Biology subjects

Cole, R. N.

Publications and source records attributed to Cole, R. N..

11 recordsLinked to original sources

bioRxiv2026

SureQuant IS-PRM Enables Cross-Species Targeted Quantification of Retinoid Metabolism and Signaling Proteins in the Heart

Retinoic acid signaling is critical for cardiac development and homeostasis. Dysregulation of all-trans retinoic acid metabolism contributes to vascular atherogenesis, restenosis, calcification, and heart failure. Therefore, assessment of proteins involved in retinoid metabolism and signaling has gained interest for identifying potential biomarkers and therapeutic targets in cardiovascular disease. However, quantifying these proteins remains challenging due to limitations of antibody-based methods. We developed a targeted proteomics approach using SureQuant internal standard-triggered parallel reaction monitoring mass spectrometry to profile these proteins. We designed a panel of 80 stable isotope-labeled (heavy) peptides representing proteins involved in retinoid signaling and metabolism, with sequences applicable to human samples and conserved across multiple species. Survey experiments using directed data-dependent acquisition on the Orbitrap Fusion Lumos mass spectrometer determined precursor and product ion masses for each heavy peptide, which were programmed into the SureQuant method for continuous monitoring. Upon detection of these heavy internal standards, the instrument transitions to a targeted PRM acquisition mode in which repeated high-resolution MS/MS spectra of both endogenous (light) and heavy peptides are acquired. Using this method, retinoid pathway-associated proteins were quantified to as low as 10 attomoles for selected targets across multiple tissues and developmental stages. Distinct tissue-specific retinoid metabolic networks were identified across lung, liver, retinal cell lines and cardiac tissues. Developmental profiling of mouse and rat hearts revealed remodeling of retinoid pathway proteins from embryonic to postnatal and adult stages, suggesting a functional transition from retinoid-driven cardiac development toward maintenance of retinoid homeostasis in the mature heart.

biochemistry↗

bioRxiv2025

CCRL2 promotes the interferon-γ signaling response in myeloid neoplasms with erythroid differentiation and mutated TP53

Patients with myeloid neoplasms with loss-of-function TP53 mutations and erythroid differentiation have poor outcomes, and a better understanding of disease biology is required. Upregulation of interferon-{gamma} (IFN-{gamma}) signaling has been associated with acute myeloid leukemia (AML) progression and chemotherapy resistance, but its drivers remain unclear. In this study, we found that the surface receptor C-C motif chemokine receptor-like 2 (CCRL2) is overexpressed in AML with erythroid differentiation and TP53 mutations compared to other AML subtypes and healthy hematopoietic cells. The knockout (KO) of CCRL2 suppressed erythroleukemia growth in vitro and in vivo. Further proteomics and transcriptomics analysis revealed IFN-{gamma} signaling response as the top CCRL2-regulated pathway in erythroleukemia. Our mechanistic studies support direct CCRL2 driven IFN-{gamma} signaling independent of exogenous IFN-{gamma}, through phosphorylation of STAT1, via JAK2-dependent and independent mechanisms. CCRL2/IFN-{gamma} signaling is upregulated in erythroid leukemias, and TP53 mutated AML without concurrent increase of IFN-{gamma} secretion in the bone marrow microenvironment and is directly induced by TP53 KO. Finally, CCRL2/IFN-{gamma} signaling is associated with the transformation of pre-leukemic single-hit TP53 clones to multi-hit TP53 mutated AML, increased resistance to venetoclax and worse survival in AML. Overall, our findings support that CCRL2 is an essential driver of cell-autonomous IFN-{gamma} signaling response in myeloid neoplasms with erythroid differentiation and TP53 mutations and highlight CCRL2 as a relevant novel target for these neoplasms. One Sentence SummaryCCRL2 is overexpressed in AML with loss-of-function TP53 mutations and erythroid differentiation and promotes IFN-{gamma} signaling response via a cell-intrinsic mechanism.

cancer biology↗

bioRxiv2024

Huntingtin interactome reveals huntingtin role in regulation of double strand break DNA damage response (DSB/DDR), chromatin remodeling and RNA processing pathways

