-
Nature Communications Jun 2024Cell polarity mechanisms allow the formation of specialized membrane domains with unique protein compositions, signalling properties, and functional characteristics. By...
Cell polarity mechanisms allow the formation of specialized membrane domains with unique protein compositions, signalling properties, and functional characteristics. By analyzing the localization of potassium channels and proteins belonging to the dystrophin-associated protein complex, we reveal the existence of distinct planar-polarized membrane compartments at the surface of C. elegans muscle cells. We find that muscle polarity is controlled by a non-canonical Wnt signalling cascade involving the ligand EGL-20/Wnt, the receptor CAM-1/Ror, and the intracellular effector DSH-1/Dishevelled. Interestingly, classical planar cell polarity proteins are not required for this process. Using time-resolved protein degradation, we demonstrate that -while it is essentially in place by the end of embryogenesis- muscle polarity is a dynamic state, requiring continued presence of DSH-1 throughout post-embryonic life. Our results reveal the unsuspected complexity of the C. elegans muscle membrane and establish a genetically tractable model system to study cellular polarity and membrane compartmentalization in vivo.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Polarity; Dystrophin; Wnt Signaling Pathway; Muscles; Dishevelled Proteins; Receptor Tyrosine Kinase-like Orphan Receptors; Cell Membrane; Dystrophin-Associated Protein Complex; Wnt Proteins; Signal Transduction
PubMed: 38858388
DOI: 10.1038/s41467-024-49154-8 -
BioRxiv : the Preprint Server For... May 2024, encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about...
BACKGROUND
, encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs).
METHODS
The hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N'-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays .
RESULTS
Confocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential (ΔΨm). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after treatment with the myosin inhibitor mavacamten (CAMZYOS®) in hCOs. Lastly, various binding assays revealed a significant loss of affinity in the presence of with myosin S2 region as a likely mechanism for hypercontraction.
CONCLUSIONS
Conceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. mutations have been implicated in hypertrophic cardiomyopathy. D389V is a polymorphic variant of predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. The molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined. The authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. The -mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. Our data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS®), a myosin inhibitor. In addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential (ΔΨm), highlighting a possible early adaptive response to primary sarcomeric changes. Early treatment of carriers with mavacamten may prevent or reduce early HCM-related pathology. A graphical abstract is available for this article.
PubMed: 38853909
DOI: 10.1101/2024.05.29.596463 -
Xenotransplantation 2024Recent clinical xenotransplantation and human decedent studies demonstrate that clinical hyperacute rejection of genetically engineered porcine organs can be reliably...
Recent clinical xenotransplantation and human decedent studies demonstrate that clinical hyperacute rejection of genetically engineered porcine organs can be reliably avoided but that antibody mediated rejection (AMR) continues to limit graft survival. We previously identified porcine glycans and proteins which are immunogenic after cardiac xenotransplantation in non-human primates, but the clinical immune response to antigens present in glycan depleted triple knockout (TKO) donor pigs is poorly understood. In this study we use fluorescence barcoded human embryonic kidney cells (HEK) and HEK cell lines expressing porcine glycans (Gal and SDa) or proteins (tetraspanin-29 [CD9], membrane cofactor protein [CD46], protectin, membrane attack complex inhibition factor [CD59], endothelial cell protein C receptor, and Annexin A2) to screen antibody reactivity in human serum from 160 swine veterinarians, a serum source with potential occupational immune challenge from porcine tissues and pathogens. High levels of anti-Gal IgM were present in all samples and lower levels of anti-SDa IgM were present in 41% of samples. IgM binding to porcine proteins, primarily CD9 and CD46, previously identified as immunogenic in pig to non-human primate cardiac xenograft recipients, was detected in 28 of the 160 swine veterinarian samples. These results suggest that barcoded HEK cell lines expressing porcine protein antigens can be useful for screening human patient serum. A comprehensive analysis of sera from clinical xenotransplant recipients to define a panel of commonly immunogenic porcine antigens will likely be necessary to establish an array of porcine non-Gal antigens for effective monitoring of patient immune responses and allow earlier therapies to reverse AMR.
Topics: Animals; Transplantation, Heterologous; Humans; Swine; Graft Rejection; HEK293 Cells; Veterinarians; Polysaccharides; Animals, Genetically Modified; Antibodies, Heterophile; Heterografts; Immunoglobulin M
PubMed: 38853364
DOI: 10.1111/xen.12865 -
Cytotherapy Mar 2024Mesenchymal stromal cells (MSCs) are attractive as a therapeutic modality in multiple disease conditions characterized by inflammation and vascular compromise....
