The CUL3–KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction

The WNK (with no lysine kinase)–SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin–RING E3 ubiquitin ligase complexes. To explore how a CUL3–KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na+/Cl− co-transporter)/NKCC1 (Na+/K+/2Cl− co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3–KLHL3 E3 ligase complex, but not a disease-causing CUL3–KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479–667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479–667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3–KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating inappropriate salt retention in the kidney by promoting activation of the NCC/NKCC2 ion co-transporters. The present study reveals how mutations that disrupt the ability of an E3 ligase to interact with and ubiquitylate a critical cellular substrate such as WNK isoforms can trigger a chronic disease such as hypertension.

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A Mammalian Ortholog of Saccharomyces cerevisiae Vac14 That Associates with and Up-Regulates PIKfyve Phosphoinositide 5-Kinase Activity

Molecular and Cellular Biology ◽

10.1128/mcb.24.23.10437-10447.2004 ◽

2004 ◽

Vol 24 (23) ◽

pp. 10437-10447 ◽

Cited By ~ 48

Author(s):

Diego Sbrissa ◽

Ognian C. Ikonomov ◽

Jana Strakova ◽

Rajeswari Dondapati ◽

Krzysztof Mlak ◽

...

Keyword(s):

Mammalian Cells ◽

Kinase Activity ◽

Ectopic Expression ◽

Multivesicular Body ◽

Hek293 Cells ◽

Lipid Kinase ◽

Morphological Alterations ◽

Protein Levels ◽

Interfering Rna

ABSTRACT Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P2, produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P2 synthesis and endomembrane homeostasis in mammalian cells.

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An empirical pipeline for personalized diagnosis of Lafora disease mutations

10.1101/2021.03.26.437206 ◽

2021 ◽

Author(s):

M. Kathryn Brewer ◽

Maria Machio-Castello ◽

Rosa Viana ◽

Jeremiah L Wayne ◽

Andrea Kuchtova ◽

...

Keyword(s):

Late Onset ◽

Missense Mutations ◽

Lafora Disease ◽

Disease Mutations ◽

Whole Exome ◽

Functional Classes ◽

Progressive Myoclonic Epilepsy ◽

Personalized Diagnosis ◽

Rapid Characterization

Lafora disease (LD) is a fatal, insidious metabolic disorder characterized by progressive myoclonic epilepsy manifesting in the teenage years, rapid neurological decline, and death typically within ten years of onset. Mutations in either EPM2A, encoding the glycogen phosphatase laforin, or EPM2B, encoding the E3 ligase malin, cause LD. Whole exome sequencing has revealed many EPM2A variants associated with late-onset or slower disease progression. We established an empirical pipeline for characterizing laforin missense mutations in vitro using complimentary biochemical approaches. Analysis of 26 mutations revealed distinct functional classes associated with different outcomes supported by multiple clinical cases. For example, F321C and G279C mutations have attenuated functional defects and are associated with slow progression. This pipeline allows rapid characterization and classification of novel EPM2A mutations, enabling clinicians and researchers to rapidly utilize genetic information to guide treatment of LD patients.

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Mutations in conserved residues of the myosin chaperone UNC-45 result in both reduced stability and chaperoning activity

10.1101/2021.05.07.443121 ◽

2021 ◽

Author(s):

Taylor Moncrief ◽

Courtney J Matheny ◽

Ivana Gaziova ◽

John Miller ◽

Hiroshi Qadota ◽

...

Keyword(s):

Protein Stability ◽

Protein Interactions ◽

Muscle Development ◽

Thick Filament ◽

Chaperone Activity ◽

Missense Mutations ◽

Protein Levels ◽

Conserved Regions

Proper muscle development and function depends on myosin being properly folded and integrated into the thick filament structure. For this to occur the myosin chaperone UNC-45, or UNC-45B, must be present and able to chaperone myosin. Here we use a combination of in vivo C. elegans experiments and in vitro biophysical experiments to analyze the effects of six missense mutations in conserved regions of UNC-45/UNC-45B. We found that the phenotype of paralysis and disorganized thick filaments in 5/6 of the mutant nematode strains can likely be attributed to both reduced steady state UNC-45 protein levels and reduced chaperone activity. Interestingly, the biophysical assays performed on purified proteins show that all of the mutations result in reduced myosin chaperone activity but not overall protein stability. This suggests that these mutations only cause protein instability in the in vivo setting and that these conserved regions may be involved in UNC-45 protein stability/ regulation via post translational modifications, protein-protein interactions, or some other unknown mechanism.

