Mutations in the Nervous System–Specific HSN2 Exon of WNK1 Cause Hereditary Sensory Neuropathy Type II
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Ubiquitin Ligase Trim32 and Chloride-Sensitive WNK1 As Regulators of Potassium Channels in the Brain Eugene Miler Cilento University of Vermont
University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2015 Ubiquitin Ligase Trim32 and Chloride-sensitive WNK1 as Regulators of Potassium Channels in the Brain Eugene Miler Cilento University of Vermont Follow this and additional works at: http://scholarworks.uvm.edu/graddis Part of the Neurosciences Commons, and the Pharmacology Commons Recommended Citation Cilento, Eugene Miler, "Ubiquitin Ligase Trim32 and Chloride-sensitive WNK1 as Regulators of Potassium Channels in the Brain" (2015). Graduate College Dissertations and Theses. Paper 431. This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. UBIQUITIN LIGASE TRIM32 AND CHLORIDE-SENSITIVE WNK1 AS REGULATORS OF POTASSIUM CHANNELS IN THE BRAIN A Dissertation Presented by Eugene Miler Cilento to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Neuroscience October, 2015 Defense Date: August 04, 2014 Dissertation Examination Committee: Anthony Morielli, Ph.D., Advisor John Green, Ph.D., Chairperson Bryan Ballif, Ph.D. Wolfgang Dostmann Ph.D. George Wellman, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT The voltage-gated potassium channel Kv1.2 impacts membrane potential and therefore excitability of neurons. Expression of Kv1.2 at the plasma membrane (PM) is critical for channel function, and altering Kv1.2 at the PM is one way to affect membrane excitability. -
A Novel Nonsense Mutation in WNK1/HSN2 Associated with Sensory Neuropathy and Limb Destruction in Four Siblings of a Large Irani
Rahmani et al. BMC Neurology (2018) 18:195 https://doi.org/10.1186/s12883-018-1201-6 RESEARCHARTICLE Open Access A novel nonsense mutation in WNK1/HSN2 associated with sensory neuropathy and limb destruction in four siblings of a large Iranian pedigree Behrouz Rahmani1,2, Fatemeh Fekrmandi3, Keivan Ahadi4, Tannaz Ahadi5, Afagh Alavi6, Abolhassan Ahmadiani2 and Sareh Asadi2* Abstract Background: Hereditary sensory and autonomic neuropathy type 2 (HSAN2) is an autosomal recessive disorder with predominant sensory dysfunction and severe complications such as limb destruction. There are different subtypes of HSAN2, including HSAN2A, which is caused by mutations in WNK1/HSN2 gene. Methods: An Iranian family with four siblings and autosomal recessive inheritance pattern whom initially diagnosed with HSAN2 underwent whole exome sequencing (WES) followed by segregation analysis. Results: According to the filtering criteria of the WES data, a novel candidate variation, c.3718C > A in WNK1/ HSN2 gene that causes p.Tyr1025* was identified. This variation results in a truncated protein with 1025 amino acids instead of the wild-type product with 2645 amino acids. Sanger sequencing revealed that the mutation segregates with disease status in the pedigree. Conclusions: The identified novel nonsense mutation in WNK1/HSN2 in an Iranian HSAN2 pedigree presents allelic heterogeneity of this gene in different populations. The result of current study expands the spectrum of mutations of the HSN2 gene as the genetic background of HSAN2A as well as further supports the hypothesis that HSN2 is a causative gene for HSAN2A. However, it seems that more research is required to determine the exact effects of this product in the nervous system. -
CUL3 Gene Cullin 3