BACKGROUND AIMS
Mesenchymal stromal cells (MSCs) are attractive as a therapeutic modality in multiple disease conditions characterized by inflammation and vascular compromise. Logistically they are advantageous because they can be isolated from adult tissue sources, such as bone marrow (BM). The phase 2a START clinical trial determined BM-MSCs to be safe in patients with moderate-to-severe acute respiratory distress syndrome (ARDS). Herein, we examine a subset of the clinical doses of MSCs generated for the phase 2a START trial from three unique donors (1-3), where one of the donors' donated BM on two separate occasions (donor 3 and 3W).
METHODS
The main objective of this study was to correlate properties of the cells from the four lots with plasma biomarkers from treated patients and relevant to ARDS outcomes. To do this we evaluated MSC donor lots for (i) post-thaw viability, (ii) growth kinetics, (iii) metabolism, (iv) surface marker expression, (v) protein expression, (vi) immunomodulatory ability and (vii) their functional effects on regulating endothelial cell permeability.
RESULTS
MSC-specific marker expression and protection of thrombin-challenged endothelial barrier permeability was similar among all four donor lots. Inter and intra-donor variability was observed in all the other in vitro assays. Furthermore, patient plasma ANG-2 and protein C levels at 6 hours post-transfusion were correlated to cell viability in an inter- and intra-donor dependent manner.
CONCLUSIONS
These findings highlight the potential of donor dependent (inter-) and collection dependent (intra-) effects in patient biomarker expression.
PubMed: 38852094
DOI: 10.1016/j.jcyt.2024.03.486 -
Nature Communications Jun 2024Age-related depletion of stem cells causes tissue degeneration and failure to tissue regeneration, driving aging at the organismal level. Previously we reported a...
Age-related depletion of stem cells causes tissue degeneration and failure to tissue regeneration, driving aging at the organismal level. Previously we reported a cell-non-autonomous DAF-16/FOXO activity in antagonizing the age-related loss of germline stem/progenitor cells (GSPCs) in C. elegans, indicating that regulation of stem cell aging occurs at the organ system level. Here we discover the molecular effector that links the cell-non-autonomous DAF-16/FOXO activity to GSPC maintenance over time by performing a tissue-specific DAF-16/FOXO transcriptome analysis. Our data show that dos-3, which encodes a non-canonical Notch ligand, is a direct transcriptional target of DAF-16/FOXO and mediates the effect of the cell-non-autonomous DAF-16/FOXO activity on GSPC maintenance through activating Notch signaling in the germ line. Importantly, expression of a human homologous protein can functionally substitute for DOS-3 in this scenario. As Notch signaling controls the specification of many tissue stem cells, similar mechanisms may exist in other aging stem cell systems.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Forkhead Transcription Factors; Germ Cells; Receptors, Notch; Stem Cells; Signal Transduction; Aging; Humans
PubMed: 38851828
DOI: 10.1038/s41467-024-49318-6 -
Acta Neuropathologica Communications Jun 2024Mitochondrial dysfunctions are key features of Alzheimer's disease (AD). The occurrence of these disturbances in the peripheral cells of AD patients and their potential...
Mitochondrial dysfunctions are key features of Alzheimer's disease (AD). The occurrence of these disturbances in the peripheral cells of AD patients and their potential correlation with disease progression are underinvestigated. We studied mitochondrial structure, function and mitophagy in fibroblasts from healthy volunteers and AD patients at the prodromal (AD-MCI) or demented (AD-D) stages. We carried out correlation studies with clinical cognitive scores, namely, (i) Mini-Mental State Examination (MMSE) and (ii) Dementia Rating-Scale Sum of Boxes (CDR-SOB), and with (iii) amyloid beta (Aβ) plaque burden (PiB-PET imaging) and (iv) the accumulation of peripheral amyloid precursor protein C-terminal fragments (APP-CTFs). We revealed alterations in mitochondrial structure as well as specific mitochondrial dysfunction signatures in AD-MCI and AD-D fibroblasts and revealed that defective mitophagy and autophagy are linked to impaired lysosomal activity in AD-D fibroblasts. We reported significant correlations of a subset of these dysfunctions with cognitive decline, AD-related clinical hallmarks and peripheral APP-CTFs accumulation. This study emphasizes the potential use of peripheral cells for investigating AD pathophysiology.