Huntingtons Disease (HD), a progressive neurodegenerative disorder with no disease-modifying therapies, is caused by a CAG repeat expansion in the HD gene encoding polyglutamine-expanded huntingtin (HTT) protein. Mechanisms of HD cellular pathogenesis and cellular functions of the normal and mutant HTT proteins are still not completely understood. HTT protein has numerous interaction partners, and it likely provides a scaffold for assembly of multiprotein complexes many of which may be altered in HD. Previous studies have implicated DNA damage response in HD pathogenesis. Gene transcription and RNA processing has also emerged as molecular mechanisms associated with HD. Here we used multiple approaches to identify HTT interactors in the context of DNA damage stress. Our results indicate that HTT interacts with many proteins involved in the regulation of interconnected DNA repair/remodeling and RNA processing pathways. We present evidence for a role for HTT in double strand break repair mechanism. We demonstrate HTT functional interaction with a major DNA damage response kinase DNA-PKcs and association of both proteins with nuclear speckles. We show that S1181 phosphorylation of HTT is regulated by DSB, and can be carried out (at least in vitro) by DNA-PK. Furthermore, we show HTT interactions with RNA binding proteins associated with nuclear speckles, including two proteins encoded by genes at HD modifier loci, TCERG1 and MED15, and with chromatin remodeling complex BAF. These interactions of HTT may position it as an important scaffolding intermediary providing integrated regulation of gene expression and RNA processing in the context of DNA repair mechanisms.

neuroscience↗

bioRxiv2024

Inhibition of Cardiac p38 Highlights the Role of the Phosphoproteome in Heart Failure Progression

Heart failure (HF) is a complex condition characterized by the inability of the heart to pump sufficient oxygen to the organs to meet their metabolic needs. Among the altered signal transduction pathways associated with HF pathogenesis, the p38 mitogen-activated protein kinase (p38 MAPK) pathway--activated in response to stress-- has attracted considerable attention for its potential role in HF progression and cardiac hypertrophy. However, the exact mechanisms by which p38 MAPK influences HF remain unclear. Addressing knowledge gaps may provide insight on why p38 inhibition has yielded inconsistent outcomes in clinical trials. Here we investigate the effects of p38 MAPK inhibition via SB203580 on cardiac remodeling in a guinea pig model of HF and sudden cardiac death. Using a well-established HF model with ascending aortic constriction and daily isoproterenol (ACi) administration, we assessed proteomic changes across three groups: sham-operated controls, untreated ACi, and ACi treated with SB203580 (ACiSB). Cardiac function was evaluated by M-mode echocardiography, while proteome and phosphoproteome profiles were analyzed using multiplexed tandem mass tag labeling and LC-MS/MS. Our findings demonstrate that chronic SB203580 treatment offers protection against progressive decline in cardiac function in HF. The proteomic data indicate that SB203580-treatment exerts broad protection of the cardiac phosphoproteome, beyond inhibiting maladaptive p38-dependent phosphorylation, extending to PKA and AMPK networks among others, ultimately protecting the phosphorylation status of critical myofibrillar and Ca2+-handling proteins. Though SB203580 had a more restricted impact on widespread protein changes in HF, its biosignature was consistent with preserved mitochondrial energetics as well as reduced oxidative and inflammatory stress.

molecular biology↗

bioRxiv2024

Transcriptomics and proteomics of projection neurons in a circuit linking hippocampus with dorsolateral prefrontal cortex in human brain

Transcriptome and proteome sequencing of brain tissue homogenate has helped unravel processes underlying schizophrenia (SCZ). However, most studies have lacked granularity at the cell type level and have focused on individual brain regions, rather than examining expression dynamics across multiple regions or illness-relevant circuitries. We used laser capture microdissection to collect excitatory neuron-enriched samples from hippocampal subregions CA1 and presubiculum (SUB), and from dorsolateral prefrontal cortex (DLPFC), a circuit prominently implicated in schizophrenia. Using RNA sequencing and quantitative proteomics, we show significantly superior discrimination of brain regional identity in the transcriptomic (>90% accuracy) and proteomic data (>97% accuracy) compared with gene-level expression data (<70% in bulk). Patients with SCZ show hippocampal-specific differential protein phosphorylation. SCZ risk co-expression gene-sets that replicate across transcript and protein networks are enriched for transmembrane transporters in the DLPFC and CA1 and postsynaptic processes in the SUB. We demonstrate a strong directional connectivity effect of SCZ risk in that excitatory synaptic genes in CA1 unidirectionally predict gene expression in SUB. Finally, parallel CA1 snRNA-seq results suggest that in SCZ excitatory efferents in CA1 are affected by interactions with glia and by downregulation of inhibitory neuropeptide inputs. Our study proposes molecular mechanisms by which hippocampal communication, previously associated with SCZ at the macroscopic level, may be altered at the inter-field and interregional circuit level.

