Papers by Miguel Van Bemmelen
The E3 ubiquitin‐protein ligase Nedd4‐2 regulates the sodium chloride cotransporter (NCC) but is not required for a potassium‐induced reduction of NCC expression
The FASEB Journal
A‐Kinase anchoring protein 2 (AKAP2) and Pendrin; cross‐talk between cell signaling and renal physiology
The FASEB Journal
Respuesta citolítica inducida por inmunización con proteínas dela fase pre-eritrocítica de las Plasmodium sp., en humanos y primates
IP 1106-04-382-98Miguel X. van Bemmelen ... [et al.]. -- En: Molecular andbiochemical parasitolog... more IP 1106-04-382-98Miguel X. van Bemmelen ... [et al.]. -- En: Molecular andbiochemical parasitology (Aug. 2000); p. 1-14. --;ISSN 0166-6851 -- Generation and characterization of malaria-specific human CD8+ lymphocyte clones : effect of;natural polymorphism on T cell recognition and endogenouscognate antigenpresentation by liver cells / Anilsa;Bonelo ... [et al.]. -- En: European journal of immunology. --no. 30 (2000); p. 3079-3088 -- HLA-A*0201;restricted CD8+ T-lymphocyte responses to malaria : identification of newPlasmodium falciparum epitopes by;IFN-Y Elispot / John Mario Gonzalez ... [et al.]. -- En: Parasite immunology. -- Vol. 22, no. 10 (Oct. 2000);p. 501-514 -- Los linfocitos T CD8+ en la respuesta inmunecelular : ?comofuncionan?, ?como se evaluan? /;John Mario Gonzalez ... [et al.]. -- En: Revista Asociacion Colombiana deAlergia, Asma e Inmunologia. -- Vol.;8, no. 2 (1999); p. 19-24.;ARTICULO(S) EN REVISTA: Expression and one-step purification ofPlasmodiumproteins in Dictyostelium
Expression and one-step purification of Plasmodium proteins in Dictyostelium
Molecular and Biochemical Parasitology, 2000
Nearly full-length Circumsporozoite protein (CSP) from Plasmodium falciparum, the C-terminal frag... more Nearly full-length Circumsporozoite protein (CSP) from Plasmodium falciparum, the C-terminal fragments from both P. falciparm and P. yoelii CSP and a fragment comprising 351 amino acids of P.vivax MSPI were expressed in the slime mold Dictyostelium discoideum. Discoidin-tag expression vectors allowed both high yields of these proteins and their purification by a nearly single-step procedure. We exploited the galactose binding activity of Discoidin Ia to separate the fusion proteins by affinity chromatography on Sepharose-4B columns. Inclusion of a thrombin recognition site allowed cleavage of the Discoidin-tag from the fusion protein. Partial secretion of the protein was obtained via an ER independent pathway, whereas routing the recombinant proteins to the ER resulted in glycosylation and retention. Yields of proteins ranged from 0.08 to 3 mg l(-1) depending on the protein sequence and the purification conditions. The recognition of purified MSPI by sera from P. vivax malaria patients was used to confirm the native conformation of the protein expressed in Dictyostelium. The simple purification procedure described here, based on Sepharose-4B, should facilitate the expression and the large-scale purification of various Plasmodium polypeptides.
PloS one, 2017
The Acid-Sensing Ion Channels (ASIC) exhibit a fast desensitizing current when activated by pH va... more The Acid-Sensing Ion Channels (ASIC) exhibit a fast desensitizing current when activated by pH values below 7.0. By contrast, non-proton ligands are able to trigger sustained ASIC currents at physiological pHs. To analyze the functional basis of the ASIC desensitizing and sustained currents, we have used ASIC1a and ASIC2a mutants with a cysteine in the pore vestibule for covalent binding of different sulfhydryl reagents. We found that ASIC1a and ASIC2a exhibit two distinct currents, a proton-induced desensitizing current and a sustained current triggered by sulfhydryl reagents. These currents differ in their pH dependency, their sensitivity to the sulfhydryl reagents, their ionic selectivity and their relative magnitude. We propose a model for ASIC1 and ASIC2 activity where the channels can function in two distinct modes, a desensitizing mode and a sustained mode depending on the activating ligands. The pore vestibule of the channel represents a functional site for binding non-proto...
