Dominant-negative Mutant Research Articles

Cell-autonomous action of Slit2 in radial migration of cortical projection neurons

Neuronal radial migration is a fundamental process for cortical development, the disruption of which causes neurological and psychiatric dysfunctions. SLIT2 plays diverse functions in brain development and is a well-known axon guidance molecule. In this study, we investigated the radial migration of projection neurons in the developing cerebral cortex by in utero knockdown (KD) of Slit2 in mice. KD of Slit2 did not interfere with the neurogenesis and fate-determination but led to the accumulation of the transfected cells in the intermediate zone (IZ), suggesting that the expression of Slit2 is crucial for the radial migration of the cortical neurons. KD of Slit2 hindered the transition of cells from a multipolar to a bipolar shape, which is necessary for glia-guided locomotion. Interestingly, reducing Slit2 did not affect the migration of neighboring untransfected cells, indicating a cell-autonomous action by SLIT2. In addition, the action of SLIT2 KD was mimicked by a dominant negative mutant of ROBO2, a canonical membrane receptor of SLIT2, supporting that SLIT2 acted locally as a secretory molecule. Our results suggest that SLIT2 is indispensable for the radial migration of cortical neurons through an autocrine signaling mechanism.

Dominant negative mutations in yeast Hsp90 indicate triage decision mechanism targeting client proteins for degradation.

Dominant negative mutations provide valuable tools for investigating protein mechanisms but can be difficult to isolate because of their toxic effects. We used a mutational scanning approach to identify dominant negative (DN) mutations in yeast Hsp90. In a previous mutational scan of the ATPase domain of Hsp90, we noticed that many mutations were at very low frequency after outgrowth in cells co-expressing WT Hsp90. Most of these depleted variants were located at the hinge of a lid that closes over ATP. To quantify toxic effects in the hinge regions, we performed mutational scanning using an inducible promoter and identified 113 variants with strong toxic effects. We analyzed individual DN mutations in detail and found that addition of the E33A mutation that prevents ATP hydrolysis by Hsp90 abrogated the dominant negative phenotype. FRET assays performed on individual DN mutants indicate the linkage between ATPase activity and formation of the closed structure is disrupted. DN Hsp90 decreased the expression level of two model Hsp90 clients, glucocorticoid receptor (GR) and v-src kinase. Using MG132, we found that GR was rapidly destabilized in a proteasome-dependent fashion. Biochemical analyses indicate that ATP hydrolysis by Hsp90 from open conformations can lead to ubiquitin-dependent client degradation.

A dominant negative Kcnd3 F227del mutation in mice causes spinocerebellar ataxia type 22 (SCA22) by impairing ER and Golgi functioning.

Spinocerebellar ataxia type 22 (SCA22) caused by KCND3 mutations is an autosomal dominant disorder. We established a mouse model carrying the Kcnd3 F227del mutation to study the molecular pathogenesis. Four findings were pinpointed. First, the heterozygous mice exhibited an early onset of defects in motor coordination and balance which mirror those of SCA22 patients. The degeneration and a minor loss of Purkinje cells, together with the concurrent presence of neuroinflammation, as well as the previous finding on electrophysiological changes, may all contribute to the development of the SCA22 ataxia phenotype in mice carrying the Kcnd3 F227del mutant protein. Second, the mutant protein is retained by the endoplasmic reticulum and Golgi, leading to activation of the unfolded protein response and a severe trafficking defect that affects its membrane destination. Intriguingly, profound damage of the Golgi is the earliest manifestation. Third, analysis of the transcriptome revealed that the Kcnd3 F227del mutation down-regulates a panel of genes involved in the functioning of synapses and neurogenesis which are tightly linked to the functioning of Purkinje cells. Finally, no ataxia phenotypes were detectable in knockout mice carrying a loss-of-function Kcnd3 mutation. Thus, Kcnd3 F227del is a dominant-negative mutation. This mouse model may serve as a preclinical model for exploring therapeutic strategies to treat patients. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

ARF6 as a Novel Activator of HIF-2α in Pulmonary Arterial Hypertension.