neuroscience↗

bioRxiv2022

Surface phenotyping and quantitative proteomics reveal differentially enriched proteins of brain-derived extracellular vesicles in Parkinson's disease

Extracellular vesicles (EVs) are produced by all cell types and are found in all tissues and biofluids. EV proteins, nucleic acids, and lipids are a "nano-snapshot" of the parent cell that may be used for novel diagnostics of various diseases, including neurodegenerative disorders. Currently, diagnosis of the most common neurodegenerative movement disorder, Parkinsons disease (PD), relies on manifestations of late-stage progression, which may furthermore associate with other neurodegenerative diseases such as progressive supranuclear palsy (PSP). Here, we profiled surface markers and other protein contents of brain-derived extracellular vesicles (bd-EVs) from PD (n= 24), PSP (n=25) and control (n=24). bdEVs displayed tetraspanins and certain microglia, astrocyte, and neuron markers, while quantitative proteomics revealed enrichment of several proteins in PD vs. control and/or PSP, including clathrin heavy chain 1 and 14-3-3 protein gamma. This characterization of EVs in the source tissue provides insights into local dynamics as well as biomarker candidates for investigation in peripheral fluids.

neuroscience↗

bioRxiv2021

Human breast milk enhances intestinal mucosal barrier function and innate immunity in a pediatric human enteroid model

Breastfeeding has been associated with long lasting health benefits. Nutrients and bioactive components of human breast milk promote cell growth, immune development, and shield the infant gut from insults and microbial threats. The molecular and cellular events involved in these processes are ill defined. We have established human pediatric enteroids and interrogated maternal milks impact on epithelial cell maturation and function in comparison with commercial infant formula. Colostrum applied apically to pediatric enteroid monolayers reduced ion permeability, stimulated epithelial cell differentiation, and enhanced tight junction function by upregulating occludin expression. Breast milk heightened the production of antimicrobial peptide -defensin 5 by goblet and Paneth cells, and modulated cytokine production, which abolished apical release of pro-inflammatory GM-CSF. These attributes were not found in commercial infant formula. Epithelial cells exposed to breast milk elevated apical and intracellular pIgR expression and enabled maternal IgA translocation. Proteomic data revealed a breast milk-induced molecular pattern associated with tissue remodeling and homeostasis. Using a novel ex vivo pediatric enteroid model, we have identified cellular and molecular pathways involved in human milk-mediated improvement of human intestinal physiology and immunity.

immunology↗

bioRxiv2019

Definition of naturally processed peptides reveals convergent presentation of autoantigenic topoisomerase-I epitopes in scleroderma

Disease-associated HLA-DRB1 alleles are thought to confer risk of developing autoimmunity by favoring the presentation of select autoantigenic epitopes. However, identification of these epitopes and the principles governing their presentation has been hindered by the imprecision of currently available methods, which cannot fully recapitulate the complexity of human pathophysiology. We present a natural antigen processing assay (NAPA), which overcomes these limitations by studying the presentation of autoantigenic CD4+ T cell epitopes by monocyte-derived dendritic cells (mo-DCs) from patients. We applied this strategy to study the processing and presentation of topoisomerase-1 (TOP1), a prevalent autoantigen in scleroderma that is associated with lung fibrosis and high mortality. We found that a common set of 10 epitopes was presented by mo-DCs from patients with diverse HLA-DR variants, including those not previously associated with the disease. Sequence analysis revealed a shared peptide-binding motif within the HLA-DR peptide binding grooves of patients who developed anti-TOP1 autoantibodies. In addition, a subset of naturally presented TOP1 peptides were characterized by immunological promiscuity, as they could bind to diverse HLA-DR peptide binding grooves. NAPA epitopes were immunorelevant: they could stimulate autoreactive CD4+ T cells in patients, and the number of epitopes recognized correlated with lung disease severity. These findings mechanistically implicate presentation of a convergent set of TOP1 epitopes in the development of scleroderma lung disease. Precise identification of autoantigenic epitopes is key to understanding the primordial mechanisms for the loss of tolerance, studying disease-propagating autoreactive T cells, and developing antigen-specific immunotherapy.\n\nOne Sentence SummaryUse of a novel natural antigen processing assay reveals a mechanism for the presentation of shared CD4+ T cell epitopes of topoisomerase-I in immunogenetically diverse patients with scleroderma.