The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface
PLOS ONE, 2015
The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here... more The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathionate (NaTT). Under these different crosslinking conditions ASIC1a migrates as four distinct oligomeric states that correspond by mass to multiples of a single ASIC1a subunit. The relative importance of each of the four ASIC1a oligomers was critically dependent on the availability of cysteines in the transmembrane domain for crosslinking, consistent with the presence of ASIC1a homo-oligomers. The expression of ASIC1a monomers, trimeric or tetrameric concatemeric cDNA constructs resulted in functional channels. The resulting ASIC1a complexes are resolved as a predominant tetramer over the other oligomeric forms, after stabilization with BMOE or NaTT and SDS-PAGE/western blot analysis. Our data identify a major ASIC1a homotetramer at the surface membrane of the cell expressing functional ASIC1a channel.
cAMP Analogues Designed for Affinity Chromatography
New Affinity Materials for cAMP-Binding Proteins
Cardiac voltage-gated sodium channel Na(v)1.5 is regulated by Nedd4-2 mediated ubiquitination
Circulation Research
Na(v)1.5, the cardiac isoform of the voltage-gated Na+ channel, is critical to heart excitability... more Na(v)1.5, the cardiac isoform of the voltage-gated Na+ channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Na(v)1.5 C-terminus contains a PY-motif (xPPxY) that is known to act as binding site for Nedd4/Nedd4-like ubiquitin-protein ligases. Because Nedd4-2 is well expressed in the heart, we investigated its role in the ubiquitination and regulation of Na(v)1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Na(v)1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Na(v)1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2-dependent ubiquitination of Na(v)1.5 was observed. To test for a functional role of Nedd4-2, patch-clamp experiments were performed on HEK cells expressing wild-type and mutant forms of both Na(v)1.5 and Nedd4-2. Na(v)1.5 current densi...
Cyclic Nucleotide Metabolism as a Target in Chemotherapy
Molecular Aspects of Chemotherapy, 1992
Journal of physiology and pharmacology : an official journal of the Polish Physiological Society, 2005
Cyclic AMP has been generally recognised as activator of cAMP-dependent protein kinases. However,... more Cyclic AMP has been generally recognised as activator of cAMP-dependent protein kinases. However, there is little evidence about role of cAMP-dependent protein kinase (PKA), in particular izoenzymes PKA-I and PKA-II, in glomeruli contractility. We measured changes of glomerular inulin space (GIS) as a marker of its contractility in the presence of phosphodiesterase resistance cAMP analogues; activators and inhibitors of PKA. Activator of PKA i.e. (Sp) 8-Cl-cAMPS (0.1-100 microM) decreased GIS. (Rp) 8-Cl-cAMPS (0.1-100 microM), inhibitor of PKA, was ineffective but shifted concentration-response curve of (Sp) 8-Cl-cAMPS to right at 50 microM. Specific A site activation by N6-benzoyl-cAMP decreased GIS with maximum at 0.1 microM. Activation of B site by 8-aminobutyloamino-cAMP (0.1-100 microM) had no effect. However, specific activation of both sites on PKA-I or PKA-II by site-selective analogue pairs e.g. 8-aminobutyloamino-cAMP plus 8-piperidino-cAMP or N6-benzoyl-cAMP plus 8-piperi...
Random insertion of green fluorescent protein into the regulatory subunit of cyclic adenosine monophosphate-dependent protein kinase
Methods in molecular biology (Clifton, N.J.), 2002
GFP in R-Subunit of cAMP-Dependent PK ... Random Insertion of Green Fluorescent Protein into the ... more GFP in R-Subunit of cAMP-Dependent PK ... Random Insertion of Green Fluorescent Protein into the Regulatory Subunit of Cyclic Adenosine Monophosphate-Dependent Protein Kinase ... Pascal J. Baehler, Ricardo M. Biondi, Miguel van Bemmelen, Michel Véron, and ...