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miR-140-5p mediates bevacizumab-induced cytotoxicity to cardiomyocytes by targeting the VEGFA/14-3-3γ signal pathway

Toxicology Research ◽

10.1039/c9tx00100j ◽

2019 ◽

Vol 8 (6) ◽

pp. 875-884

Author(s):

Xuan-Ying Chen ◽

Wei-Lin Huang ◽

Xiao-Ping Peng ◽

Yan-Ni Lv ◽

Jun-He Li ◽

...

Keyword(s):

Cardiac Myocyte ◽

Annexin V ◽

Signal Pathway ◽

Luciferase Reporter ◽

Protein Levels ◽

Humanized Monoclonal Antibody ◽

Novel Target ◽

Commercial Kits ◽

Interfering Rna

Abstract Bevacizumab (BVZ) is the first recombinant humanized monoclonal antibody against vascular endothelial growth factor (VEGFA) approved by the FDA for the treatment of different kinds of cancers, especially colorectal cancer. Although the anti-tumor effects have been verified, the side effects of BVZ are also noteworthy, among which, cardiotoxicity may be the most serious side effect of BVZ. However, the exact mechanisms of cardiotoxicity induced by BVZ have been little explored. This study was conducted in vitro in a human cardiac myocyte (HCM) model. MTT assay was conducted to determine BVZ-stimulated cell viability. For testing the function and mechanism, the cells were transfected with miR-140-5p mimics, miR-140-5p inhibitor and/or VEGFA small interfering RNA (si-VEGFA). Then, apoptosis of the cells was detected via annexin V/propidium iodide (AV-PI) staining followed by flow cytometry. qRT-PCR and western blot assays were applied to measure gene expression (i.e. mRNA) and protein levels, respectively. The CK, LDH, SOD, CAT and GSH-Px activities and MDA level were determined using commercial kits. ROS levels were determined by DCFH-DA assay. Mitochondrial membrane potential was measured by JC-1 assay. Dual-luciferase reporter assay was used to detect the interaction between miR-140-5p and VEGFA. BVZ could inhibit HCM proliferation and induce apoptosis. miR-140-5p was upregulated in response to BVZ treatment and miR-140-5p restraint could alleviate HCM damage caused by BVZ treatment. In contrast, VEGFA and 14-3-3γ expressions were down-regulated by BVZ, and miR-140-5p could inhibit the expression of 14-3-3γ by directly targeting VEGFA. Moreover, VEGFA suppression enhanced HCM injury stimulated by BVZ and partially reversed the functional role of the miR-140-5p inhibitor in BVZ-treated cells. Taken together, miR-140-5p promoted BVZ-treated cardiomyocyte toxicity by targeting the VEGFA/14-3-3γ signal pathway. Collectively, miR-140-5p mediated the BVZ-induced cytotoxicity to cardiomyocytes by targeting the VEGFA/14-3-3γ signal pathway, indicating that miR-140-5p may be a novel target for treating BVZ-induced cardiotoxicity.

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Exploration of Aberrant E3 Ligases Implicated in Alzheimer’s Disease and Development of Chemical Tools to Modulate Their Function

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.768655 ◽

2021 ◽

Vol 15 ◽

Author(s):

