Biology subjects

James, C. A.

Publications and source records attributed to James, C. A..

4 recordsLinked to original sources

bioRxiv2026

Damaging RBM20 E-rich domain variants are not rescued by gene replacement

The promise of precision therapeutics in genetic cardiomyopathies relies on linking specific therapies to variant mechanisms. Missense variants in the cardiac splice regulator RBM20 cause a highly penetrant and arrhythmogenic dilated cardiomyopathy. Disease-causing variants in RBM20s arginine-serine rich (RS) domain act via formation of toxic gain of function cytoplasmic granules, but this is not true for a small number of clinically adjudicated pathogenic variants in its glutamate(E)-rich domain. To better define the effects of E-rich domain variants, we developed a scalable screen based on induced pluripotent stem cell (iPSC) cardiomyocyte differentiation that identified several additional damaging variants. Several of these reduced RBM20 protein abundance and stability. We therefore hypothesized that, unlike RS domain variants, these E-rich variants might be rescued by RBM20 overexpression. To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with a pathogenic E-rich domain variant (p.E913K), and confirmed reduced RBM20 protein expression in these RBM20+/p.E913K cells after differentiation to iPSC-derived cardiomyocytes (iPSC-CM, vs. engineered isogenic RBM20+/+). These iPSC-CMs also displayed aberrant transcriptional splicing, reduced contractility, increased calcium-induced calcium release, and nuclear localization of RBM20 protein, often with more than the two expected RBM20-centric splice factories. AAV-based overexpression of RBM20 reversed some, but not all of the mis-splicing events identified in RBM20+/p.E913K iPSC-CMs, and did not improve their abnormal contractility, calcium handling or supernumerary RBM20 nuclear granules. In summary, our data indicate that pathogenic E-rich domain variants reduce RBM20 protein abundance, but that their mechanism is unlikely to be explained by haploinsufficiency alone.

genetics↗

bioRxiv2024

Desmoplakin mutations in cardiac fibroblasts cause TGF?1-mediated pathological fibrogenesis in desmoplakin cardiomyopathy via beclin-1 regulation

BackgroundPathological fibrosis is a major finding in cardiovascular diseases and can result in arrhythmia and heart failure. Desmosome gene mutations can lead to arrhythmogenic cardiomyopathy (ACM). Among ACM, pathogenic desmoplakin (DSP) variants cause a distinctive cardiomyopathy with excessive cardiac fibrosis that could precede ventricular dysfunction. DSP variants are also linked to other fibrotic diseases. Whether DSP plays any role in pathological fibrosis remain unknown. MethodsMesenchymal stromal cells (MSCs) are resident fibroblast-like cells that are responsible for fibrogenesis in most organs, including hearts. We first used unbiased genome-wide analyses to generate cardiac fibroblasts-like, induced pluripotent stem cell-derived MSCs from normal donors and ACM patients with DSP mutations. We then studied the fibrogenic responses of cardiac MSCs to transforming growth factor beta-1 (TGF-{beta}1) using Western/Co-IP, autophagy assay, gene knockdowns/over-expressions, genomic analyses, mouse DSP knockdown models, immunostaining, and qPCR. ResultsTGF{beta}1 induced excessive accumulations of vimentin (VIM)/fibrillar collagens, and over-activated fibrotic genes in DSP-mutant MSCs when compared to normal MSCs. In normal MSCs, VIMs bind to wild-type DSP during normal fibrogenesis after TGF{beta}1. DSP-mutant MSCs exhibited a haplo-insufficient phenotype with increased DSP-unbound VIMs that sequestered beclin-1 (BECN1) from activating autophagy and caveolin-1 (CAV1)-mediated endocytosis. Decreased autophagy caused collagen accumulations and diminished CAV1 endocytosis resulted in abnormal CAV1 plaque formation that over-activated fibrotic genes [COL1A1, COL3A1, and fibronectin (FN)] via heightened p38 activities after TGF{beta}1. Genome-wide analysis and DSP knockdown in mouse fibroblasts confirmed this novel role of DSP mutations in pathological fibrosis. Overexpression of VIM-binding domains of DSP could suppress pathological fibrosis by increasing collagen autophagic degradation and decreasing fibrotic gene expressions. ConclusionsOur data reveal that DSP deficiency in MSCs/fibroblasts leads to exaggerated fibrogenesis in DSP-cardiomyopathy by decreasing BECN1 availability for autophagy and CAV1-endocytosis. Overexpression of VIM binding domains of DSP could be a new strategy to treat pathological fibrosis.

cell biology↗

bioRxiv2022

A Universal Language for Finding Mass Spectrometry Data Patterns

Even though raw mass spectrometry data is information rich, the vast majority of the data is underutilized. The ability to interrogate these rich datasets is handicapped by the limited capability and flexibility of existing software. We introduce the Mass Spec Query Language (MassQL) that addresses these issues by enabling an expressive set of mass spectrometry patterns to be queried directly from raw data. MassQL is an open-source mass spectrometry query language for flexible and mass spectrometer manufacturer-independent mining of MS data. We envision the flexibility, scalability, and ease of use of MassQL will empower the mass spectrometry community to take fuller advantage of their mass spectrometry data and accelerate discoveries.

bioinformatics↗

bioRxiv2020

CD4 and CD8 co-receptors modulate functional avidity of CD1b-restricted T cells.

CD4 and CD8 co-receptors define distinct lineages of T cells restricted by major histocompatibility complex (MHC) Class II and I molecules, respectively. Co-receptors interact with the T cell receptor (TCR) at the surface of MHC-restricted T cells to facilitate antigen recognition, thymic selection, and functional differentiation. T cells also recognize lipid antigens presented by CD1 molecules, but the role that CD4 and CD8 play in lipid antigen recognition is unknown. We studied the effect of CD4 and CD8 on the avidity, activation, and function of T cells specific for two CD1b-presented mycobacterial lipid antigens, glucose monomycolate (GMM) and diacylated sulfoglycolipids (SGL). In a human cohort study using SGL-loaded CD1b tetramers, we discovered a hierarchy among SGL-specific T cells in which T cells expressing the CD4 or CD8 co-receptor stain with a higher tetramer mean fluorescence intensity (MFI) than CD4-CD8- T cells. To determine the role of the TCR co-receptor in lipid antigen recognition, we exogenously expressed GMM and SGL-specific TCRs in Jurkat or polyclonal T cells and quantified tetramer staining and activation thresholds. Transduced CD4+ primary T cells bound the lipid-loaded CD1b tetramer with a higher MFI than CD8+ primary T cells, and transduced CD8+ Jurkat cells bound the SGL-CD1b tetramer with higher MFI than CD4-CD8- Jurkat cells. The presence of either co-receptor also decreased the threshold for IFN-{gamma} secretion. Further, co-receptor expression increased surface expression of CD3{varepsilon}, suggesting a mechanism for increased tetramer binding and activation. Finally, we used single-cell sequencing to define the TCR repertoire and ex vivo functional profiles of SGL-specific T cells from individuals with M.tb disease. We found that CD8+ T cells specific for SGL express canonical markers associated with cytotoxic T lymphocytes, while CD4+ T cells could be classified as T regulatory or T follicular helper cells. Among SGL-specific T cells, only those expressing the CD4 co-receptor also expressed Ki67, suggesting that they were actively proliferating at the time of sample collection. Together, these data reveal that expression of CD4 and CD8 co-receptor modulates TCR avidity for lipid antigen, leading to functional diversity and differences in in vivo proliferation during M.tb disease.

immunology↗