immunology↗

bioRxiv2019

Human Rotavirus Diarrhea Is Associated with Altered Trafficking and Expression of Apical Membrane Transport Proteins

BackgroundRotavirus (RV) is the 5th leading cause of death in children <5 years old but the leading cause of diarrhea related deaths in this age group. The mechanism of RV diarrhea involves decreased activity of Na+-dependent solute transporters with increased luminal secretion of Cl- in the absence of significant histologic damage. While our understanding of RV diarrhea has come from studies in animal models and cancer cell lines, the mechanism of the diarrhea and the transport proteins affected in human RV disease remains only partially understood. This understanding is likely to impact drug development therapy for RV diarrhea.\n\nMethodsFormalin-fixed paraffin-embedded small intestinal specimens from patients diagnosed with RV diarrhea (confirmed by anti-RV antibodies) were analyzed by immunofluorescence for changes in apical/basolateral ion/nutrient transporters/channels as well as tight junctional and cytoskeletal proteins. Proximal small intestinal enteroids generated from biopsies obtained from healthy human subjects were grown as monolayers, differentiated to resemble villus epithelial cells, and infected with human RV.\n\nResultsRV diarrhea was associated with reduced expression and intracellular localization of transport proteins normally found in the brush border membranes, including SGLT1, NHE3, NHE2, the Na+-dependent amino acid transporter SLC6A19, and CFTR. In contrast, basolateral proteins, including Na+/K+-ATPase, NKCC1, and {beta}-catenin, the brush border marker ezrin, as well as the tight junction protein, ZO-1, were expressed and localized normally. RV-induced mislocalization of NHE3, SGLT1, SLC6A19 and CFTR was also seen when human small intestinal enteroids were infected with RV.\n\nConclusionsThese data demonstrate a new pathophysiologic mechanism of acute diarrhea in which expression of multiple apical transport proteins are reduced. This acute diarrhea is likely to be caused by an effect on a common apical trafficking pathway, as exemplified by RV diarrhea, and its contribution to other enteric pathogen-induced diarrheal diseases should be determined.

cell biology↗

bioRxiv2019

The structural unit of melanin in the cell wall of the fungal pathogen Cryptococcus neoformans

Melanins are synthesized macromolecules that are found in all biological kingdoms. These pigments have a myriad of roles that range from microbial virulence to key components of the innate immune response in invertebrates. Melanins also exhibit unique properties with potential applications in physics and material sciences, ranging from electrical batteries to novel therapeutics. In the fungi, melanins such as eumelanins, are components of the cell wall that provide protection against biotic and abiotic elements. Elucidation of the smallest fungal cell wall-asociated melanin unit that serves as a building block is critical to understand the architecture of these polymers, its interaction with surrounding components, and their functional versatility. In this study, we used isopycnic gradient sedimentation, NMR, EPR, high-resolution microscopy, and proteomics to analyze the melanin in the cell wall of the human pathogenic fungus Cryptococcus neoformans. We observed that melanin is assembled into the cryptococcal cell wall in spherical structures of [~]200 nm in diameter, termed melanin granules, which are in turn composed of nanospheres of [~]30 nm in diameter, the fungal melanosomes. We noted that melanin granules are closely associated with proteins that may play critical roles in the fungal melanogenesis and the supramolecular structure of this polymer. Using this structural information, we propose a model for C. neoformans melanization that is similar to the process used in animal melanization and is consistent with the phylogenetic relatedness of the fungal and animal kingdoms.

microbiology↗

bioRxiv2018

Epithelial WNT2B and Desert Hedgehog are necessary for human colonoid regeneration after bacterial cytotoxin injury

Intestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagic E. coli secreted cytotoxin, EspP to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.

physiology↗