Francoise Haeseleer - Academia.edu (original) (raw)

Papers by Francoise Haeseleer

Investigative Ophthalmology & Visual Science, Apr 22, 2011

The Journal of General Physiology, Oct 24, 2018

Rockefeller University Press Ca 2+ influx through Ca v 1.4 L-type Ca 2+ channels supports the sus... more Rockefeller University Press Ca 2+ influx through Ca v 1.4 L-type Ca 2+ channels supports the sustained release of glutamate from photoreceptor synaptic terminals in darkness, a process that is critical for vision. Consistent with this role, Ca v 1.4 exhibits weak Ca 2+-dependent inactivation (CDI)-a negative feedback regulation mediated by Ca 2+-bound calmodulin (CaM). CaM binds to a conserved IQ domain in the proximal C-terminal domain of Ca v channels, but in Ca v 1.4, a C-terminal modulatory domain (CTM) disrupts interactions with CaM. Exon 47 encodes a portion of the CTM and is deleted in a Ca v 1.4 splice variant (Ca v 1.4Δex47) that is highly expressed in the human retina. Ca v 1.4Δex47 exhibits CDI and enhanced voltage-dependent activation, similar to that caused by a mutation that is associated with congenital stationary night blindness type 2, in which the CTM is deleted (K1591X). The presence of CDI and very negative activation thresholds in a naturally occurring variant of Ca v 1.4 are perplexing considering that these properties are expected to be maladaptive for visual signaling and result in night blindness in the case of K1591X. Here we show that Ca v 1.4Δex47 and K1591X exhibit fundamental differences in their regulation by CaM. In Ca v 1.4Δex47, CDI requires both the N-terminal (N lobe) and C-terminal (C lobe) lobes of CaM to bind Ca 2+ , whereas CDI in K1591X is driven mainly by Ca 2+ binding to the C lobe. Moreover, the CaM N lobe causes a Ca 2+-dependent enhancement of activation of Ca v 1.4Δex47 but not K1591X. We conclude that the residual CTM in Ca v 1.4Δex47 enables a form of CaM N lobe regulation of activation and CDI that is absent in K1591X. Interaction with the N lobe of CaM, which is more sensitive to global elevations in cytosolic Ca 2+ than the C lobe, may allow Ca v 1.4Δex47 to be modulated by a wider range of synaptic Ca 2+ concentrations than K1591X; this may distinguish the normal physiological function of Ca v 1.4Δex47 from the pathological consequences of K1591X.

ENeuro, Sep 1, 2016

Significance Statement Electrical signals generated by the photoreceptors in response to incident... more Significance Statement Electrical signals generated by the photoreceptors in response to incident light are processed by diverse retinal neurons before being sent to the brain. Ca 2ϩ signaling controls both cellular and synaptic mechanisms that shape signals as they are transmitted through the retina. Ca 2ϩ-binding proteins (CaBPs), including the calmodulin-like CaBPs, exert Ca 2ϩ-dependent effects on specific target proteins-e.g., ion channels. To determine whether CaBP1/caldendrin and CaBP2 are important for normal retinal function, we used CaBP1/caldendrin-and CaBP2-deficient mice. Although these proteins are not required for retinal development and maintenance, CaBP1/caldendrin and CaBP2 are important for normal transfer of light signals through the retina.

Investigative Ophthalmology & Visual Science, Sep 26, 2016

Molecular therapy. Methods & clinical development, Mar 1, 2023

Adoptive therapy with genetically engineered T cells offers potential for infectious disease trea... more Adoptive therapy with genetically engineered T cells offers potential for infectious disease treatment in immunocompromised persons. HIV/simian immunodeficiency virus (SIV)-infected cells express phosphatidylserine (PS) early post infection. We tested whether chimeric engulfment receptor (CER) T cells designed to recognize PS-expressing cells could eliminate SIVinfected cells. Lentiviral CER constructs composed of the extracellular domain of T cell immunoglobulin and mucin domain containing 4 (TIM-4), the PS receptor, and engulfment signaling domains were transduced into primary rhesus macaque (RM) T cells. We measured PS binding and T cell engulfment of RM CD4+ T cells infected with SIV expressing GFP and in vitro, TIM-4 CER CD4+ T cells effectively killed SIV-infected cells, which was dependent on TIM-4 binding to PS. Enhanced killing of SIV-infected CD4+ T cells by CER and chimeric antigen receptor T cell combinations was also observed. This installation of innate immune functions into T cells presents an opportunity to enhance elimination of SIV-infected cells, and studies to evaluate their effect in vivo are warranted.