Frances M. Potjewyd ◽

Alison D. Axtman

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

E3 Ligase ◽

Amyloid Β ◽

3D Cell Culture ◽

Ubiquitin Proteasome System ◽

E3 Ligases ◽

Protein Levels ◽

Chemical Tools

The Ubiquitin Proteasome System (UPS) is responsible for the degradation of misfolded or aggregated proteins via a multistep ATP-dependent proteolytic mechanism. This process involves a cascade of ubiquitin (Ub) transfer steps from E1 to E2 to E3 ligase. The E3 ligase transfers Ub to a targeted protein that is brought to the proteasome for degradation. The inability of the UPS to remove misfolded or aggregated proteins due to UPS dysfunction is commonly observed in neurodegenerative diseases, such as Alzheimer’s disease (AD). UPS dysfunction in AD drives disease pathology and is associated with the common hallmarks such as amyloid-β (Aβ) accumulation and tau hyperphosphorylation, among others. E3 ligases are key members of the UPS machinery and dysfunction or changes in their expression can propagate other aberrant processes that accelerate AD pathology. The upregulation or downregulation of expression or activity of E3 ligases responsible for these processes results in changes in protein levels of E3 ligase substrates, many of which represent key proteins that propagate AD. A powerful way to better characterize UPS dysfunction in AD and the role of individual E3 ligases is via the use of high-quality chemical tools that bind and modulate specific E3 ligases. Furthermore, through combining gene editing with recent advances in 3D cell culture, in vitro modeling of AD in a dish has become more relevant and possible. These cell-based models of AD allow for study of specific pathways and mechanisms as well as characterization of the role E3 ligases play in driving AD. In this review, we outline the key mechanisms of UPS dysregulation linked to E3 ligases in AD and highlight the currently available chemical modulators. We present several key approaches for E3 ligase ligand discovery being employed with respect to distinct classes of E3 ligases. Where possible, specific examples of the use of cultured neurons to delineate E3 ligase biology have been captured. Finally, utilizing the available ligands for E3 ligases in the design of proteolysis targeting chimeras (PROTACs) to degrade aberrant proteins is a novel strategy for AD, and we explore the prospects of PROTACs as AD therapeutics.

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Anti-c-myc cholesterol based lipoplexes as onco-nanotherapeutic agents in vitro

F1000Research ◽

10.12688/f1000research.25142.2 ◽

2021 ◽

Vol 9 ◽

pp. 770

Author(s):

Saffiya Habib ◽

Aliscia Daniels ◽

Mario Ariatti ◽

Moganavelli Singh

Keyword(s):

Sirna Delivery ◽

Cationic Lipid ◽

Lipid Vesicles ◽

Breast Adenocarcinoma ◽

Protein Levels ◽

Anti Cancer ◽

Attractive Option ◽

Interfering Rna ◽

Mcf 7

Background: Strategies aimed at inhibiting the expression of the c-myc oncogene could provide the basis for alternative cancer treatment. In this regard, silencing c-myc expression using small interfering RNA (siRNA) is an attractive option. However, the development of a clinically viable, siRNA-based, c-myc silencing system is largely dependent upon the design of an appropriate siRNA carrier that can be easily prepared. Nanostructures formed by the electrostatic association of siRNA and cationic lipid vesicles represent uncomplicated siRNA delivery systems. Methods: This study has focused on cationic liposomes prepared with equimolar quantities of the cytofectin, N,N-dimethylaminopropylamido-succinylcholesteryl-formylhydrazide (MS09), and cholesterol (Chol) for the development of a simple, but effective anti- c-myc onco-nanotherapeutic agent. Liposomes formulated with dioleoylphosphatidylethanolamine (DOPE) in place of Chol as the co-lipid were included for comparative purposes. Results: Liposomes successfully bound siRNA forming lipoplexes of less than 150 nm in size, which assumed bilamellar aggregrates. The liposome formulations were well tolerated in the human breast adenocarcinoma (MCF-7) and colon carcinoma (HT-29) cells, which overexpress c-myc. Lipoplexes directed against the c-myc transcript mediated a dramatic reduction in c-myc mRNA and protein levels. Moreover, oncogene knockdown and anti-cancer effects were superior to that of Lipofectamine™ 3000. Conclusion: This anti- c-myc MS09:Chol lipoplex exemplifies a simple anticancer agent with enhanced c-myc gene silencing potential in vitro

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The cryoEM structure of the fibril-forming low-complexity domain of hnRNPA2 reveals distinct differences from pathogenic amyloid and shows how mutation converts it to the pathogenic form

10.1101/2020.05.24.113621 ◽

2020 ◽

Cited By ~ 1

Author(s):

Jiahui Lu ◽

Qin Cao ◽

Michael P. Hughes ◽

Michael R. Sawaya ◽

David R. Boyer ◽

...