CUL3 gene cullin 3 Normal Function The CUL3 gene provides instructions for making a protein called cullin-3. This protein plays a role in the cell machinery that breaks down (degrades) unwanted proteins, called the ubiquitin-proteasome system. Cullin-3 is a core piece of a complex known as an E3 ubiquitin ligase. E3 ubiquitin ligases function as part of the ubiquitin-proteasome system by tagging damaged and excess proteins with molecules called ubiquitin. Ubiquitin serves as a signal to specialized cell structures known as proteasomes, which attach (bind) to the tagged proteins and degrade them. The ubiquitin-proteasome system acts as the cell's quality control system by disposing of damaged, misshapen, and excess proteins. This system also regulates the level of proteins involved in several critical cell activities such as the timing of cell division and growth. E3 ubiquitin ligases containing the cullin-3 protein tag proteins called WNK1 and WNK4 with ubiquitin. These proteins are involved in controlling blood pressure in the body. By regulating the amount of WNK1 and WNK4 available, cullin-3 plays a role in blood pressure control. Health Conditions Related to Genetic Changes Pseudohypoaldosteronism type 2 At least 17 mutations in the CUL3 gene can cause pseudohypoaldosteronism type 2 ( PHA2), a condition characterized by high blood pressure (hypertension) and high levels of potassium in the blood (hyperkalemia). These mutations lead to production of an abnormally short cullin-3 protein that is missing a region. Studies show that this change alters the function of the E3 ubiquitin ligase complex. The change leads to impaired degradation of the WNK4 protein, although the exact mechanism is unclear. -
WNK1 Activates SGK1 to Regulate the Epithelial Sodium Channel
WNK1 activates SGK1 to regulate the epithelial sodium channel Bing-e Xu*, Steve Stippec*, Po-Yin Chu†, Ahmed Lazrak†, Xin-Ji Li†, Byung-Hoon Lee*, Jessie M. English*‡, Bernardo Ortega†, Chou-Long Huang†§, and Melanie H. Cobb*§ Departments of *Pharmacology and †Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041 Communicated by David L. Garbers, University of Texas Southwestern Medical Center, Dallas, TX, May 27, 2005 (received for review April 20, 2005) WNK (with no lysine [K]) kinases are serine–threonine protein (15). ENaC is the rate-limiting step for sodium transport across kinases with an atypical placement of the catalytic lysine. Intronic high-resistance epithelia and is regulated by insulin and mineralo- deletions increase the expression of WNK1 in humans and cause corticoid hormones. The activity of SGK1 depends on phosphati- pseudohypoaldosteronism type II, a form of hypertension. WNKs dylinositol 3-kinase (PI-3 kinase), an essential mediator of insulin have been linked to ion carriers, but the underlying regulatory signaling. PI-3 kinase is also required for SGK1-dependent stimu- mechanisms are unknown. Here, we report a mechanism for the lation of ENaC by mineralocorticoids (16). The impact of SGK1 on control of ion permeability by WNK1. We show that WNK1 acti- ENaC, together with its induction by aldosterone and insulin, places vates the serum- and glucocorticoid-inducible protein kinase SGK1, this protein kinase in a key position to regulate sodium homeostasis leading to activation of the epithelial sodium channel. Increased and blood pressure (14). channel activity induced by WNK1 depends on SGK1 and the E3 Ubiquitinylation controls the cell-surface expression of many ubiquitin ligase Nedd4-2. -
Kelch-Like 3 and Cullin 3 Regulate Electrolyte Homeostasis Via Ubiquitination and Degradation of WNK4
Kelch-like 3 and Cullin 3 regulate electrolyte homeostasis via ubiquitination and degradation of WNK4 Shigeru Shibataa,b, Junhui Zhanga,b, Jeremy Puthumanaa,b, Kathryn L. Stonec, and Richard P. Liftona,b,1 aDepartment of Genetics, bHoward Hughes Medical Institute, and cW. M. Keck Facility, Yale University School of Medicine, New Haven, CT 06510 Contributed by Richard P. Lifton, March 8, 2013 (sent for review February 25, 2013) Pseudohypoaldosteronism type II (PHAII) is a rare Mendelian syn- Mutations in four genes have been identified to cause PHAII drome featuring hypertension and hyperkalemia resulting from (4, 5). Two encode the serine-threonine kinases WNK1 (with no constitutive renal salt reabsorption and impaired K+ secretion. Re- lysine kinase 1) and WNK4 (4). Disease-causing mutations in cently, mutations in Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), compo- WNK4 are missense mutations that cluster in a short, highly acidic nents of an E3 ubiquitin ligase complex, were found to cause PHAII, domain of the protein, whereas mutations in WNK1 are large suggesting