Biology subjects

Phillips, T. A.

Publications and source records attributed to Phillips, T. A..

7 recordsLinked to original sources

bioRxiv2026

Neprilysin mediated cleavage of phospholamban dysregulates SERCA in heart failure

BackgroundNeprilysin (NEP) is a zinc-dependent metalloprotease targeted in heart failure therapy to prevent it degrading circulating cardioprotective vasoactive peptides. NEP can also cleave sarcolipin (SLN), the skeletal- and atrial muscle-specific micropeptide regulator of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). A direct pathophysiological role of NEP in ventricular muscle has not been established. MethodsProteomics and immunoblot analysis of human myocardial specimens were used to quantify NEP abundance in failing and non-failing hearts. Heterologous protein expression and biochemical binding assays assessed NEP-mediated cleavage of phospholamban (PLB) and its impact on PLB-SERCA interactions. Functional consequences of NEP expression or inhibition were evaluated in neonatal rat ventricular myocytes and in a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of heart failure. ResultsWe observed increased NEP abundance in failing human myocardium relative to non-failing controls. We demonstrated that NEP cleaves phospholamban (PLB), disrupting PLB-SERCA interactions. Mutation of PLB (V49A), prevented NEP cleavage and preserved PLB-SERCA binding, indicating V49 is critical for NEP substrate recognition. In neonatal rat ventricular myocytes, NEP expression was associated with faster Ca2+ transient decay kinetics and increased SR Ca2+ load, consistent with reduced SERCA inhibition. Inhibition of NEP in a hiPSC-CM heart failure model attenuated the hypertrophic transcriptional responses and reversed Ca2+-transport dysregulation. ConclusionsThese findings implicate increased NEP expression in the sarcoplasmic reticulum of cardiomyocytes as previously unrecognized maladaptive consequence of heart failure contributing to cardiac dysfunction. In this novel pathophysiological mechanism, increased NEP results in PLB cleavage and loss of regulation of SERCA. While this may relieve SERCA inhibition and augment cellular Ca2+ handling, loss of PLB chronically disrupts hearts dynamic response to adrenergic stress, changing heart rate, or other physiological challenges. The data provide new insight into the cardioprotective effects of pharmacological NEP inhibition in clinical practice, reveal a novel mechanism of action of neprilysin inhibition in cardiomyocytes and may help inform future therapeutic strategies for patients with heart failure. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/732949v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@5ddc5dorg.highwire.dtl.DTLVardef@2e746corg.highwire.dtl.DTLVardef@1dd8273org.highwire.dtl.DTLVardef@19f5635_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2026

A Novel Mechanism of Cardiomyopathy: Toxic Peptides Dysregulate Calcium Transport

A hallmark of dilated cardiomyopathy (DCM) is calcium mishandling, including reduced transport activity of the SERCA calcium pump in cardiac muscle cells. This has focused attention on SERCA as mechanism of disease and potential therapeutic target. Previously, diminished SERCA activity has been attributed to decreased protein expression, but recent studies suggest SERCA levels are unchanged in DCM. Thus, another mechanism must be responsible for the deficit. Since proteolysis is increased and proteosome function is impaired in DCM, we reasoned that accumulation of toxic protein fragments may contribute to SERCA dysfunction. In particular, previous studies showed diverse species of hydrophobic -helices can inhibit SERCA, so we hypothesized that SERCA may become congested with transmembrane peptides that mimic endogenous regulatory partners. We purified cell membranes from non-failing and DCM human ventricles and subjected them to mass spectrometry to identify protein species upregulated in DCM. Select candidates were screened for binding and inhibition of SERCA. Several small membrane proteins and membrane protein fragments bound avidly to SERCA and significantly reduced cellular calcium stores. The data suggest a novel pathophysiological mechanism in which transmembrane protein debris obstructs SERCA function and regulation, contributing to cardiac muscle dysfunction in heart failure.

physiology↗

bioRxiv2025

Spatially distinct chromatin compaction states predict neoadjuvant chemotherapy resistance in Triple Negative Breast Cancer

Organisation and dynamics of chromatin play a key role in regulation of cell state and function. In cancer, chromatin plasticity is known to be important in control of drug resistance, but the relationship between chromatin compaction and chemotherapy response within complex tissue settings remains unclear. Here, we measured single nuclei chromatin compaction using fluorescence lifetime imaging microscopy (FLIM) in situ in whole biopsies from 53 pre treatment and 14 post surgery tissue samples of human triple-negative breast cancer (TNBC) patients to determine whether single nuclei spatial chromatin compaction state can predict resistance to neoadjuvant chemotherapy (NACT). Bulk chromatin compaction across 53 pre-treatment core biopsies did not predict patient outcome. However, machine learning analysis revealed that a subset of patients exhibited distinct distributions of single nuclei with more open chromatin states, which was predictive of NACT-resistance. Graph neural network analysis established that the spatial arrangement of chromatin compaction contributed to prediction of NACT resistance and that chromatin compaction signatures were preserved in tissue state transitions from pre- to 14 post-NACT samples. Our findings shed new light on spatial control of chromatin structure and relationship to therapeutic resistance and establish a foundation for further molecular analysis of chromatin states in complex biological tissues.