Autophagy
Despite abundant evidence for autophagic cell death as a morphological type, the notion that auto... more Despite abundant evidence for autophagic cell death as a morphological type, the notion that autophagy can actually contribute mechanistically to the cell's death is controversial. In cells capable of apoptosis, autophagic cell death has been dismissed by some authors as a morphologically unusual form of apoptosis. But strong recent evidence for autophagy-mediated death of cells rendered incapable of apoptosis has been criticized on the grounds that this cell death is too artificial to be relevant to normal cells. We here argue from our own and other recent evidence that autophagy can mediate the death even of apoptosis-competent cells.
European Journal of Biochemistry, 1997
The C subunit of Dictyostelium CAMP-dependent protein kinase (PKA) is unusually large (73 kDa) du... more The C subunit of Dictyostelium CAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core. The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI.
Mechanisms of Development, 1998
The catalytic subunit of the cAMP-dependent protein kinase (PKA) from Dictyostelium discoideum co... more The catalytic subunit of the cAMP-dependent protein kinase (PKA) from Dictyostelium discoideum contains several domains, including an unusually long N-terminal extension preceding a highly conserved catalytic core. We transformed the aggregationless PkaC-null strain with several deletion constructs of both domains. Strains transformed with genes expressing catalytically-inactive polypeptides could not rescue development. Cotransformation with constructs encoding the N-terminal extension and the catalytic core, both unable to rescue development by themselves, yielded transformants able to proceed to late development. A 27-amino acid long hydrophobic region, immediately upstream of the catalytic core, was found indispensable for PKA function. A putative role of this sequence in the acquisition of the active conformation of the protein is discussed.
Congenital ataxia and hemiplegic migraine with cerebral edema associated with a novel gain of function mutation in the calcium channel CACNA1A
Journal of the Neurological Sciences, 2014
Mutations in the CACNA1A gene, encoding the α1 subunit of the voltage-gated calcium channel Ca(V)... more Mutations in the CACNA1A gene, encoding the α1 subunit of the voltage-gated calcium channel Ca(V)2.1 (P/Q-type), have been associated with three neurological phenotypes: familial and sporadic hemiplegic migraine type 1 (FHM1, SHM1), episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 (SCA6). We report a child with congenital ataxia, abnormal eye movements and developmental delay who presented severe attacks of hemiplegic migraine triggered by minor head traumas and associated with hemispheric swelling and seizures. Progressive cerebellar atrophy was also observed. Remission of the attacks was obtained with acetazolamide. A de novo 3 bp deletion was found in heterozygosity causing loss of a phenylalanine residue at position 1502, in one of the critical transmembrane domains of the protein contributing to the inner part of the pore. We characterized the electrophysiology of this mutant in a Xenopus oocyte in vitro system and showed that it causes gain of function of the channel. The mutant Ca(V)2.1 activates at lower voltage threshold than the wild type. These findings provide further evidence of this molecular mechanism as causative of FHM1 and expand the phenotypic spectrum of CACNA1A mutations with a child exhibiting severe SHM1 and non-episodic ataxia of congenital onset.
Early Aldosterone-Induced Gene Product Regulates the Epithelial Sodium Channel by Deubiquitylation
Journal of the American Society of Nephrology, 2007
The mineralocorticoid hormone aldosterone controls sodium reabsorption and BP largely by regulati... more The mineralocorticoid hormone aldosterone controls sodium reabsorption and BP largely by regulating the cell-surface expression and function of the epithelial sodium channel (ENaC) in target kidney tubules. Part of the stimulatory effect of aldosterone on ENaC is mediated by the induction of serum- and glucocorticoid-regulated kinase 1 (Sgk1), a kinase that interferes with the ubiquitylation of ENaC by ubiquitin-protein ligase Nedd4-2. In vivo early aldosterone-regulated mRNA now has been identified in microselected mouse distal nephron by microarray. From 22 mRNA that displayed a two-fold or more change, 13 were downregulated and nine were upregulated. Besides Sgk1, the induced mRNA include Grem2 (protein related to DAN and cerebrus [PRDC]), activating transcription factor 3, cAMP responsive element modulator, and the ubiquitin-specific protease Usp2-45. The induction of this last enzyme isoform was verified in mouse distal nephron tubule at the protein level. With the use of Hek293 cells, Xenopus oocytes, and mpkCCD(c14) cells as expression systems, it was shown that Usp2-45 deubiquitylates ENaC and stimulates ENaC-mediated sodium transport, an effect that is not additive to that of Sgk1. A deubiquitylating enzyme that targets ENaC in vitro and thus may play a role in sodium transport regulation was identified within a series of new in vivo early aldosterone-regulated gene products.