Molecular therapy. Methods & clinical development, Sep 1, 2021

Chimeric antigen receptor (CAR) T cell therapies are being investigated as potential HIV cures an... more Chimeric antigen receptor (CAR) T cell therapies are being investigated as potential HIV cures and designed to target HIV reservoirs. Monoclonal antibodies (mAbs) targeting the simian immunodeficiency virus (SIV) envelope allowed us to investigate the potency of single-chain variable fragment (scFv)-based anti-SIV CAR T cells. In vitro, CAR T cells expressing the scFv to both the variable loop 1 (V1) or V3 of the SIV envelope were highly potent at eliminating SIV-infected T cells. However, in preclinical studies, in vivo infusion of these CAR T cells in rhesus macaques (RMs) resulted in lack of expansion and no detectable in vivo antiviral activity. Injection of envelope-expressing antigen-presenting cells (APCs) 1 week post-CAR T cell infusion also failed to stimulate CAR T cell expansion in vivo. To investigate this in vitro versus in vivo discrepancy, we examined host immune responses directed at CAR T cells. A humoral immune response against the CAR scFv was detected post-infusion of the anti-SIV CAR T cells; anti-SIV IgG antibodies present in plasma of SIV-infected animals were associated with inhibited CAR T cell effector functions. These data indicate that lack of in vivo expansion and efficacy of CAR T cells might be due to antibodies blocking the interaction between the CAR scFv and its epitope.

Cette thèse de doctorat a été numérisée par l'Université libre de Bruxelles. L'auteur qui s'oppos... more Cette thèse de doctorat a été numérisée par l'Université libre de Bruxelles. L'auteur qui s'opposerait à sa mise en ligne dans DI-fusion est invité à prendre contact avec l'Université

Investigative Ophthalmology & Visual Science, Feb 12, 2013

PURPOSE. CaBP4 is a neuronal Ca 2þ-binding protein that is expressed in the retina and in the coc... more PURPOSE. CaBP4 is a neuronal Ca 2þ-binding protein that is expressed in the retina and in the cochlea, and is essential for normal photoreceptor synaptic function. CaBP4 is phosphorylated by protein kinase C zeta (PKCf) in the retina at serine 37, which affects its interaction with and modulation of voltage-gated Ca v 1 Ca 2þ channels. In this study, we investigated the potential role and functional significance of protein phosphatase 2A (PP2A) in CaBP4 dephosphorylation. METHODS. The effect of protein phosphatase inhibitors, light, and overexpression of PP2A subunits on CaBP4 dephosphorylation was measured in in vitro assays. Pull-down experiments using retinal or transfected HEK293 cell lysates were used to investigate the association between CaBP4 and PP2A subunits. Electrophysiologic recordings of cotransfected HEK293 cells were performed to analyze the effect of CaBP4 dephosphorylation in modulating Ca v 1.3 currents. RESULTS. PP2A inhibitors, okadaic acid (OA), and fostriecin, but not PP1 selective inhibitors, NIPP-1, and inhibitor 2, block CaBP4 dephosphorylation in retinal lysates. Increased phosphatase activity in light-dependent conditions reverses phosphorylation of CaBP4 by PKCf. In HEK293 cells, overexpression of PP2A enhances the rate of dephosphorylation of CaBP4. In addition, inhibition of protein phosphatase activity by OA increases CaBP4 phosphorylation and potentiates the modulatory effect of CaBP4 on Ca v 1.3 Ca 2þ channels in HEK293T cells. CONCLUSIONS. This study provides evidence that CaBP4 is dephosphorylated by PP2A in the retina. Our findings reveal a novel role for protein phosphatases in regulating CaBP4 function in the retina, which may fine tune presynaptic Ca 2þ signals at the photoreceptor synapse.

PLOS ONE, 2016

CaBPs are a family of EF-hand Ca 2+ binding proteins that are structurally similar to calmodulin.... more CaBPs are a family of EF-hand Ca 2+ binding proteins that are structurally similar to calmodulin. CaBPs can interact with, and yet differentially modulate, effectors that are regulated by calmodulin, such as Ca v 1 voltage-gated Ca 2+ channels. Immunolabeling studies suggest that multiple CaBP family members (CaBP1, 2, 4, and 5) are expressed in the cochlea. To gain insights into the respective auditory functions of these CaBPs, we characterized the expression and cellular localization of CaBPs in the mouse cochlea. By quantitative reverse transcription PCR, we show that CaBP1 and CaBP2 are the major CaBPs expressed in mouse cochlea both before and after hearing onset. Of the three alternatively spliced variants of CaBP1 (caldendrin, CaBP1-L, and CaBP1-S) and CaBP2 (CaBP2-alt, CaBP2-L, CaBP2-S), caldendrin and CaBP2-alt are the most abundant. By in situ hybridization, probes recognizing caldendrin strongly label the spiral ganglion, while probes designed to recognize all three isoforms of CaBP1 weakly label both the inner and outer hair cells as well as the spiral ganglion. Within the spiral ganglion, caldendrin/CaBP1 labeling is associated with cells resembling satellite glial cells. CaBP2-alt is strongly expressed in inner hair cells both before and after hearing onset. Probes designed to recognize all three variants of CaBP2 strongly label inner hair cells before hearing onset and outer hair cells after the onset of hearing. Thus, CaBP1 and CaBP2 may have overlapping roles in regulating Ca 2+ signaling in the hair cells, and CaBP1 may have an additional function in the spiral ganglion. Our findings provide a framework for understanding the role of CaBP family members in the auditory periphery.