ADP-ribosylation factor 6 (ARF6), a GTPase associated with cancer metastasis, is activated in the lung endothelium in pulmonary arterial hypertension (PAH). To identify ARF6-regulated pathways relevant to PAH, we performed a state-of-the-art proteomic analysis of human pulmonary artery endothelial cells (HPAECs) overexpressing the wildtype, constitutively active, fast-cycling and dominant negative mutants of ARF6. The analysis revealed a novel link of ARF6 with hypoxia-inducible factor (HIF), in addition to endocytotic vesicle trafficking, cell proliferation, angiogenesis, oxidative stress and lipid metabolism. Active ARF6 markedly increased expression and activity of HIF-2, critical in PAH, with HIF-1 relatively unaffected. Hypoxic ARF6 activation was a prerequisite for HIF-2 activation and HIF-dependent gene expression in HPAECs, PAH blood-derived late outgrowth endothelial colony forming cells (ECFCs) and hypoxic mouse lungs in vivo. A novel ARF6 inhibitor, chlortetracycline (CTC), reduced hypoxia-induced HIF-2 activation, proliferation and angiogenesis in HPAECs and reduced HIF-2 expression in lung and heart tissues of hypoxic mice. PAH ECFCs showed elevated expression and activity of ARF6 and HIF2, which was attenuated by CTC, and oral CTC attenuated development of PH in chronically hypoxic mice. We identify epidermal growth factor receptor (EGFR) as a direct interactor of ARF6, and EGFR signalling as a crucial mechanism linking ARF6 and HIF activation. In conclusion, we are first to demonstrate a key role of ARF6 in the regulation of HIF-2α activation in vitro and in vivo and show that HIF-2α, a master-regulator of vascular remodelling in PAH, can be targeted by a clinically approved antibiotic chlortetracycline.

The role of the essential GTPase ObgE in regulating lipopolysaccharide synthesis in Escherichia coli

During growth, cells need to synthesize and expand their envelope, a process that requires careful regulation. Here, we show that the GTPase ObgE of E. coli contributes to the regulation of lipopolysaccharide (LPS) synthesis, an essential component of the Gram-negative outer membrane. Using a dominant-negative mutant (named ‘ObgE*’), we show a direct interaction between ObgE and LpxA, which catalyzes the first step in LPS synthesis. This interaction is enhanced by the mutation in ObgE* which, when bound to GTP, leads to inhibition of LpxA, decreased LPS synthesis, and cell death. Although wild-type ObgE does not exert the same strong effects as ObgE* on LpxA or LPS synthesis, our data indicate that ObgE participates in the regulation of cell envelope synthesis in E. coli. Because ObgE also influences other cellular functions (i.e., ribosome assembly, DNA replication, etc.), it seems increasingly plausible that this GTPase coordinates several processes to finetune cell growth.

Dominant-negative TP53 mutations potentiated by the HSF1-regulated proteostasis network.

Protein mutational landscapes are sculpted by the impacts of the resulting amino acid substitutions on the proteins stability and folding or aggregation kinetics. These properties can, in turn, be modulated by the composition and activities of the cellular proteostasis network. Heat shock factor 1 (HSF1) is the master regulator of the cytosolic and nuclear proteostasis networks, dynamically tuning the expression of cytosolic and nuclear chaperones and quality control factors to meet demand. Chronic increases in HSF1 levels and activity are prominent hallmarks of cancer cells. One plausible explanation for this observation is that the consequent upregulation of proteostasis factors could biophysically facilitate the acquisition of oncogenic mutations. Here, we experimentally evaluate the impacts of chronic HSF1 activation on the mutational landscape accessible to the quintessential oncoprotein p53. Specifically, we apply quantitative deep mutational scanning of p53 to assess how HSF1 activation shapes the mutational pathways by which p53 can escape cytotoxic pressure conferred by the small molecule nutlin-3, which is a potent antagonist of the p53 negative regulator MDM2. We find that activation of HSF1 broadly increases the fitness of dominant-negative substitutions within p53. This effect of HSF1 activation was particularly notable for non-conservative, biophysically unfavorable amino acid substitutions within buried regions of the p53 DNA-binding domain. These results indicate that chronic HSF1 activation profoundly shapes the oncogenic mutational landscape, preferentially supporting the acquisition of cancer-associated substitutions that are biophysically destabilizing. Along with providing the first experimental and quantitative insights into how HSF1 influences oncoprotein mutational spectra, these findings also implicate HSF1 inhibition as a strategy to reduce the accessibility of mutations that drive chemo-therapeutic resistance and metastasis.

Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases.

Background: Oncogenic mutations in the KRAS gene are detected in >90% of pancreatic cancers (PC). In genetically engineered mouse models of PC, oncogenic KRAS drives the formation of precursor lesions and their progression to invasive PC. The Yes-associated Protein (YAP) is a transcriptional coactivator required for transformation by the RAS oncogenes and the development of PC. In Ras-driven tumors, YAP can also substitute for oncogenic KRAS to drive tumor survival after the repression of the oncogene. Ras oncoproteins exert their transforming properties through their downstream effectors, including the PI3K kinase, Rac1 GTPase, and MAPK pathways. Methods: To identify Ras effectors that regulate YAP, YAP levels were measured in PC cells exposed to inhibitors of oncogenic K-Ras and its effectors. Results: In PC cells, the inhibition of Rac1 leads to a time-dependent decline in YAP protein, which could be blocked by proteosome inhibitor MG132. This YAP degradation after Rac1 inhibition was observed in a range of cell lines using different Rac1 inhibitors, Rac1 siRNA, or expression of dominant negative Rac1T17N mutant. Several E3 ubiquitin ligases, including SCFβTrCP, regulate YAP protein stability. To be recognized by this ligase, the βTrCP degron of YAP (amino acid 383-388) requires its phosphorylation by casein kinase 1 at Ser384 and Ser387, but these events must first be primed by the phosphorylation of Ser381 by LATS1/2. Using Flag-tagged mutants of YAP, we show that YAP degradation after Rac1 inhibition requires the integrity of this degron and is blocked by the silencing of βTrCP1/2 and by the inhibition of casein kinase 1. Unexpectedly, YAP degradation after Rac1 inhibition was still observed after the silencing of LATS1/2 or in cells carrying a LATS1/2 double knockout. Conclusions: These results reveal Rac1 as an oncogenic KRAS effector that contributes to YAP stabilization in PC cells. They also show that this regulation of YAP by Rac1 requires the SCFβTrCP ligase but occurs independently of the LATS1/2 kinases.

DNA-PK inhibition shows differential radiosensitization in orthotopic GBM PDX models based on DDR pathway deficits.

Glioblastoma (GBM) remains one of the most therapy-resistant malignancies with frequent local failures despite aggressive surgery, chemotherapy, and ionizing radiation (IR). Small molecule inhibitors of DNA-dependent protein kinase (DNA-PKi's) are potent radiosensitizers currently in clinical trials. Determining which patients may benefit from radiosensitization with DNA-PKi's is critical to avoid unnecessary increased risk of normal tissue toxicity. In this study we used GBM patient derived xenografts (PDXs) in orthotopic murine models to study the relationship between molecular features, pharmacokinetics, and the radiosensitizing potential of the DNA-PKi peposertib. We show that peposertib radiosensitizes established and PDX GBM lines in vitro at 300nM and above, with significant increase in radiosensitization by maintaining post-IR exposure for >12 hours. Radiosensitization by peposertib is mediated by catalytic inhibition of DNA-PK, and knock-down of DNA-PK by short hairpin RNA (shRNA) largely abolished the radiosensitizing effect. Peposertib decreased auto-phosphorylation of DNA-PKcs after IR in a dose-dependent manner with delay in resolution of γH2AX foci at 24 hours. The addition of peposertib to IR significantly increased survival in GBM120 orthotopic xenografts, but not in GBM10. There was no difference in plasma or average tumor concentrations of peposertib in the two cohorts. While the mechanism underpinning this discordant effect in vitro vs. in vivo is not clear, there was an association for greater sensitization in TP53 mutant lines. Transfection of a dominant-negative TP53 mutant in baseline TP53 wildtype GBM lines significantly delayed growth and decreased NHEJ efficiency (but not Homologous Recombination), after peposertib exposure.

