GlialCAM cytoplasmic domain in glial cells of the brain results in vacuolization within white matter regions without disrupting neurovascular barrier integrity. Consequently, mutant mice exhibited selective deficits in motor coordination, muscular strength, and memory. Single cell transcriptome sequencing identifies GlialCAM-dependent defects in ECM remodeling pathways in white matter tracts. In situ spatial profiling revealed robust activation of astrocytes and microglia in the mutant brain. Proteomic analysis identified GlialCAM cytoplasmic tail interactors with links to MAPK signaling and cytoskeletal regulatory networks. These data reveal important functions for the GlialCAM cytoplasmic tail in homeostasis of white matter tracts in the adult murine brain. The GlialCAM {Delta}CT model may also be useful for studying the pathogenesis and possible treatment of neurological diseases linked to white matter degeneration.

neuroscience↗

bioRxiv2025

An interaction between the transmembrane domains of Streptococcus pyogenes sortase A and its endogenous substrate M protein revealed by molecular dynamics simulations

Sortase enzymes are cysteine transpeptidases at the cell surface of gram-positive bacteria. Localized to distinct foci on the cell membrane, class A sortases (SrtAs) recognize a cell wall sorting signal (CWSS), and following cleavage at this specific binding motif, target proteins are ligated to precursors of the growing peptidoglycan layer. This activity of SrtA enzymes is utilized extensively in sortase-mediated ligation (SML) strategies, for a variety of protein engineering applications. Typically, engineered variants of SrtA are used for SML experiments considering the relatively low catalytic efficiency of this enzyme. Understandably, most biochemical studies are conducted with the isolated catalytic domain of SrtA enzymes from various bacteria, and the stereochemistry of the endogenous interaction between SrtA and substrate is not well understood. Here, we used AlphaFold2 to create a model of the full-length SrtA enzyme from Streptococcus pyogenes (spySrtA) with or without either a peptide substrate or a portion of M protein, a cellular target. We ran triplicate 500 ns molecular dynamics simulations for each model embedded in a lipid bilayer, which revealed several stereochemical features of this system. Contact map analyses revealed specific interactions between catalytic domain positions of spySrtA and the lipid bilayer, as well as between the enzyme and M protein residues outside the canonical LPXTG pentapeptide CWSS. We also characterized a potential transmembrane domain interaction between spySrtA and M protein that we predict orients and stabilizes substrate binding. Taken together, these interactions likely increase the catalytic efficiency of the enzyme for its substrates in vivo, and may provide important stereochemical insights for SML uses.

biochemistry↗

bioRxiv2025

Single Cell RNA Sequencing and Spatial Profiling Identify Mechanisms of Neonatal Brain Hemorrhage Development and Resolution

Precise control of cell-cell communication networks within brain neurovascular units (NVUs) promotes normal tissue physiology, and dysregulation of these networks can lead to pathologies including intracerebral hemorrhage (ICH). The cellular and molecular mechanisms underlying ICH development and subsequent tissue repair processes remain poorly understood. Here we employed quantitative single cell RNA sequencing coupled with spatial in situ gene expression profiling to characterize NVU signaling pathways associated with ICH in neonatal mouse brain tissue. The initial stages of ICH pathogenesis are characterized by downregulation of extracellular matrix (ECM)-associated signaling factors (Adamtsl2, Htra3, and Lama4) that functionally connect to canonical TGF{beta} activation and signaling in vascular endothelial cells. Conversely, the progressive resolution of ICH involves upregulation of neuroinflammatory signaling networks (Gas6 and Axl) alongside activation of iron metabolism pathway components (Hmox1, Cp, and Slc40a1) in astrocytes and microglial cells. Integrated computational modeling identifies additional ligand-receptor signaling networks between perivascular glial cells and endothelial cells during both ICH pathogenesis and resolution. Collectively, these findings illuminate the molecular signaling networks that promote NVU maturation and provide novel mechanistic insights into the pathways controlling ICH pathogenesis and repair.

developmental biology↗

bioRxiv2024

Biochemical characterization of Bacillus anthracis sortase B: Use in sortase mediated ligation and substrate recognition dependent on residues beyond the canonical pentapeptide binding motif for sortase enzymes