Journal of Biological Chemistry, 2007
The putative selectivity filter of the epithelial sodium channel (ENaC) comprises a three-residue... more The putative selectivity filter of the epithelial sodium channel (ENaC) comprises a three-residue sequence G/SXS, but it remains uncertain whether the backbone atoms of this sequence or whether their side chains are lining the pore. It has been reported that the S589C mutation in the selectivity filter of alphaENaC renders the channel sensitive to block by externally applied Cd2+; this was interpreted as evidence for Cd2+ coordination with the thiol group of the side chain of alpha589C, pointing toward the pore lumen. Because the alphaS589C mutation alters the monovalent to divalent cation selectivity ratio of ENaC and because internally applied Cd2+ blocks wild-type ENaC with high affinity, we hypothesized that the inhibition of alphaS589C ENaC by Cd2+ results rather from the coordination of this cation with native cysteine residues located in the internal pore of ENaC. We show here that Cd2+ inhibits not only ENaC alphaS589C and alphaS589D but also alphaS589N mutants and that Ca2+ weakly interacts with the S589D mutant. The block of alphaS589C, -D, and -N mutants is characterized by a slow on-rate, is nearly irreversible, is voltage-dependent, and can be prevented by amiloride. The C546S mutation in the second transmembrane helix of gamma subunit in the background of the ENaC alphaS589C, -D, or -N mutants reduces the sensitivity to block by Cd2+ and renders the block rapidly reversible. We conclude therefore that the block by Cd2+ of the alphaS589C, -D, and -N mutants results from the trapping of Cd2+ ions in the internal pore of the channel and involves Cys-546 in the second transmembrane helix of the gammaENaC subunit.
A Gating Mutation in the Internal Pore of ASIC1a
Journal of Biological Chemistry, 2006
Using a substituted cysteine accessibility scan, we have investigated the structures that form th... more Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore.
FEBS Letters, 1990
The activation of the cGMP-dependent protein kinase and CAMP-dependent protein kinase by the dias... more The activation of the cGMP-dependent protein kinase and CAMP-dependent protein kinase by the diastereomers of guanosine 3',5'-monophosphorothioate, (Sp)-cGMPS and (Rp)-cGMPS, and I-chloroguanosine 3',5'-monophosphorothioate, (Sp)&CltGMPS and (Rp)-8-Cl-cGMPS, was investigated using the peptide Kemptide as substrate. The (Sp)-diastereomers, which have an axial exocyclic sulfur atom, bound to the &IMPdependent protein kinase and stimulated its phosphotransferase activity. In contrast, the (Rp)-isomers, which have an equatorial exocyclic sulfur atom, bound to the enzyme without stimulation of its activity. (Rp)-cGMPS and (Rp)-8-Cl-cGMPS antagonized the activation of the cGMPdependent protein kinase with a Ki of 20 PM and 1.5 PM, respectively. (Rp)-cGMPS also antagonized the activation of CAMP-dependent protein kinase with a Ki of 20 PM. In contrast, (Rp)-8-Cl-cGMPS was a weak inhibitor of the CAMP-dependent protein kinase with a Ki of 100 pM. (Rp)-8-Cl-cGMPS appears to be a rather selective inhibitor of the cGMP-dependent protein kinase and may be a useful tool for studying the role of cGMP in broken and intact cell systems.
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Papers by Miguel Van Bemmelen