Enhancing CRISPR-Cas-based gene targeting in tomato using a dominant-negative ku80

Abstract The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical nonhomologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented an approach to shift the repair pathway preference toward HR by using a dominant-negative ku80 mutant protein (KUDN) to disrupt the initiation of cNHEJ. The employment of KUDN conferred a 1.71- to 3.55-fold improvement in GT efficiency at the callus stage. When we screened transformants, there was a more remarkable increase in GT efficiency, ranging from 1.62- to 9.84-fold, at two specific tomato loci, SlHKT1;2 and SlEPSPS1. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated a 9-bp addition at an additional locus, SlCAB13. These findings provide another promising method for more efficient and precise plant breeding.

PAK4 is Required for Meiotic Resumption, Spindle Assembly, and Cortical Migration in Mouse Oocytes During Meiotic Maturation.

Oocyte meiotic errors can cause infertility, miscarriage, and birth defects. Here the role and the underlying mechanism of p21 activated kinase 4 (PAK4) in mouse oocyte meiosis is evaluated. It is found that PAK4 expression and its phosphorylation are detected in high level at germinal vesicle (GV) stage, and gradually decreased after meiotic resumption in oocytes. PAK4 has direct physical interaction with both mitogen-activated protein kinases 1/2 (MEK1/2) and Paxillin, they are colocalized on the spindle structure during metaphases I and II. Phospho-PAK4 is distributed beneath the cytoplasmic membrane and on the chromosomes, and colocalized with the microtubule organizing center (MTOC) proteins, Pericentrin and γ-tubulin, as well as phosphor-MEK1/2 and phosphor-Paxillin on spindle poles. PAK4 inhibition by chemical inhibitor LCH-7749944, specific Pak4 morpholino oligo or the dominant negative mutant Pak4K350, 351M influence the meiotic resumption, spindle assembly and its cortical migration, and associated with the downregulation in the dephosphorylation of cyclin dependent kinase 1 (CDK1) and the levels of Cyclin B1, MEK1/2, Paxillin, g-tubulin, acetylated a-tubulin, Arp3, and Cofilin phosphorylation in oocytes. In sum, PAK4 functions to sustain the rational levels of Cyclin B1, MEK1/2, Paxillin, y-tubulin, acetylated a-tubulin, Arp3, and phosphor-Cofilin in mouse oocytes, thereby promotes the meiotic resumption, spindle assembly, and migration during meiotic maturation.

A new-disease-causing dominant-negative variant in CARD11 gene in a Chinese case with recurrent fever

Immunodeficiency 11B with atopic dermatitis (IMD11B, OMIM:617638) is rare primary immunodeficiency disease caused by germline dominant negative (DN) mutations in the CARD11 gene. Affected patients present with immune dysfunction, recurrent infections and atopic dermatitis. In this study, we sought to identify and characterize the genetic variant in one patient with periodic fever, recurrent infections, and eczema. Trio whole-exome sequencing (WES) was employed in this patient and her parents, and Sanger sequencing validated the potential pathogenic variant. In vitro functional study was performed to evaluate the pathogenicity of genetic variant identified. A very rare missense mutation (c.2324C > T, p.S775L) in CARD11 gene (NM_032415) was identified by WES in the patient but not her parents. Luciferase reporter assays and co-immunoprecipitation demonstrated mutation exerts a dominant-interfering effect on wild-type CARD11, inhibiting the activity of NF-κB. RNA sequencing analysis also confirmed that mutant CARD11 inhibited down-stream transcriptional activity of NF-κB. A review of literature doesn’t found significant genotype–phenotype correlation. We identified a vary rare DN CARD11 mutation, expanding the genetic and phenotypic spectrum of CARD11.