Sortases are cysteine transpeptidases located on the surface of Gram-positive bacteria. These critical enzymes facilitate the attachment of proteins to the cell wall, and are potential targets for novel antibiotic development, as well as versatile tools in protein engineering applications. Although there are six classes of sortases recognized, class A sortases (SrtA) are the most widely studied and utilized. SrtA enzymes recognize the canonical Cell Wall Sorting Signal (CWSS), LPXTG, where X=any amino acid, although work in recent years identified additional promiscuity in multiple positions of this recognition motif. Much less is known about Class B sortases (SrtB), which target a distinct sequence, typically with an N-terminal Asn, e.g., variations of NPXTG or NPQTN. Although understudied overall, two SrtB enzymes have previously been shown to be specific for heme transporter proteins, and in vitro experiments with the catalytic domains of these enzymes reveal activities significantly worse than SrtA from the same organisms. Here, we use protein biochemistry, structural analyses, and computational simulations to better understand and characterize these enzymes, specifically investigating Bacillus anthracis SrtB (baSrtB) as a model SrtB protein. Structural modeling predicts a plausible enzyme-substrate complex, which is verified by mutagenesis of binding cleft residues at several positions. Furthermore, residues N- and C-terminal to the pentapeptide recognition motif are critical for observed activity. We also use chimeric proteins to identify a single site that improves baSrtB activity by [~]4-fold and use purified protein substrates to validate sortase-mediated ligation of two proteins using SrtB enzymes for the first time. Taken together, these studies provide insight into SrtB-target binding as well as evidence that SrtB enzymes can be modified to be of potential use in protein engineering.

biochemistry↗

bioRxiv2020

The Proline-rich Domain Promotes Tau Liquid Liquid Phase Separation in Cells

Tau protein in vitro can undergo liquid liquid phase separation (LLPS); however, observations of this phase transition in living cells are limited. To investigate protein state transitions in living cells we found that Cry2 can optogentically increase the association of full lengh tau with microtubules. To probe this mechanism, we identified tau domains that drive tau clustering on microtubules in living cells. The polyproline rich domain (PRD) drives LLPS and does so under the control of phosphorylation. These readily observable cytoplasmic condensates underwent fusion and fluorescence recovery after photobleaching consistent with the ability of the PRD to undergo LLPS in vitro. In absence of the MTBD, the tau PRD co-condensed with EB1, a regulator of plus-end microtubule dynamic instability. The specific domain properties of the MTBD and PRD serve distinct but mutually complementary roles that utilize LLPS in a cellular context to implement emergent functionalities that scale their relationship from binding alpha-beta tubulin heterodimers to the larger proportions of microtubules.

neuroscience↗

bioRxiv2019

Killer Immunoglobulin-like Receptor-Ligand Interactions Predict Clinical Outcomes Following Unrelated Donor Transplants

Killer immunoglobulin-like receptor (KIR) and KIR-ligand (KIRL) interactions play an important role in natural killer (NK) cell mediated graft versus leukemia effect following hematopoietic cell transplantation (HCT). However, there is considerable heterogeneity in the KIR gene and KIRL content in individuals, making it difficult to estimate the full clinical impact of NK cell reconstitution following HCT. Herein a novel mathematical model designed to quantify these interactions is presented to better assess the influence of NK cell-mediated alloreactivity on transplant outcomes. Ninety-eight HLA matched unrelated donor (URD) HCT recipients were retrospectively studied. The KIR-KIRL interactions were quantified using a system of matrix equations. Unit values were ascribed to each KIR-KIRL interaction and directionality of interactions was denoted by, either a positive (activating) or negative symbol (inhibition); these interactions were then summed. The absolute values of both the missing KIRL as well as inhibitory KIR-KIRL interactions were significantly associated with overall survival and relapse. These score components were initially used to develop a weighted (w-KIR Score) and subsequently a simplified, non-weighted KIR-KIRL interaction scores (IM-KIR Score). Increased w-KIR Score and IM-KIR Score were both predictive of all-cause mortality and relapse; w-KIR score HR of 0.37 (P=0.001) and 0.44 (P=0.044) respectively; IM-KIR score HR of 0.5 (P=0.049) and 0.44 (P=0.002) respectively. IM-KIR score was also associated with NK cell reconstitution post HCT. KIR-KIRL interactions as reflected by the w-KIR and IM-KIR scores influence both relapse risk and survival in recipients of HLA matched URD HCT with hematological malignancies.\n\nHighlightsO_LIKiller immunoglobulin-like receptor (KIR) - KIR-ligand (KIRL) interactions display a high degree of variability in HLA matched stem cell transplant (SCT) recipients.\nC_LIO_LIIndividually, the magnitude of inhibitory and missing KIR-KIRL interactions predicts overall survival and relapse risk following SCT.\nC_LIO_LITaken together the known KIR-KIRL interactions also predict clinical outcomes after HLA matched unrelated donor stem cell transplantation.\nC_LI

