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Circulation Research Jul 2023Systemic defects in intestinal iron absorption, circulation, and retention cause iron deficiency in 50% of patients with heart failure. Defective subcellular iron uptake...
BACKGROUND
Systemic defects in intestinal iron absorption, circulation, and retention cause iron deficiency in 50% of patients with heart failure. Defective subcellular iron uptake mechanisms that are independent of systemic absorption are incompletely understood. The main intracellular route for iron uptake in cardiomyocytes is clathrin-mediated endocytosis.
METHODS
We investigated subcellular iron uptake mechanisms in patient-derived and CRISPR/Cas-edited induced pluripotent stem cell-derived cardiomyocytes as well as patient-derived heart tissue. We used an integrated platform of DIA-MA (mass spectrometry data-independent acquisition)-based proteomics and signaling pathway interrogation. We employed a genetic induced pluripotent stem cell model of 2 inherited mutations ( []-R141W and []-L185F) that lead to dilated cardiomyopathy (DCM), a frequent cause of heart failure, to study the underlying molecular dysfunctions of DCM mutations.
RESULTS
We identified a druggable molecular pathomechanism of impaired subcellular iron deficiency that is independent of systemic iron metabolism. Clathrin-mediated endocytosis defects as well as impaired endosome distribution and cargo transfer were identified as a basis for subcellular iron deficiency in DCM-induced pluripotent stem cell-derived cardiomyocytes. The clathrin-mediated endocytosis defects were also confirmed in the hearts of patients with DCM with end-stage heart failure. Correction of the mutation in DCM patient-derived induced pluripotent stem cells, treatment with a peptide, Rho activator II, or iron supplementation rescued the molecular disease pathway and recovered contractility. Phenocopying the effects of the mutation into WT induced pluripotent stem cell-derived cardiomyocytes could be ameliorated by iron supplementation.
CONCLUSIONS
Our findings suggest that impaired endocytosis and cargo transport resulting in subcellular iron deficiency could be a relevant pathomechanism for patients with DCM carrying inherited mutations. Insight into this molecular mechanism may contribute to the development of treatment strategies and risk management in heart failure.
Topics: Humans; Myocytes, Cardiac; Mutation; Cardiomyopathy, Dilated; Induced Pluripotent Stem Cells; Iron Deficiencies; Heart Failure; Iron; Clathrin
PubMed: 37313752
DOI: 10.1161/CIRCRESAHA.122.321157 -
JCI Insight Dec 2023Transmembrane and tetratricopeptide repeat 4 (Tmtc4) is a deafness gene in mice. Tmtc4-KO mice have rapidly progressive postnatal hearing loss due to overactivation of...
Transmembrane and tetratricopeptide repeat 4 (Tmtc4) is a deafness gene in mice. Tmtc4-KO mice have rapidly progressive postnatal hearing loss due to overactivation of the unfolded protein response (UPR); however, the cellular basis and human relevance of Tmtc4-associated hearing loss in the cochlea was not heretofore appreciated. We created a hair cell-specific conditional KO mouse that phenocopies the constitutive KO with postnatal onset deafness, demonstrating that Tmtc4 is a hair cell-specific deafness gene. Furthermore, we identified a human family in which Tmtc4 variants segregate with adult-onset progressive hearing loss. Lymphoblastoid cells derived from multiple affected and unaffected family members, as well as human embryonic kidney cells engineered to harbor each of the variants, demonstrated that the human Tmtc4 variants confer hypersensitivity of the UPR toward apoptosis. These findings provide evidence that TMTC4 is a deafness gene in humans and further implicate the UPR in progressive hearing loss.
Topics: Animals; Humans; Mice; Cochlea; Deafness; Hair; Hair Cells, Auditory; Hearing Loss
PubMed: 37943620
DOI: 10.1172/jci.insight.172665 -
BioRxiv : the Preprint Server For... Sep 2023Hypoxic cancer cells resist many anti-neoplastic therapies and can seed recurrence. We found previously that PTP1B deficiency promotes HER2+ breast cancer cell death in...