Keyword(s):

Low Complexity ◽

Atomic Level ◽

Missense Mutations ◽

Cryo Electron Microscopy ◽

Fibril Structure ◽

Disease Mutations ◽

Structural Differences ◽

Energetic State ◽

Protein Fibril

AbstracthnRNPA2 is one of a group of human ribonucleoproteins (RNPs) involved in RNA metabolism which form fibrils both under cellular stress and in mutated form in neurodegenerative conditions. Previous work established that the C-terminal low-complexity domain (LCD) of hnRNPA2 fibrillizes under stress, and that missense mutations in this domain are found in the disease multisystem proteinopathy (MSP) with symptoms indistinguishable from ALS and FTD. However, little is known at the atomic level about the hnRNPA2 LCD structure that is involved in those processes and how disease mutations cause structural change. Here we present the cryo-electron microscopy (cryoEM) structure of hnRNPA2 LCD fibril core and demonstrate its capability to form a reversible hydrogel in vitro containing amyloid-like fibrils. Whereas these fibrils, like pathogenic amyloid, are formed from protein chains stacked into β-sheets by backbone hydrogen bonds, they display distinct structural differences: the chains are kinked, enabling non-covalent cross-linking of fibrils and disfavoring formation of pathogenic steric zippers. Both their reversibility and energetic calculations suggest these fibrils are less stable than pathogenic amyloid. Moreover, the crystal structure of the disease-mutation-containing segment of hnRNPA2 suggests that the replacement fundamentally alters the fibril structure to a more stable energetic state. These findings illuminate how molecular interactions promote protein fibril networks and how mutation can transform fibril structure from functional to pathogenic form.

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E3 ligase MKRN3 is a tumor suppressor regulating PABPC1 ubiquitination in non–small cell lung cancer

Journal of Experimental Medicine ◽

10.1084/jem.20210151 ◽

2021 ◽

Vol 218 (8) ◽

Author(s):

Ke Li ◽

Xufen Zheng ◽

Hua Tang ◽

Yuan-Sheng Zang ◽

Chunling Zeng ◽

...

Keyword(s):

Lung Cancer ◽

Tumor Suppressor ◽

E3 Ligase ◽

Central Precocious Puberty ◽

Small Cell ◽

Missense Mutations ◽

Small Cell Lung ◽

Kras Mutations

Central precocious puberty (CPP), largely caused by germline mutations in the MKRN3 gene, has been epidemiologically linked to cancers. MKRN3 is frequently mutated in non–small cell lung cancers (NSCLCs) with five cohorts. Genomic MKRN3 aberrations are significantly enriched in NSCLC samples harboring oncogenic KRAS mutations. Low MKRN3 expression levels correlate with poor patient survival. Reconstitution of MKRN3 in MKRN3-inactivated NSCLC cells directly abrogates in vitro and in vivo tumor growth and proliferation. MKRN3 knockout mice are susceptible to urethane-induced lung cancer, and lung cell–specific knockout of endogenous MKRN3 accelerates NSCLC tumorigenesis in vivo. A mass spectrometry–based proteomics screen identified PABPC1 as a major substrate for MKRN3. The tumor suppressor function of MKRN3 is dependent on its E3 ligase activity, and MKRN3 missense mutations identified in patients substantially compromise MKRN3-mediated PABPC1 ubiquitination. Furthermore, MKRN3 modulates cell proliferation through PABPC1 nonproteolytic ubiquitination and subsequently, PABPC1-mediated global protein synthesis. Our integrated approaches demonstrate that the CPP-associated gene MKRN3 is a tumor suppressor.

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Abstract 054: Mutations In Pde3a Explain Mendelian Hypertension With Brachydactyly

Hypertension ◽

10.1161/hyp.64.suppl_1.054 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Philipp Maass ◽

Atankan Aydin ◽

Friedrich C Luft ◽

Hakan Toka ◽

Irene Hollfinger ◽

...