cancer biology↗

bioRxiv2018

Narrow equilibrium window for complex coacervation of tau and RNA under cellular conditions

The conditions that lead to the liquid-liquid phase separation (LLPS) of the tau protein, a microtubule associated protein whose pathological aggregation has been implicated in neurodegenerative disorders, are not well understood. Establishing a phase diagram that delineates the boundaries of phase co-existence is key to understanding its LLPS. Using a combination of EPR, turbidity measurements, and microscopy, we show that tau and RNA form complex coacervates with lower critical solution temperature (LCST) behavior. The coacervates are reversible, and the biopolymers can be driven to the supernatant phase or coacervate phase by varying the experimental conditions (temperature, salt concentration, tau:RNA charge ratio, total polymer concentration and osmotic stress). Furthermore, the coacervates can be driven to a fibrillar state through the addition of heparin. The equilibrium phase diagram of the tau/RNA complex coacervate system can be described by a Flory-Huggins model, augmented by an approximate Voorn Overbeek electrostatic term (FH-VO), after fitting the experimental data to an empirical Flory interaction parameter divided into an entropic and enthalpic term. However, a more advanced model in which tau and RNA are treated as discrete bead-spring chains with a temperature-dependent excluded volume interaction and electrostatic interactions between charged residues, investigated through field theoretic simulations (FTS), provided direct and unique insight into the thermodynamic driving forces of tau/RNA complexation. FTS corroborated the experimental finding that the complex coacervation of tau and RNA is has an entropy-driven contribution, with a transition temperature around the physiological temperature of 37 {degrees}C and salt concentrations around 100-150 mM. Together, experiment and simulation show that LLPS of tau can occur under physiological cellular conditions, but has a narrow equilibrium window over experimentally tunable parameters including temperature, salt and tau concentrations. Guided by our phase diagram, we show that tau can be driven towards LLPS under live cell coculturing conditions with rationally chosen experimental parameters.

biophysics↗

bioRxiv2018

Th17/regulatory T cells balance is predictive of Coccidioides infection outcome in pediatric patients

BackgroundProtective immunity against the fungal pathogen Coccidioides requires specific T helper responses. Mouse vaccine and infection studies have defined CD4+ T helper (Th)1 and Th17 cells in the resolution of infection and in effective protection. Patients with persistent Coccidioides infection demonstrate reduced cellular responses.\n\nMethodsPeripheral blood and serum were collected from 30 pediatric Coccidioides-infected patients and 20 healthy controls in the California San Joaquin Valley. Samples were evaluated by flow cytometry for innate and adaptive immune populations and cytokines to define the early immune response and identify clinically useful biomarkers for predicting disease outcome. Clinical and flow data were evaluated according to disease outcome (resolved or persistent) using principal component analysis, high-dimensional flow cytometry analysis tools, chi-square automatic interaction detection, and individual cell population comparisons.\n\nResultsPatients with persistent infection had lower Th17 and higher Treg frequencies, but similar Th1 responses, relative to patients that resolved disease. Treg frequency, eosinophil numbers and neutrophil numbers together distinguish patients that resolve infection from those that develop persistent infection.\n\nConclusionsThe inability to resolve Coccidioides infection may be a result of elevated Treg frequency and functional capacity, and Treg frequency may predict patient disease outcome at diagnosis. In our study, Th1 responses were similar in persistent and resolved infection, in contrast to prior human studies. Instead, our data suggest that Th17 cells provide an effective protection during Coccidioides infection, and that elevated Treg frequency inhibits protective immunity.

immunology↗