systems biology↗

bioRxiv2025

Identifying tissue states by spatial protein patterns related to chemotherapy response in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive malignancy with limited targeted therapies and variable responses to conventional chemotherapy, influenced by intratumoral heterogeneity and complex tumor microenvironment (TME) interactions. Understanding spatiotemporal cellular interplay and tissue organization is crucial for advancing tumor biology and improving patient stratification. Spatially resolved proteomics, such as Imaging Mass Cytometry (IMC), offers a powerful approach to dissect the TME. We present an end-to-end computational pipeline for robust quantitative analysis of large-scale IMC datasets, addressing the challenge of batch effects through image-level contrast adjustment. Applying this framework to 813 tissue regions encompassing over 4 million cells from 63 TNBC patients, we revealed distinct spatial arrangements of cell types between chemotherapy responders and non-responders. Non-responders showed reduced cytotoxic T-cell infiltration into tumor regions and increased spatial co-localization between fibroblasts and macrophages, a pattern that persisted and intensified after chemotherapy treatment. To integrate these complex spatial-molecular relationships, we used graph neural networks (GNNs) to predict treatment response from pre-treatment samples with AUROC=0.71. Interpretability analysis identified B7H4, CD11b, CD366, and FOXP3 as the most predictive protein markers, with fibroblasts, cancer cells, and CD8+ T cells being the most informative cell types. This study introduces a scalable analytical framework for spatial proteomics with interpretable predictions, suggesting features of tissue state that could guide treatment decisions in TNBC and further our understanding of the spatial determinants of therapeutic response.

cancer biology↗

bioRxiv2025

SMART: A Spatio-Molecular Atlas of Response Trajectories in Triple-Negative Breast Cancer

A major challenge in treating Triple-Negative Breast Cancer (TNBC) lies in its molecular, morphological and clinical heterogeneity, which hampers accurate prediction of responses to neoadjuvant treatment. To address this, we introduce SMART: Spatio-Molecular Atlas of Response Trajectories, a comprehensive, multimodal resource compiled from 129 TNBC samples across 89 patients, obtained before, during, and after neoadjuvant chemotherapy (NACT). SMART comprises of 5,096 high quality manually selected spatial transcriptomic profiles enriched for epithelial, immune, or stromal compartments; paralleled with histological annotations, imagebased network analysis and protein expression. Seven novel spatial epithelial archetypes (EAs), seven tumour-immune microenvironments (TIMEs) and their co-localisation patterns were defined, revealing an opposing prevalence of functionally divergent EAs between response groups and the prognostic significance of B-cell enriched TIMEs, in particular those surrounding histologically normal epithelium adjacent to the tumour. The SMART dataset and analytical tools are publicly available via the PharosAI platform, providing the research community with the most comprehensive, manually annotated spatio-molecular transcriptomics atlas of NACT-treated TNBC to date.

cancer biology↗

bioRxiv2023

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here we used fluorescence lifetime (FLT)-measurements of Forster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multi-exponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent-verticilide, yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.

biophysics↗

bioRxiv2023

Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca2+ stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and dynamic exchange of PLB between pentamers and the regulatory complex with SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic mutations of PLB, a cysteine substitution of arginine 9 (R9C) and an in-frame deletion of arginine 14 (R14del). Both mutations are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized that this mutation may also alter PLB homo-oligomerization and disrupt the PLB-SERCA regulatory interaction. SDS-PAGE revealed a significantly increased pentamer:monomer ratio for R14del-PLB when compared to WT-PLB. In addition, we quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed an increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT, suggesting that, like R9C, the R14del mutation stabilizes PLB in its pentameric form, decreasing its ability to regulate SERCA. Moreover, the R14del mutation reduces the rate of PLB unbinding from the pentamer after a transient Ca2+ elevation, limiting the rate of re-binding to SERCA. A computational model predicted that hyperstabilization of PLB pentamers by R14del impairs the ability of cardiac Ca2+ handling to respond to changing heart rates between rest and exercise. We postulate that impaired responsiveness to physiological stress contributes to arrhythmogenesis in human carriers of the R14del mutation.

physiology↗