Biology subjects

Price, J. C.

Publications and source records attributed to Price, J. C..

11 recordsLinked to original sources

bioRxiv2026

Kinetic Lipidomics: Quantifying in vivo changes in lipid metabolism using metabolic labeling

Lipid metabolism reflects the dynamic balance between metabolic turnover and concentration. Kinetic mass spectrometry (MS) enables direct quantification of molecular turnover in vivo. Previous work has shown that MS-based kinetic proteomics has provided powerful insights into proteome regulation. Analogous lipidome-wide kinetic measurements remain limited by challenges in defining molecule-specific labeling behavior. Here, we extend kinetic MS to untargeted lipidomics. Isotope labeling with deuterated water (2H2O) is commonly used for monitoring turnover of palmitate and other select lipids by measuring labeling of stable C-H positions with deuterium (2H). Here, we extend the deuterium-incorporation model underlying these targeted lipid turnover assays to support untargeted analysis of all detectable lipids. This allows us to empirically quantify the effective fraction of endogenous synthesis (Asyn) and the turnover rate (k) across hundreds of lipid species simultaneously. One central barrier to lipidome-wide kinetic modeling is determining the endogenous number of deuterium-labeling sites for each molecule (nL) which is required to estimate Asyn and k accurately. The nL value is an essential component of biological kinetic assays. In kinetic proteomics, curated amino acid nL libraries enable peptide-level modeling by summing sequence-specific labeling-site values, but comparable resources are lacking for lipids and may not generalize across metabolic states or non-mammalian systems. Yet, gaps remain for lipids and for amino acids in modified metabolic conditions or non-mammalian biologies. Here, we empirically determine lipid nL values and validate the process with peptides against an nL library. To evaluate this strategy in a biologically relevant setting, we applied it to brain tissue from transgenic mice expressing human ApoE isoforms, where altered lipid transport and metabolism are implicated in Alzheimers disease risk. These data validate the method in a clinically relevant context and suggest that genotype-dependent metabolism can alter empirically determined lipid nL values.

biochemistry↗

bioRxiv2026

Ontological Analysis of Brain Proteostasis Highlights the Sex-Dependent Trajectory of ApoE Isoform-Specific Regulation

Apolipoprotein E (ApoE) is the strongest genetic predictor of Alzheimers disease (AD) risk, with ApoE4 increasing and ApoE2 decreasing risk relative to ApoE3. Using a global LC-MS proteomic approach, we integrated protein abundance and kinetics in Human-APOE knock-in mice for young (3-month) and aged (18-month) cohorts to quantify the changes in steady-state proteostasis. By mapping 6,052 identified proteins and 3,986 associated turnover rates into ontological groups, we observed that vesicle trafficking and mitochondrial dysregulation occur as early as 3 months in ApoE4 mice accompanied by hyperactive metabolism that eventually reduces with age. In contrast, young and old ApoE2 mice retain similar signatures to ApoE3 mice in metabolic, mitochondrial, cellular regulation, and membrane trafficking ontologies. We found that females had more isoform-induced ontological changes relative to ApoE3, providing insight into sex-dependent vulnerabilities. Our global proteomic approach for ApoE proteostasis crucially unifies independent literature observations while providing turnover kinetics to uncover the underlying mechanism behind abundance changes. Data are available via ProteomeXchange with identifier PXD079261. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/735293v1_ufig2.gif" ALT="Figure 1000"> View larger version (45K): org.highwire.dtl.DTLVardef@28ebfforg.highwire.dtl.DTLVardef@9dc6f2org.highwire.dtl.DTLVardef@704577org.highwire.dtl.DTLVardef@e0b0e2_HPS_FORMAT_FIGEXP M_FIG C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/735293v1_ufig1.gif" ALT="Figure 1001"> View larger version (48K): org.highwire.dtl.DTLVardef@17d0474org.highwire.dtl.DTLVardef@af771borg.highwire.dtl.DTLVardef@1b49f67org.highwire.dtl.DTLVardef@14d3aa2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Human Cerebral Cortex Organization Characterized by Functional PET-FDG "Metabolic Connectivity"