Topics: Humans; Alzheimer Disease; Fibroblasts; Aged; Female; Mitochondria; Male; Mitophagy; Middle Aged; Aged, 80 and over; Amyloid beta-Protein Precursor; Cognitive Dysfunction; Autophagy
PubMed: 38851733
DOI: 10.1186/s40478-024-01807-x -
Indian Dermatology Online Journal 2024Imatinib and nilotinib are inhibitors of tyrosine kinases (TKIs) generated from the bcr-abl fusion protein, c-Kit, and platelet-derived growth factor receptors....
Imatinib and nilotinib are inhibitors of tyrosine kinases (TKIs) generated from the bcr-abl fusion protein, c-Kit, and platelet-derived growth factor receptors. Cutaneous adverse effects (AEs) of TKI are the most frequent non-hematological sequelae. In our case, the common molecular target raises the possibility that cross-intolerance, in which similar AEs occur with both agents, can arise. We hereby report a rare case report on cross-intolerance of cutaneous AEs of imatinib and nilotinib in chronic myeloid leukemia.
PubMed: 38845649
DOI: 10.4103/idoj.idoj_229_23 -
Science (New York, N.Y.) Jun 2024Successive cleavages of amyloid precursor protein C-terminal fragment with 99 residues (APP-C99) by γ-secretase result in amyloid-β (Aβ) peptides of varying lengths....
Successive cleavages of amyloid precursor protein C-terminal fragment with 99 residues (APP-C99) by γ-secretase result in amyloid-β (Aβ) peptides of varying lengths. Most cleavages have a step size of three residues. To elucidate the underlying mechanism, we determined the atomic structures of human γ-secretase bound individually to APP-C99, Aβ49, Aβ46, and Aβ43. In all cases, the substrate displays the same structural features: a transmembrane α-helix, a three-residue linker, and a β-strand that forms a hybrid β-sheet with presenilin 1 (PS1). Proteolytic cleavage occurs just ahead of the substrate β-strand. Each cleavage is followed by unwinding and translocation of the substrate α-helix by one turn and the formation of a new β-strand. This mechanism is consistent with existing biochemical data and may explain the cleavages of other substrates by γ-secretase.
Topics: Humans; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Crystallography, X-Ray; Models, Molecular; Peptide Fragments; Presenilin-1; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Proteolysis; Substrate Specificity
PubMed: 38843321
DOI: 10.1126/science.adn5820 -
Indian Journal of Dermatology,... Apr 2024
A novel homozygous missense mutation in exon 3 at codon 42 c.125G>A (p.Arg42His) in the PROC gene causing protein C deficiency and presenting as neonatal purpura fulminans.
PubMed: 38841958
DOI: 10.25259/IJDVL_618_2023 -
Nature Jun 2024Increasing rates of autoimmune and inflammatory disease present a burgeoning threat to human health. This is compounded by the limited efficacy of available treatments...
Increasing rates of autoimmune and inflammatory disease present a burgeoning threat to human health. This is compounded by the limited efficacy of available treatments and high failure rates during drug development, highlighting an urgent need to better understand disease mechanisms. Here we show how functional genomics could address this challenge. By investigating an intergenic haplotype on chr21q22-which has been independently linked to inflammatory bowel disease, ankylosing spondylitis, primary sclerosing cholangitis and Takayasu's arteritis-we identify that the causal gene, ETS2, is a central regulator of human inflammatory macrophages and delineate the shared disease mechanism that amplifies ETS2 expression. Genes regulated by ETS2 were prominently expressed in diseased tissues and more enriched for inflammatory bowel disease GWAS hits than most previously described pathways. Overexpressing ETS2 in resting macrophages reproduced the inflammatory state observed in chr21q22-associated diseases, with upregulation of multiple drug targets, including TNF and IL-23. Using a database of cellular signatures, we identified drugs that might modulate this pathway and validated the potent anti-inflammatory activity of one class of small molecules in vitro and ex vivo. Together, this illustrates the power of functional genomics, applied directly in primary human cells, to identify immune-mediated disease mechanisms and potential therapeutic opportunities.
Topics: Female; Humans; Male; Anti-Inflammatory Agents; Cells, Cultured; Chromosomes, Human, Pair 21; Databases, Factual; Gene Expression Regulation; Genome-Wide Association Study; Genomics; Haplotypes; Inflammation; Inflammatory Bowel Diseases; Macrophages; Proto-Oncogene Protein c-ets-2; Reproducibility of Results; Tumor Necrosis Factors; Interleukin-23
PubMed: 38839969
DOI: 10.1038/s41586-024-07501-1