Keyword(s):

Blood Pressure ◽

Genome Project ◽

Sodium Reabsorption ◽

Missense Mutations ◽

Gain Of Function ◽

Gene Encoding ◽

Arterial Blood ◽

The Mean ◽

Camp Hydrolysis

We identified Mendelian type E brachydactyly (BDE) with hypertension in six different families from Turkey, America, France, Canada and South Africa. The two phenotypes, hypertension and BDE, invariably coincide. The blood pressure in affected individuals increases with increasing age, the mean arterial blood pressure at 50 years exceeds 150 mm Hg, resulting in stroke. Earlier we suggested that a chromosomal rearrangement on 12p could be responsible. We now used whole-genome sequencing and discovered six different so far unknown missense mutations in the gene encoding PDE3A. The mutations are not identical, but adjacent to each other. Each is responsible for an amino acid substitution in a conserved portion of the protein. The mutations were not present in any non-affected family members, in more than 200 additional controls, and not found in the “1000 genome” project. We performed in vitro cell transfections and found that mutated PDE3A showed gain-of-function with increased cAMP hydrolysis. Mesenchymal stromal cell-derived vascular smooth muscle cells and chondrocytes gave insight into the molecular pathogenesis; PDE3A and vasodilator-stimulated phosphoprotein (VASP) were differently phosphorylated and parathyroid hormone-related peptide (PTHrP) was dysregulated. Our preliminary results reveal that the mutated PDE3A enzymes show gain-of-function with increased cAMP hydrolysis and sensitivity to cGMP inhibition. We suggest that the mutations are responsible for both phenotypes. This Mendelian hypertension is the first that is not attributable to increased sodium reabsorption in the distal nephron.

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Abstract 15555: Potential Cure for Hypertension? The Effect of Crispr Genome Editing

Circulation ◽

10.1161/circ.142.suppl_3.15555 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Hualing Sun ◽

Conrad Hodgkinson ◽

Richard E Pratt ◽

Victor J Dzau

Keyword(s):

Blood Pressure ◽

Systolic Blood Pressure ◽

Pressure Control ◽

Systemic Delivery ◽

Protein Levels ◽

Cardiovascular Stress ◽

Medical Compliance ◽

Agt Gene

Hypertension is recognized as one of the largest contributors to the global burden of disease. Unfortunately, for the majority of patients, hypertension is poorly controlled due, in part, to poor medical compliance resulting from the need to take one or more drugs, often several times daily. An ideal therapy would be administered a single time but yield long-term blood pressure control. Under the appropriate condition, we hypothesize that this modality could potentially lead to a cure for hypertension. In this study, we investigated whether that sustained, and possibly life-long, reductions in blood pressure could be achieved via Crispr-Cas9 mediated disruption of a key gene in the RAAS, Angiotensinogen (AGT). In vitro, we demonstrated that expression of Crispr-Cas9 system in the BRL 3A rat liver cell line led to a significant reduction in AGT protein levels (75% reduction, N=3, P<0.01). Delivery of the Crispr-Cas9 system into the liver via systemic delivery of the hepatocyte-targeting AAV8 reduced both AGT mRNA (70% reduction, N=5, P<0.01) and circulating AGT protein (50% reduction, N=5, P<0.01) and Ang II levels (33% reduction, n=5, P<0.01). In the SHR model of hypertension, Crispr-Cas9 mediated loss of AGT expression reduced systolic blood pressure in adult animals with established hypertension (30-35mmHg decrease in both females and males, N=12, P<0.001). Importantly, AGT gene disruption also prevented the spontaneous development of hypertension in young pre-hypertensive SHR (SHR control: 55-60mmHg increase in systolic blood pressure both female and male SHR; SHR plus Crispr-Cas9: 30-33 mmHg increase. N=12. P<0.001). Importantly, in all the models studied, reductions in blood pressure were sustained for at least 4 months. Importantly, the partial disruption of the hepatic AGT gene was sufficient to control hypertension but did not block the renin-angiotensin response to cardiovascular stress such as sodium depletion. In summary, we have demonstrated that targeting the Crispr-Cas9 system to RAAS reduces blood pressure and may be a potential therapy to achieve safe and sustained, possibly life-long, control of hypertension and this approach maybe a cure for hypertension.

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