PurposeIn this study, we characterize the spatiotemporal organization of resting-state metabolic connectivity (RSMC) in the human brain, as measured by [18F]- fluorodeoxyglucose (FDG) functional PET (fPET-FDG). We examine the relationship between RSMC organization and resting-state functional connectivity (RSFC) derived from functional magnetic resonance imaging and other known cortical organizational principles. MethodsResting-state fPET-FDG data from 24 individuals were obtained from a publicly available repository. We characterized local metabolic organization using connectivity-based boundary mapping, with adaptations to account for the low signal-to-noise ratio of fPET-FDG data. We then estimated global metabolic organization through community detection-based network and principal gradient analyses. Furthermore, we examined how metabolic connectivity is shaped by temporal-frequency-specific components of fPET-FDG signal. Finally, we contextualized metabolic organization by relating metabolic gradients to anatomical, functional, and energetic reference measures. ResultsAt the local scale, boundary mapping results indicated structured transitions shaped by a combination of both fast and slow fPET-FDG signals, partly overlapping with RSFC boundary maps. Globally, RSMC analyses revealed a robust metabolic structure organized along a superior-inferior cortical gradient. This pattern remained consistent across network community detection and principal gradient analyses and was primarily driven by low-frequency, minute-scale fPET-FDG dynamics. The identified large-scale metabolic profile aligns closely with several known anatomical and energetic constraints. ConclusionThis study characterizes the spatiotemporal organizational principles of RSMC, deepening insight into the brains energetic framework and providing a basis for future cognitive and clinical investigations of metabolic connectivity organization.

neuroscience↗

bioRxiv2024

On the analysis of functional PET (fPET)-FDG: baseline mischaracterization can introduce artifactual metabolic (de)activations

Functional Positron Emission Tomography (fPET) with (bolus plus) constant infusion of [18F]-fluorodeoxyglucose FDG), known as fPET-FDG, is a recently introduced technique in human neuroimaging, enabling the detection of dynamic glucose metabolism changes within a single scan. However, the statistical analysis of fPET-FDG data remains challenging because its signal and noise characteristics differ from both classic bolus-administration FDG PET and from functional Magnetic Resonance Imaging (fMRI), which together compose the primary sources of inspiration for analytical methods used by fPET-FDG researchers. In this study, we present an investigate of how inaccuracies in modeling baseline FDG uptake can introduce artifactual patterns to detrended TAC residuals, potentially introducing spurious (de)activations to general linear model (GLM) analyses. By combining simulations and empirical data from both constant infusion and bolus-plus-constant infusion protocols, we evaluate the effects of various baseline modeling methods, including polynomial detrending, regression against the global mean time-activity curve, and two analytical methods based on tissue compartment model kinetics. Our findings indicate that improper baseline removal can introduce statistically significant artifactual effects, although these effects characterized in this study ([~]2-8%) are generally smaller than those reported by previous literature employing robust sensory stimulation ([~]10-30%). We discuss potential strategies to mitigate this issue, including informed baseline modeling, optimized tracer administration protocols, and careful experimental design. These insights aim to enhance the reliability of fPET-FDG in capturing true metabolic dynamics in neuroimaging research.

neuroscience↗

bioRxiv2024

Quantitative and Kinetic Proteomics Reveal ApoE Isoform-dependent Proteostasis Adaptations in Mouse Brain

Apolipoprotein E (ApoE) polymorphisms modify the risk of neurodegenerative disease with the ApoE4 isoform increasing and ApoE2 isoform decreasing risk relative to the wild-type control ApoE3 isoform. To elucidate how ApoE isoforms alter the proteome, we measured relative protein abundance and turnover in transgenic mice expressing a human ApoE gene (isoform 2, 3, or 4). This data provides insight into how ApoE isoforms affect the in vivo synthesis and degradation of a wide variety of proteins. We identified 4849 proteins and tested for ApoE isoform-dependent changes in the homeostatic regulation of [~]2700 ontologies. In the brain, we found that ApoE4 and ApoE2 both lead to modified regulation of mitochondrial membrane proteins relative to the wild-type control ApoE3. In ApoE4 mice, this regulation is not cohesive suggesting that aerobic respiration is impacted by proteasomal and autophagic dysregulation. ApoE2 mice exhibited a matching change in mitochondrial matrix proteins and the membrane which suggests coordinated maintenance of the entire organelle. In the liver, we did not observe these changes suggesting that the ApoE-effect on proteostasis is amplified in the brain relative to other tissues. Our findings underscore the utility of combining protein abundance and turnover rates to decipher proteome regulatory mechanisms and their potential role in biology.