that loss of this complex’s ability to target specific sub- deletions of the first intron that increase WNK1 expression. Bio- strates for ubiquitination leads to PHAII. By MS and coimmunopre- chemistry, cell biology, and animal model studies have demon- cipitation, we show that KLHL3 normally binds to WNK1 and WNK4, strated that WNK4 regulates the balance between renal Na-Cl + members of WNK (with no lysine) kinase family that have previously reabsorption and K secretion, with missense mutations found in been found mutated in PHAII. We show that this binding leads to patients with PHAII promoting increased levels of the renal Na-Cl ubiquitination, including polyubiquitination, of at least 15 specific cotransporter NCC and decreased levels of renal outer medullary + + sites in WNK4, resulting in reduced WNK4 levels. -
Antagonistic Regulation of ROMK by Long and Kidney-Specific WNK1 Isoforms
Antagonistic regulation of ROMK by long and kidney-specific WNK1 isoforms Ahmed Lazrak*, Zhen Liu*, and Chou-Long Huang† Department of Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-8856 Communicated by Steven C. Hebert, Yale University School of Medicine, New Haven, CT, December 8, 2005 (received for review November 8, 2005) WNK kinases are serine-threonine kinases with an atypical place- of hypertension (7, 8). Others have reported that WNK4 phos- ment of the catalytic lysine. Intronic deletions with increased phorylates claudins 1–4, the tight-junction proteins involved in expression of a ubiquitous long WNK1 transcript cause pseudohy- the regulation of paracellular ion permeability (9, 10). The poaldosteronism type 2 (PHA II), characterized by hypertension and paracellular chloride permeability is greater in cells expressing hyperkalemia. Here, we report that long WNK1 inhibited ROMK1 WNK4 mutants than in cells expressing wild-type proteins. Thus, by stimulating its endocytosis. Inhibition of ROMK by long WNK1 hypertension in patients with WNK4 mutations may be caused by was synergistic with, but not dependent on, WNK4. A smaller an increase in NaCl reabsorption through the Na-Cl cotrans- transcript of WNK1 lacking the N-terminal 1–437 amino acids is porter and the paracellular pathway. Wild-type WNK4 inhibits expressed highly in the kidney. Whether expression of the KS- the ROMK1 channel and WNK4 mutants that cause disease WNK1 (kidney-specific, KS) is altered in PHA II is not known. We exhibit increased inhibition of ROMK (11), suggesting that found that KS-WNK1 did not inhibit ROMK1 but reversed the WNK4 mutations cause hyperkalemia by inhibiting ROMK. -
WNK4 Kinase Is a Physiological Intracellular Chloride Sensor
WNK4 kinase is a physiological intracellular chloride sensor Jen-Chi Chena,b, Yi-Fen Loa, Ya-Wen Linb, Shih-Hua Lina, Chou-Long Huangc, and Chih-Jen Chenga,1 aDivision of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan; bGraduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei 114, Taiwan; and cDivision of Nephrology, Department of Medicine, University of Iowa Carver College of Medicine, Iowa City, IA 52242-1081 Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved January 11, 2019 (received for review October 7, 2018) With-no-lysine (WNK) kinases regulate renal sodium-chloride cotrans- unclear. In transporting epithelia, changes in concentrations of porter (NCC) to maintain body sodium and potassium homeostasis. ions from exit across one membrane (e.g., basolateral) will be Gain-of-function mutations of WNK1 and WNK4 in humans lead to a coupled by parallel entry on the other membrane (e.g., apical). Mendelian hypertensive and hyperkalemic disease pseudohypoal- The tight coupling between apical and basolateral transport to dosteronism type II (PHAII). X-ray crystal structure and in vitro minimize fluctuations of intracellular concentration of solutes − studies reveal chloride ion (Cl ) binds to a hydrophobic pocket and cell volume is a fundamental homeostatic feature of trans- − within the kinase domain of WNKs to inhibit its activity. The mech- porting epithelia. Whether the intracellular concentration of Cl anism is thought to be important for physiological regulation of − ([Cl ]i) in DCT under physiological conditions is within the dy- NCC by extracellular potassium. -