Hypoxic cancer cells resist many anti-neoplastic therapies and can seed recurrence. We found previously that PTP1B deficiency promotes HER2+ breast cancer cell death in hypoxia by activating RNF213, an ∼600kDa protein containing AAA-ATPase domains and two ubiquitin ligase domains (RING and RZ) that also is implicated in Moyamoya disease (MMD), lipotoxicity, and innate immunity. Here we report that PTP1B and ABL1/2 reciprocally control RNF213 phosphorylation on tyrosine-1275. This phosphorylation promotes RNF213 oligomerization and RZ domain activation. The RZ domain ubiquitylates CYLD/SPATA2, and together with the LUBAC complex, induces their degradation. Decreased CYLD/SPATA2 causes NF-κB activation, which together with hypoxia-induced ER-stress triggers GDSMD-dependent pyroptosis. Mutagenesis experiments show that the RING domain negatively regulates the RZ domain. -deleted HER2+ cell-derived xenografts phenocopy the effects of PTP1B deficiency, and reconstituting knockout lines with RNF213 mutants shows that the RZ domain mediates PTP1B-dependent tumor cell death. Our results identify a novel, potentially targetable PTP1B/RNF213/CYCLD/SPATA pathway critical for controlling inflammatory cell death in hypoxic tumors that could be exploited to target hypoxic tumor cells, potentially turning "cold" tumors "hot". Our findings also reveal new insights into RNF213 regulation, and have potentially important implications for the pathogenesis of MMD, atherosclerosis, and inflammatory and auto-immune disorders.
PubMed: 37808759
DOI: 10.1101/2023.08.05.552118 -
Cell Reports Dec 2023Neurodegenerative disorders, such as Alzheimer's disease (AD) or Huntington's disease (HD), are linked to protein aggregate neurotoxicity. According to the "cholinergic...
Neurodegenerative disorders, such as Alzheimer's disease (AD) or Huntington's disease (HD), are linked to protein aggregate neurotoxicity. According to the "cholinergic hypothesis," loss of acetylcholine (ACh) signaling contributes to the AD pathology, and therapeutic restoration of ACh signaling is a common treatment strategy. How disease causation and the effect of ACh are linked to protein aggregation and neurotoxicity remains incompletely understood, thus limiting the development of more effective therapies. Here, we show that BAZ-2, the Caenorhabditis elegans ortholog of human BAZ2B, limits ACh signaling. baz-2 mutations reverse aggregation and toxicity of amyloid-beta as well as polyglutamine peptides, thereby restoring health and lifespan in nematode models of AD and HD, respectively. The neuroprotective effect of Δbaz-2 is mediated by choline acetyltransferase, phenocopied by ACh-esterase depletion, and dependent on ACh receptors. baz-2 reduction or ectopic ACh treatment augments proteostasis via induction of the endoplasmic reticulum unfolded protein response and the ubiquitin proteasome system.
Topics: Animals; Humans; Acetylcholine; Alzheimer Disease; Amyloid beta-Peptides; Bromodomain Containing Proteins; Caenorhabditis elegans; Huntington Disease; Proteostasis; Transcription Factors, General
PubMed: 38100354
DOI: 10.1016/j.celrep.2023.113577 -
Life Science Alliance Sep 2023Phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 5-phosphate (PI5P) are low-abundance phosphoinositides crucial for key cellular events such as endosomal...
Phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 5-phosphate (PI5P) are low-abundance phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P in vitro. In this study, we report a role for PIP4K in regulating PI3P levels in Loss-of-function mutants of the only PIP4K gene show reduced cell size in salivary glands. PI3P levels are elevated in and reverting PI3P levels back towards WT, without changes in PI5P levels, can rescue the reduced cell size. mutants also show up-regulation in autophagy and the reduced cell size can be reverted by depleting Atg8a that is required for autophagy. Lastly, increasing PI3P levels in WT can phenocopy the reduction in cell size and associated autophagy up-regulation seen in Thus, our study reports a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size.
Topics: Animals; Autophagy; Cell Size; Drosophila; Endosomes
PubMed: 37316298
DOI: 10.26508/lsa.202301920 -
Journal of Lipid Research Feb 2024Familial hypercholesterolemia (FH) is a common genetic disorder of lipid metabolism caused by pathogenic/likely pathogenic variants in LDLR, APOB, and PCSK9 genes....