biochemistry↗

bioRxiv2023

The formation of ubiquitin rich condensates triggers recruitment of the ATG9A lipid transfer complex to initiate basal autophagy

Autophagy is an essential cellular recycling process that maintains protein and organelle homeostasis. ATG9A vesicle recruitment is a critical early step in autophagy to initiate autophagosome biogenesis. The mechanisms of ATG9A vesicle recruitment are best understood in the context of starvation-induced non-selective autophagy, whereas less is known about the signals driving ATG9A vesicle recruitment to autophagy initiation sites in the absence of nutrient stress. Here we demonstrate that loss of ATG9A or the lipid transfer protein ATG2 leads to the accumulation of phosphorylated p62 aggregates in the context of basal autophagy. Furthermore, we show that p62 degradation requires the lipid scramblase activity of ATG9A. Lastly, we present evidence that poly-ubiquitin is an essential signal that recruits ATG9A and mediates autophagy foci assembly in nutrient replete cells. Together, our data support a ubiquitin-driven model of ATG9A recruitment and autophagosome formation during basal autophagy.

cell biology↗

bioRxiv2023

Micrococcin cysteine-to-thiazole conversion through transient interactions between a scaffolding protein and two modification enzymes

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a broad group of compounds mediating microbial competition in nature. Azole/azoline heterocycle formation in the peptide backbone is a key step in the biosynthesis of many RiPPs. Heterocycle formation in RiPP precursors is often carried out by a scaffold protein, an ATP-dependent cyclodehydratase, and an FMN-dependent dehydrogenase. It has generally been assumed that the orchestration of these modifications is carried out by a stable complex including the scaffold, cyclodehydratase and dehydrogenase. The antimicrobial RiPP micrococcin begins as a precursor peptide (TclE) with a 35-amino acid N-terminal leader and a 14-amino acid C-terminal core containing six Cys residues that are converted to thiazoles. The putative scaffold protein (TclI) presumably presents the TclE substrate to a cyclodehydratase (TclJ) and a dehydrogenase (TclN) to accomplish the two-step installation of the six thiazoles. In this study, we identify a minimal TclE leader region required for thiazole formation, we demonstrate complex formation between TclI, TclJ and TclN, and further define regions of these proteins required for complex formation. Our results point to a mechanism of thiazole installation in which TclI associates with the two enzymes in a mutually exclusive fashion, such that each enzyme competes for access to the peptide substrate in a dynamic equilibrium, thus ensuring complete modification of each Cys residue in the TclE core. IMPORTANCEThiopeptides are a family of antimicrobial peptides characterized for having sulfur-containing heterocycles and for being highly post-translationally modified. Numerous thiopeptides have been identified; almost all of which inhibit protein synthesis in gram-positive bacteria. These intrinsic antimicrobial properties make thiopeptides promising candidates for the development of new antibiotics. The thiopeptide micrococcin is synthesized by the ribosome and undergoes several post-translational modifications (PTMs) to acquire its bioactivity. In this study, we identify key interactions within the enzymatic complex that carries out cysteine to thiazole conversion in the biosynthesis of micrococcin.

biochemistry↗

bioRxiv2023

Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase cassettes, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of structures of Cdc48 affinity purified from lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data reveal new elements of Shp1-Cdc48 binding and support a "hand-over-hand" mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by D2 in substrate translocation/unfolding.