Potential Applications for Mrna and Peptide-Based Vaccines
viruses Article An Epitope Platform for Safe and Effective HTLV-1-Immunization: Potential Applications for mRNA and Peptide-Based Vaccines Guglielmo Lucchese 1,*,†, Hamid Reza Jahantigh 2,3,† , Leonarda De Benedictis 2, Piero Lovreglio 2 and Angela Stufano 2,3 1 Department of Neurology, Medical University of Greifswald, 17475 Greifswald, Germany 2 Interdisciplinary Department of Medicine-Section of Occupational Medicine, University of Bari, 70124 Bari, Italy; [email protected] (H.R.J.); [email protected] (L.D.B.); [email protected] (P.L.); [email protected] (A.S.) 3 Animal Health and Zoonosis Doctoral Program, Department of Veterinary Medicine, University of Bari, 70010 Bari, Italy * Correspondence: [email protected] † These authors contributed equally to the work. Abstract: Human T-cell lymphotropic virus type 1 (HTLV-1) infection affects millions of individuals worldwide and can lead to severe leukemia, myelopathy/tropical spastic paraparesis, and numerous other disorders. Pursuing a safe and effective immunotherapeutic approach, we compared the viral polyprotein and the human proteome with a sliding window approach in order to identify oligopeptide sequences unique to the virus. The immunological relevance of the viral unique oligopeptides was assessed by searching them in the immune epitope database (IEDB). We found that Citation: Lucchese, G.; Jahantigh, HTLV-1 has 15 peptide stretches each consisting of uniquely viral non-human pentapeptides which H.R.; De Benedictis, L.; Lovreglio, P.; Stufano, A. An Epitope Platform for are ideal candidate for a safe and effective anti-HTLV-1 vaccine. Indeed, experimentally validated Safe and Effective HTLV-1 epitopes, as retrieved from the IEDB, contain peptide sequences also present in a vast number HTLV-1-Immunization: Potential of human proteins, thus potentially instituting the basis for cross-reactions. -
Mutations Affecting the Conserved Acidic Wnk1 Motif Cause Inherited Hyperkalemic Hyperchloremic Acidosis Supplemental Informatio
MUTATIONS AFFECTING THE CONSERVED ACIDIC WNK1 MOTIF CAUSE INHERITED HYPERKALEMIC HYPERCHLOREMIC ACIDOSIS SUPPLEMENTAL INFORMATIONS OF METHODS IN VITRO FUNCTIONAL CHARACTERIZATION Vectors The pGH19-L- and KS-WNK1-∆11 plasmids carrying the A634T, D635E, Q636E, Q636R or D635N mutations were generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) and checked by direct sequencing. The primers used are referenced in Supplemental Table 7B. We subcloned the smaller fragment of the SnaB1-BamH1 digestion from pGH19-L-WNK1- ∆11 and pGH19-KS-WNK1-∆11 plasmids carrying the mutation into pCDNA3-L and KS-WNK1- ∆11 plasmids linearised by SnaB1-BamH1. The LigaFast Rapid DNA Ligation System (Promega) was used according to the manufacturer's instructions. A pCMV6-mKLHL3 (Origene) plasmid encoded Myc- and Flag-tagged mouse KLHL3. We deleted the Flag tag by subcloning the HindIII- EcoRV digestion fragment of pCMV6-mKLHL3 into the same plasmid digested with HindIII-PmeI. pcDNA-hCUL3 encodes HA-tagged human CUL3 is a gift from C. Rochette-Egly (Department of Functional Genomics and Cancer, University of Strasbourg, Illkirch, France). Flag-hKLHL3 cDNA was purchased from the MRC-PPU facility of the University of Dundee and was subcloned into pCDNA5/FRT/(His)6-Protein C vector (derived from pCDNA5/FRT/V5-His (Invitrogen) as described by Derivery and Gautreau (1). All tags are fused at the N-terminus of KLHL3 cDNA. The Ubiquitin-HA expressing vector was a gift from Gervaise Loirand (INSERM UMR1087, Nantes, France) Mutant proteins in HEK293T cells Cell culture conditions The stable and inducible cell lines derived were grown in DMEM medium (Gibco) supplemented with 10% (v/v) Fetal Calf Serum (Biological Industries), Penicillin/Streptomycin (0.1 mg/ml each) (Gibco), Hygromycin B 200 µg/ml (Invivogen), Blasticidin 7,5 µg/ml (Invivogen). -
LINGO-1 and AMIGO3, Potential Therapeutic Targets for Neurological and Dysmyelinating Disorders?