7615 Genomic Consequences of GRHL2 Overexpression in ER+ Breast Cancer Cells

Abstract Disclosure: K.O. Allen: None. C. Zheng: None. N. Solodin: None. M.S. Ozers: Owner/Co-Owner; Self; Proteovista, LLC. C.L. Warren: None. P.E. Messa: None. D.T. Vogt: None. E.T. Alarid: None. Breast cancer progression is characterized by the presence and prevalence of metastatic disease. At a genomic level, metastasis can be facilitated via rearrangement of the chromatin landscape to expose oncogenic genes for transcription. Grainyhead like protein 2 (GRHL2) is a nuclear transcription factor implicated in epithelial cell differentiation. The GRHL2 motif has been found near activated estrogen receptor (ER) binding sites, and elevated levels of GRHL2 have been linked to worse prognosis in hormone receptor positive breast cancer patients. In its capacity to contribute to a more severe disease state when overexpressed, GRHL2 may function as either a transcription or pioneer factor to aid in the expression of metastasis-associated genes. Our lab previously showed that GRHL2 genomic binding precedes ER binding at co-regulated binding sites. Also, overexpression of GRHL2 in MCF7 cells for 24 hours did not appreciably alter gene expression by RNA-Seq. These data taken together indicate that GRHL2 may be exhibiting pioneer factor activity. However, the specific role of GRHL2 overexpression in hormone positive breast cancer progression remains unknown. In order to examine the mechanism of GRHL2 action, GRHL2 genomic binding was investigated using a tetracycline-inducible MCF7 model that allows controlled overexpression of GRHL2. ChIP studies were performed to determine GRHL2 binding intensity at specific and genome wide GRHL2 binding sites. Specific focus was given to established GRHL2 binding sites near oncogenic genes responsible for epithelial to mesenchymal transition (EMT), dormancy, and proto-oncogenic behavior. A similar analysis examined H3K27 acetylation to validate active gene transcription. An orthogonal technology, Specificity and Affinity for Protein (SNAP) microarrays, which characterizes direct GRHL2 binding to tiled genomic regions identified in ChIP-Seq analyses from multiple labs, was utilized to discriminate direct and indirect GRHL2 binding sites. The requirement for GRHL2 transactivation function in regulation of EMT related genes was further interrogated using transactivation GRHL2 dominant negative mutants. This work aims to distinguish the transcriptional and non-transcriptional activities of GRHL2 in breast cancer cells on a genome wide level and thereby, provide a deeper understanding of how overexpressed GRHL2 contributes to hormone positive breast cancer. Presentation: 6/3/2024

The dimer of human SVCT1 is key for transport function

Humans and other primates lack the ability to synthesize the essential nutrient, Vitamin C, which is derived exclusively from the diet. Crucial for effective vitamin C uptake are the Na+ dependent Vitamin C transporters, SVCT1 and SVCT2, members of the nucleobase ascorbate transporter (NAT) family. SVCT1 and 2 actively transport the reduced form of Vitamin C, ascorbic acid, into key tissues. The recent structure of the mouse SVCT1 revealed the molecular basis of substrate binding and that, like the other structurally characterised members of the NAT family, it exists as a closely associated dimer. SVCT1 is likely to function via the elevator mechanism with the core domain of each protomer able to bind substrate and move through the membrane carrying the substrate across the membrane. Here we explored the function of a range of variants of the human SVCT1, revealing a range of residues involved in substrate selection and binding, and confirming the importance of the C-terminus in membrane localisation. Furthermore, using a dominant negative mutant we show that the dimer is essential for transport function, as previously seen in the fungal homologue, UapA. In addition, we show that a localisation deficient C-terminal truncation of SVCT1 blocks correct localisation of co-expressed, associated wildtype SVCT1. These results clearly show the importance of the dimer in both correct SVCT1 trafficking and transport activity.

Epigenetic Reprogramming and Inheritance of the Cellular Differentiation Status Following Transient Expression of a Nonfunctional Dominant-Negative Retinoblastoma Mutant in Murine Mesenchymal Stem Cells.

The retinoblastoma gene product (Rb1), a master regulator of the cell cycle, plays a prominent role in cell differentiation. Previously, by analyzing the differentiation of cells transiently overexpressing the ΔS/N DN Rb1 mutant, we demonstrated that these cells fail to differentiate into mature adipocytes and that they constitutively silence Pparγ2 through CpG methylation. Here, we demonstrate that the consequences of the transient expression of ΔS/N DN Rb1 are accompanied by the retention of Cebpa promoter methylation near the TSS under adipogenic differentiation, thereby preventing its expression. The CGIs of the promoters of the Rb1, Ezh2, Mll4, Utx, and Tet2 genes, which are essential for adipogenic differentiation, have an unmethylated status regardless of the cell differentiation state. Moreover, Dnmt3a, a de novo DNA methyltransferase, is overexpressed in undifferentiated ΔS/N cells compared with wild-type cells and, in addition to Dnmt1, Dnmt3a is significantly upregulated by adipogenic stimuli in both wild-type and ΔS/N cells. Notably, the chromatin modifier Ezh2, which is also involved in epigenetic reprogramming, is highly induced in ΔS/N cells. Overall, we demonstrate that two major genes, Pparγ2 and Cebpa, which are responsible for terminal adipocyte differentiation, are selectively epigenetically reprogrammed to constitutively silent states. We hypothesize that the activation of Dnmt3a, Rb1, and Ezh2 observed in ΔS/N cells may be a consequence of a stress response caused by the accumulation and malfunctioning of Rb1-interacting complexes for the epigenetic reprogramming of Pparγ2/Cebpa and prevention of adipogenesis in an inappropriate cellular context. The failure of ΔS/N cells to differentiate and express Pparγ2 and Cebpa in culture following the expression of the DN Rb1 mutant may indicate the creation of epigenetic memory for new reprogrammed epigenetic states of genes.