Familial hypercholesterolemia (FH) is a common genetic disorder of lipid metabolism caused by pathogenic/likely pathogenic variants in LDLR, APOB, and PCSK9 genes. Variants in FH-phenocopy genes (LDLRAP1, APOE, LIPA, ABCG5, and ABCG8), polygenic hypercholesterolemia, and hyperlipoprotein (a) [Lp(a)] can also mimic a clinical FH phenotype. We aim to present a new diagnostic tool to unravel the genetic background of clinical FH phenotype. Biochemical and genetic study was performed in 1,005 individuals with clinical diagnosis of FH, referred to the Portuguese FH Study. A next-generation sequencing panel, covering eight genes and eight SNPs to determine LDL-C polygenic risk score and LPA genetic score, was validated, and used in this study. FH was genetically confirmed in 417 index cases: 408 heterozygotes and 9 homozygotes. Cascade screening increased the identification to 1,000 FH individuals, including 11 homozygotes. FH-negative individuals (phenotype positive and genotype negative) have Lp(a) >50 mg/dl (30%), high polygenic risk score (16%), other monogenic lipid metabolism disorders (1%), and heterozygous pathogenic variants in FH-phenocopy genes (2%). Heterozygous variants of uncertain significance were identified in primary genes (12%) and phenocopy genes (7%). Overall, 42% of our cohort was genetically confirmed with FH. In the remaining individuals, other causes for high LDL-C were identified in 68%. Hyper-Lp(a) or polygenic hypercholesterolemia may be the cause of the clinical FH phenotype in almost half of FH-negative individuals. A small part has pathogenic variants in ABCG5/ABCG8 in heterozygosity that can cause hypercholesterolemia and should be further investigated. This extended next-generation sequencing panel identifies individuals with FH and FH-phenocopies, allowing to personalize each person's treatment according to the affected pathway.
Topics: Humans; Proprotein Convertase 9; Hypercholesterolemia; Cholesterol, LDL; Hyperlipoproteinemia Type II; Phenotype; Genetic Background; Receptors, LDL; Mutation
PubMed: 38122934
DOI: 10.1016/j.jlr.2023.100490 -
Protein Science : a Publication of the... Sep 2023BAZ2A promotes migration and invasion in prostate cancer. Two chemical probes, the specific BAZ2-ICR, and the BAZ2/BRD9 cross-reactive GSK2801, interfere with the...
BAZ2A promotes migration and invasion in prostate cancer. Two chemical probes, the specific BAZ2-ICR, and the BAZ2/BRD9 cross-reactive GSK2801, interfere with the recognition of acetylated lysines in histones by the bromodomains of BAZ2A and of its BAZ2B paralog. The two chemical probes were tested in prostate cancer cell lines with opposite androgen susceptibility. BAZ2-ICR and GSK2801 showed different cellular efficacies in accordance with their unequal selectivity profiles. Concurrent inhibition of BAZ2 and BRD9 did not reproduce the effects observed with GSK2801, indicating possible off-targets for this chemical probe. On the other hand, the single BAZ2 inhibition by BAZ2-ICR did not phenocopy genetic ablation, demonstrating that bromodomain interference is not sufficient to strongly affect BAZ2A functionality and suggesting a PROTAC-based chemical ablation as an alternative optimization strategy and a possible therapeutic approach. In this context, we also present the crystallographic structures of BAZ2A in complex with the above chemical probes. Binding poses of TP-238 and GSK4027, chemical probes for the bromodomain subfamily I, and two ligands of the CBP/EP300 bromodomains identify additional headgroups for the development of BAZ2A ligands.