[Downloaded free from http://www.nrronline.org on Wednesday, February 28, 2018, IP: 147.188.108.81] NEURAL REGENERATION RESEARCH August 2017, Volume 12, Issue 8 www.nrronline.org INVITED REVIEW LINGO-1 and AMIGO3, potential therapeutic targets for neurological and dysmyelinating disorders? Simon Foale, Martin Berry, Ann Logan, Daniel Fulton, Zubair Ahmed* Neuroscience and Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, UK How to cite this article: Foale S, Berry M, Logan A, Fulton D, Ahmed Z (2017) LINGO-1 and AMIGO3, potential therapeutic targets for neu- rological and dysmyelinating disorders?. Neural Regen Res 12(8):1247-1251. Funding: This work was supported by a grant from The University of Birmingham. Abstract *Correspondence to: Zubair Ahmed, Ph.D., Leucine rich repeat proteins have gained considerable interest as therapeutic targets due to their expres- [email protected]. sion and biological activity within the central nervous system. LINGO-1 has received particular attention since it inhibits axonal regeneration after spinal cord injury in a RhoA dependent manner while inhibiting orcid: leucine rich repeat and immunoglobulin-like domain-containing protein 1 (LINGO-1) disinhibits neuron 0000-0001-6267-6492 outgrowth. Furthermore, LINGO-1 suppresses oligodendrocyte precursor cell maturation and myelin (Zubair Ahmed) production. Inhibiting the action of LINGO-1 encourages remyelination both in vitro and in vivo. Accord- ingly, LINGO-1 antagonists show promise as therapies for demyelinating diseases. An analogous protein doi: 10.4103/1673-5374.213538 to LINGO-1, amphoterin-induced gene and open reading frame-3 (AMIGO3), exerts the same inhibitory effect on the axonal outgrowth of central nervous system neurons, as well as interacting with the same re- Accepted: 2017-07-17 ceptors as LINGO-1. -
WNK1 Affects Surface Expression of the ROMK Potassium Channel Independent of WNK4
WNK1 Affects Surface Expression of the ROMK Potassium Channel Independent of WNK4 Georgina Cope, Meena Murthy, Amir P. Golbang, Abbas Hamad, Che-Hsiung Liu, Alan W. Cuthbert, and Kevin M. O’Shaughnessy Department of Medicine, University of Cambridge, Cambridge, United Kingdom The WNK (with no lysine kinase) kinases are a novel class of serine/threonine kinases that lack a characteristic lysine residue for ATP docking. Both WNK1 and WNK4 are expressed in the mammalian kidney, and mutations in either can cause the rare familial syndrome of hypertension and hyperkalemia (Gordon syndrome, or pseudohypoaldosteronism type 2). The molecular basis for the action of WNK4 is through alteration in the membrane expression of the NaCl co-transporter (NCCT) and the renal outer-medullary K channel KCNJ1 (ROMK). The actions of WNK1 are less well defined, and evidence to date suggests that it can affect NCCT expression but only in the presence of WNK4. The results of co-expressing WNK1 with ROMK in Xenopus oocytes are reported for the first time. These studies show that WNK1 is able to suppress total current directly through ROMK by causing a marked reduction in its surface expression. The effect is mimicked by a kinase-dead mutant of WNK1 (368D>A), suggesting that it is not dependent on its catalytic activity. Study of the time course of ROMK expression further suggests that WNK1 accelerates trafficking of ROMK from the membrane, and this effect seems to be dynamin dependent. Using fragments of full-length WNK1, it also is shown that the effect depends on residues in the middle section of the protein (502 to 1100 WNK1) that contains the acidic motif. -
WNK1 Activates Large-Conductance Ca -Activated K Channels
BASIC RESEARCH www.jasn.org WNK1 Activates Large-Conductance Ca2+-Activated K+ Channels through Modulation of ERK1/2 Signaling † ‡ | Yingli Liu,* Xiang Song, Yanling Shi,* Zhen Shi,§ Weihui Niu,§ Xiuyan Feng,* Dingying Gu,§ | Hui-Fang Bao,¶ He-Ping Ma,¶ Douglas C. Eaton,¶ Jieqiu Zhuang,§ and Hui Cai* ¶ *Renal Division, Department of Medicine, and ¶Department of Physiology, Emory University School of Medicine, Atlanta, Georgia; §Department of Nephrology, The Second Affiliated Hospital, Wenzhou Medical University, Zhejiang, China; †Department of Nephrology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine; ‡Department of Cardiology, The Fourth Affiliated Hospital, Harbin Medical University, Heilongjiang, China; and |Renal Section, Atlanta Veterans Affairs Medical Center, Decatur, Georgia ABSTRACT With no lysine (WNK) kinases are members of the serine/threonine kinase family. We previously showed that WNK4 inhibits renal large-conductance Ca2+-activated K+ (BK) channel activity by enhancing its degradation through a lysosomal pathway. In this study, we investigated the effect of WNK1 on BK channel activity. In HEK293 cells stably expressing the a subunit of BK (HEK-BKa cells), siRNA-mediated knockdown of WNK1 expression significantly inhibited both BKa channel activity and open probability. Knockdown of WNK1 expression also significantly inhibited BKa protein expression and increased ERK1/2 phosphorylation, whereas overexpression of WNK1 significantly enhanced BKa expression and decreased ERK1/2 phosphor- ylation in a dose-dependent manner in HEK293 cells. Knockdown of ERK1/2 prevented WNK1 siRNA-mediated inhibition of BKa expression. Similarly, pretreatment of HEK-BKa cells with the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of WNK1 siRNA on BKa expression in a dose-dependent manner.