The mitochondrial redistribution of ENOS is regulated by AKT1 and dimer status

Previously, we have shown that endothelial nitric-oxide synthase (eNOS) dimer levels directly correlate with the interaction of eNOS with hsp90 (heat shock protein 90). Further, the disruption of eNOS dimerization correlates with its redistribution to the mitochondria. However, the causal link between these events has yet to be investigated and was the focus of this study. Our data demonstrates that simvastatin, which decreases the mitochondrial redistribution of eNOS, increased eNOS-hsp90 interactions and enhanced eNOS dimerization in cultured pulmonary arterial endothelial cells (PAEC) from a lamb model of pulmonary hypertension (PH). Our data also show that the dimerization of a monomeric fraction of human recombinant eNOS was stimulated in the presence of hsp90 and ATP. The over-expression of a dominant negative mutant of hsp90 (DNHsp90) decreased eNOS dimer levels and enhanced its mitochondrial redistribution. We also found that the peroxynitrite donor3-morpholinosydnonimine (SIN-1) increased the mitochondrial redistribution of eNOS in PAEC and this was again associated with decreased eNOS dimer levels. Our data also show in COS-7 cells, the SIN-1 mediated mitochondrial redistribution of wildtype eNOS (WT-eNOS) is significantly higher than a dimer stable eNOS mutant protein (C94R/C99R-eNOS). Conversely, the mitochondrial redistribution of a monomeric eNOS mutant protein (C96A-eNOS) was enhanced. Finally, we linked the SIN-1-mediated mitochondrial redistribution of eNOS to the Akt1-mediated phosphorylation of eNOS at Serine(S)617 and showed that the accessibility of this residue to phosphorylation is regulated by dimerization status. Thus, our data reveal a novel mechanism of pulmonary endothelial dysfunction mediated by mitochondrial redistribution of eNOS, regulated by dimerization status and the phosphorylation of S617.

Damaging mutations in liver X receptor-α are hepatotoxic and implicate cholesterol sensing in liver health

Liver X receptor-α (LXRα) regulates cellular cholesterol abundance and potently activates hepatic lipogenesis. Here we show that at least 1 in 450 people in the UK Biobank carry functionally impaired mutations in LXRα, which is associated with biochemical evidence of hepatic dysfunction. On a western diet, male and female mice homozygous for a dominant negative mutation in LXRα have elevated liver cholesterol, diffuse cholesterol crystal accumulation and develop severe hepatitis and fibrosis, despite reduced liver triglyceride and no steatosis. This phenotype does not occur on low-cholesterol diets and can be prevented by hepatocyte-specific overexpression of LXRα. LXRα knockout mice exhibit a milder phenotype with regional variation in cholesterol crystal deposition and inflammation inversely correlating with steatosis. In summary, LXRα is necessary for the maintenance of hepatocyte health, likely due to regulation of cellular cholesterol content. The inverse association between steatosis and both inflammation and cholesterol crystallization may represent a protective action of hepatic lipogenesis in the context of excess hepatic cholesterol.

Pathways to precision medicine: deciphering the secrets of physiological and pathological atrial enlargement.