Topics: Male; Humans; Ligands; Chromosomal Proteins, Non-Histone; Indolizines; Prostatic Neoplasms; Transcription Factors; Transcription Factors, General
PubMed: 37574751
DOI: 10.1002/pro.4752 -
The American Journal of Cardiology Feb 2024Hypertrophic cardiomyopathy (HCM) is increasingly recognized and may benefit from the recent approval of new, targeted medical therapy. Successful management of HCM is... (Review)
Review
Hypertrophic cardiomyopathy (HCM) is increasingly recognized and may benefit from the recent approval of new, targeted medical therapy. Successful management of HCM is dependent on early and accurate diagnosis. The lack of a definitive diagnostic test, the wide variation in phenotype and the commonness of phenocopy conditions, and the presence of normal or hyperdynamic left ventricular function in most patients makes HCM a condition that is highly dependent on imaging for all aspects of management including, diagnosis, classification, predicting risk of complications, detecting complications, identifying risk for ventricular arrhythmias, evaluating choice of therapy and monitoring therapy, intraprocedural guidance, and screening family members. Although echocardiographic imaging remains the mainstay in the diagnosis and subsequent management of HCM, this disease clearly requires multimethod imaging for various aspects of optimal patient care. Advances in echocardiography hardware and techniques, development and refinement of imaging with computed tomography, magnetic resonance, and nuclear scanning, and the emergence of very focused assessments such as diastology and fibrosis imaging have all advanced the diagnosis and management of HCM. In this review, we discuss the relative utility and evidence support for these imaging approaches to contribute to improve patient outcomes.
Topics: Humans; Cardiomyopathy, Hypertrophic; Magnetic Resonance Imaging; Echocardiography; Arrhythmias, Cardiac; Ventricular Function, Left
PubMed: 38368033
DOI: 10.1016/j.amjcard.2023.10.081 -
Nature Communications Sep 2023HELLS/LSH (Helicase, Lymphoid Specific) is a SNF2-like chromatin remodelling protein involved in DNA methylation. Its loss-of-function in humans causes humoral...
HELLS/LSH (Helicase, Lymphoid Specific) is a SNF2-like chromatin remodelling protein involved in DNA methylation. Its loss-of-function in humans causes humoral immunodeficiency, called ICF4 syndrome (Immunodeficiency, Centromeric Instability, Facial anomalies). Here we show by our newly generated B-cell-specific Hells conditional knockout mouse model that HELLS plays a pivotal role in T-dependent B-cell responses. HELLS deficiency induces accelerated decay of germinal center (GC) B cells and impairs the generation of high affinity memory B cells and circulating antibodies. Mutant GC B cells undergo dramatic DNA hypomethylation and massive de-repression of evolutionary recent retrotransposons, which surprisingly does not directly affect their survival. Instead, they prematurely upregulate either memory B cell markers or the transcription factor ATF4, which is driving an mTORC1-dependent metabolic program typical of plasma cells. Treatment of wild type mice with a DNMT1-specific inhibitor phenocopies the accelerated kinetics, thus pointing towards DNA-methylation maintenance by HELLS being a crucial mechanism to fine-tune the GC transcriptional program and enable long-lasting humoral immunity.
Topics: Animals; Humans; Mice; B-Lymphocytes; DNA; DNA Helicases; DNA Methylation; Germinal Center; Plasma Cells
PubMed: 37709749
DOI: 10.1038/s41467-023-41317-3 -
The Journal of Clinical Investigation Dec 2023Foxp3-expressing Tregs employ multiple suppressive mechanisms to curtail conventional T cell (Tconv) responses and establish tissue homeostasis. How Foxp3 coordinates...
Foxp3-expressing Tregs employ multiple suppressive mechanisms to curtail conventional T cell (Tconv) responses and establish tissue homeostasis. How Foxp3 coordinates Treg contact-dependent suppressive function is not fully resolved. In this issue of the JCI, Wang and colleagues revealed that Foxp3-mediated inhibition of ryanodine receptor 2 (RyR2) led to strong Treg-DC interactions and enhanced immunosuppression. RyR2 depletion in Tconvs phenocopied this effect and equipped Tconvs with Treg-like suppressive function in multiple inflammatory or autoimmune contexts. This study provides molecular and therapeutic insights underlying how cell-cell contact limits immune reactivity.
Topics: Mice; Animals; T-Lymphocytes, Regulatory; Ryanodine Receptor Calcium Release Channel; Mice, Inbred C57BL; Immunosuppression Therapy; Forkhead Transcription Factors
PubMed: 38099491
DOI: 10.1172/JCI172986