biochemistry↗

bioRxiv2022

Structural stability of Human serum albumin is modified in rheumatoid arthritis

Differential scanning calorimetry (DSC) can interrogate changes in structure and/or concentration of the most abundant proteins in a biological sample via heat denaturation curves (HDCs). In blood serum for example, HDC changes are a result of either concentration or altered thermal stabilities for 7-10 proteins and has previously been shown capable of differentiating between sick and healthy human subjects. Here, we compare HDCs and proteomic profiles of 50 patients experiencing joint-inflammatory symptoms, 27 of which were clinically diagnosed with rheumatoid arthritis (RA). The HDC of all 50 subjects appeared significantly different from expected healthy curves, but comparison of additional differences between the RA the non-RA subjects allowed more specific understanding of RA samples. We used mass spectrometry (MS) to investigate the reasons behind the additional HDC changes in RA patients. The HDC differences do not appear to be directly related to differences in the concentrations of abundant serum proteins. Rather, the differences can be attributed to modified thermal stability of the most abundant protein, human serum albumin (HSA). By quantifying differences in the frequency of artificially induced post translational modifications (PTMs), we found that HSA in RA subjects had a much lower surface accessibility, indicating potential ligand or protein binding partners in certain regions that could explain the shift in HSA melting temperature in the RA HDCs. Several low abundance proteins were found to have significant changes in concentration in RA subjects and could be involved in or related to binding of HSA. Certain amino acid sites clusters were found to be less accessible in RA subjects, suggesting changes in HSA structure that may be related to changes in protein-protein interactions. These results all support a change in behavior of HSA which may give insight into mechanisms of RA pathology.

biochemistry↗

bioRxiv2020

Self-activated photoblinking of nitrogen vacancy centers in nanodiamonds (sandSTORM): A method for rapid single molecule localization microscopy with unlimited observation time

Stochastic optical reconstruction microscopy (STORM) is one of the most commonly used super-resolution microscopy techniques. Popular implementations of STORM utilize aromatic fluorophores and consist of a number of intrinsic limitations such the finite photostability of the dyes, the reliance upon non-physiological redox buffers and speed which is ultimately limited by the off-rates of the photoblinking. Self-activated nanodiamond-based STORM (sandSTORM) has been developed as an accelerated STORM protocol which harvests the rapid, high quantum-yield and sustained photoblinking of nanodiamonds (ND). Photoluminescence emanating from the stochastic charge-state interconversion of Nitrogen Vacancy (NV) centers between NV0and NV- is localized using conventional STORM-optimized hardware and image processing protocols over an unlimited duration of imaging. This produces super-resolution images of matching resolution at [~] 3-times the speed and [~] 100 times less light exposure to the sample compared to traditional STORM. The enabling NDs have been used to map arrays of ryanodine receptor in skeletal muscle tissues via immunolabelling and directly visualize the internal spaces of living neurons via endocytosis of NDs. This paper details the physical basis of sandSTORM, factors which optimize its performance, and key characteristics which make it a powerful STORM protocol suitable for imaging nanoscale sub-cellular structures.

biophysics↗

bioRxiv2019

Effect of PEI-coated MNPs on the Regulation of Cellular Focal Adhesions and Actin Stress Fibres

The biocompatibility of surface coated/functionalised magnetic nanoparticles (MNPs) is key to their successful incorporation and application in biological systems. Polyethylene imine (PEI) -coated MNPs provide improved in vitro transfection efficiency compared to conventional chemical methods such as Lipofectamine and cationic polymers, and are also safer than viral transduction. Commercial cell toxicity assays are useful for end-point and high-throughput screening, providing fast results and an overview of cell health. However these assays only take into account cells that have undergone an extreme toxic response leading to cell death. Cell toxicity is a complex process which can be expressed in many forms, through morphological, metabolic, and epigenetic changes. A common indicator of cell stress and toxic response is increased cell adhesion and stress fibre formation. It is important to identify these changes in cells as it may affect downstream results and applications in biomedicine. This study explores the effect of the nanomagnetic transfection agent PEI-coated MNPs (MNP-PEIs) and an external magnetic field on cell behaviour, by studying particle internalization, changes in cellular morphology, and cell adhesion. We found that MNP-PEIs induced cell stress through a dose-dependent increase in cell adhesion via the overexpression of vinculin and formation of actin stress fibres. While the presence of PEI was the main contributor to increased cell stress, free PEI polyplexes induced higher toxicity compared to PEI bound to MNPs. MNPs without PEI coating however did not adversely affect cells suggesting a chemical effect instead of a mechanical one. In addition, genes identified as being associated with actin fibre regulation and cell adhesion, showed significant increases in expression due to the internalization of the MNP-PEI complex. From these results, we identify anomalous cell behaviour, morphology, and gene expression after interaction with MNP-PEIs, as well as a safe dosage to reduce acute cell toxicity.

bioengineering↗