Cardiac functional, morphological, and histological analysis, coupled with liquid chromatography and mass spectrometry, of two transgenic mouse models with cardiomyocyte-specific overexpression of insulin-like growth factor 1 receptor (IGF1R) or a dominant-negative PI3K mutant (DCM-dnPI3K) revealed distinctive functional and molecular profiles during physiological (driven by IGF1R overexpression) and pathological (driven by dn-PI3K overexpression) atrial remodeling. The current study confirmed previously reported findings, including ventricular dilatation and enhanced systolic function with no evidence of arrhythmia in IGF1R model, as well as ventricular hypertrophy and decreased systolic function with intermittent atrial fibrillation in DCM-dnPI3K model. Novel findings obtained from the left atrial (LA) characterization of female mice revealed that physiological atrial enlargement resulted from increased atrial myocyte size and was associated with preserved atrial function, as determined by maintained LA ejection fraction (EF). The proteomic profile of IGF1R transgenic (Tg) mice was enriched for metabolic remodeling and showed a protein expression pattern similar to that of healthy human atria; on the other hand, pathological atrial enlargement resulted from increased atrial fibrosis with normal myocyte size and was associated with impaired atrial function due to a reduced LA EF. The proteomic profile of DCM-dnPI3K mice was enriched to both metabolic and structural remodeling and showed a protein expression pattern similar to that of human AF atria.

Drosophila model to clarify the pathological significance of OPA1 in autosomal dominant optic atrophy.

Autosomal dominant optic atrophy (DOA) is a progressive form of blindness caused by degeneration of retinal ganglion cells and their axons, mainly caused by mutations in the OPA1 mitochondrial dynamin like GTPase (OPA1) gene. OPA1 encodes a dynamin-like GTPase present in the mitochondrial inner membrane. When associated with OPA1 mutations, DOA can present not only ocular symptoms but also multi-organ symptoms (DOA plus). DOA plus often results from point mutations in the GTPase domain, which are assumed to have dominant-negative effects. However, the presence of mutations in the GTPase domain does not always result in DOA plus. Therefore, an experimental system to distinguish between DOA and DOA plus is needed. In this study, we found that loss-of-function mutations of the dOPA1 gene in Drosophila can imitate the pathology of optic nerve degeneration observed in DOA. We successfully rescued this degeneration by expressing the human OPA1 (hOPA1) gene, indicating that hOPA1 is functionally interchangeable with dOPA1 in the fly system. However, mutations previously identified did not ameliorate the dOPA1 deficiency phenotype. By expressing both WT and DOA plus mutant hOPA1 forms in the optic nerve of dOPA1 mutants, we observed that DOA plus mutations suppressed the rescue, facilitating the distinction between loss-of-function and dominant-negative mutations in hOPA1. This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies.

Dysregulated Airway Host Defense in Hyper IgE Syndrome due to STAT3 Mutations.

Hyper IgE syndrome (STAT3-HIES), also known as Job's syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES syndrome is characterized by chronic pulmonary infection and inflammation, suggesting impairment of pulmonary innate host defense. To identify airway epithelial host defense defects consequent to STAT3 mutations that, in addition to reported mutant STAT3 immunologic abnormalities, produce pulmonary infection. STAT3-HIES sputum was evaluated for biochemical/biophysical properties. STAT3-HIES excised lungs were harvested for histology; bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific mutation (R382W), expressed by lentiviruses, and a STAT3 knockout, generated by CRISPR/Cas9, were maintained in normal human bronchial epithelia under basal or inflammatory (IL1β) conditions. Effects of STAT3 deficiency on transcriptomics, and epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed. Mucus concentrations and viscoelasticity were increased in STAT3-HIES sputum. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1β expression. STAT3 deficiency impaired CFTR-dependent fluid and mucin secretion, inhibited expression of antimicrobial peptides, cytokines, and chemokines, and acidified airway surface liquid at baseline and post-IL1β exposure in vitro. Notably, mutant STAT3 suppressed IL1R1 expression. STAT3 mutations also inhibited ciliogenesis in vivo and impaired mucociliary transport in vitro, a process mediated via HES6 suppression. Administration of a γ-secretase inhibitor increased HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells. STAT3 dysfunction leads to multi-component defects in airway epithelial innate defense, which, in conjunction with STAT3-HIES immune deficiency, contributes to chronic pulmonary infection.

HSP60 chaperone deficiency disrupts the mitochondrial matrix proteome and dysregulates cholesterol synthesis

ObjectiveMitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. MethodsWe studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. ResultsWe show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. ConclusionsOur data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.