The Journal of biological chemistry, Jan 7, 2014

CaBP4 modulates Ca(2+)-dependent activity of L-type voltage-gated Ca(2+) channels (Cav1.4) in ret... more CaBP4 modulates Ca(2+)-dependent activity of L-type voltage-gated Ca(2+) channels (Cav1.4) in retinal photoreceptor cells. Mg(2+) binds to the first and third EF-hands (EF1 and EF3), and Ca(2+) binds to EF1, EF3, and EF4 of CaBP4. Here we present NMR structures of CaBP4 in both Mg(2+)-bound and Ca(2+)-bound states and model the CaBP4 structural interaction with Cav1.4. CaBP4 contains an unstructured N-terminal region (residues 1-99) and four EF-hands in two separate lobes. The N-lobe consists of EF1 and EF2 in a closed conformation with either Mg(2+) or Ca(2+) bound at EF1. The C-lobe binds Ca(2+) at EF3 and EF4 and exhibits a Ca(2+)-induced closed-to-open transition like that of calmodulin. Exposed residues in Ca(2+)-bound CaBP4 (Phe(137), Glu(168), Leu(207), Phe(214), Met(251), Phe(264), and Leu(268)) make contacts with the IQ motif in Cav1.4, and the Cav1.4 mutant Y1595E strongly impairs binding to CaBP4. We conclude that CaBP4 forms a collapsed structure around the IQ motif in C...

Journal of Biological Chemistry, 2014

The Journal of Physiology, 2007

Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter ... more Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter release, triggered by sustained activation of presynaptic Ca v 1.3 voltage-gated Ca 2+ channels. Central to their role in this regard, Ca v 1.3 channels in inner hair cells show little Ca 2+-dependent inactivation, a fast negative feedback regulation by incoming Ca 2+ ions, which depends on calmodulin association with the Ca 2+ channel α 1 subunit. Ca 2+-dependent inactivation characterizes nearly all voltage-gated Ca 2+ channels including Ca v 1.3 in other excitable cells. The mechanism underlying the limited autoregulation of Ca v 1.3 in inner hair cells remains a mystery. Previously, we established calmodulin-like Ca 2+-binding proteins in the brain and retina (CaBPs) as essential modulators of voltage-gated Ca 2+ channels. Here, we demonstrate that CaBPs differentially modify Ca 2+ feedback to Ca v 1.3 channels in transfected cells and explore their significance for Ca v 1.3 regulation in inner hair cells. Of multiple CaBPs detected in inner hair cells (CaBP1, CaBP2, CaBP4 and CaBP5), CaBP1 most efficiently blunts Ca 2+-dependent inactivation of Ca v 1.3. CaBP1 and CaBP4 both interact with calmodulin-binding sequences in Ca v 1.3, but CaBP4 more weakly inhibits Ca 2+-dependent inactivation than CaBP1. Ca 2+-dependent inactivation is marginally greater in inner hair cells from CaBP4 −/− than from wild-type mice, yet CaBP4 −/− mice are not hearing-impaired. In contrast to CaBP4, CaBP1 is strongly localized at the presynaptic ribbon synapse of adult inner hair cells both in wild-type and CaBP4 −/− mice and therefore is positioned to modulate native Ca v 1.3 channels. Our results reveal unexpected diversity in the strengths of CaBPs as Ca 2+ channel modulators, and implicate CaBP1 rather than CaBP4 in conferring the anomalous slow inactivation of Ca v 1.3 Ca 2+ currents required for auditory transmission.

Research in Microbiology, 1994

ABSTRACT

Neuroscience, 2014

Ca 2+ binding protein 1 (CaBP1) and caldendrin are alternatively spliced variants of a subfamily ... more Ca 2+ binding protein 1 (CaBP1) and caldendrin are alternatively spliced variants of a subfamily of Ca 2+ binding proteins with high homology to calmodulin. Although CaBP1 and caldendrin regulate effectors including plasma membrane and intracellular Ca 2+ channels in heterologous expression systems, little is known about their functions in vivo. Therefore, we generated mice deficient in CaBP1/ caldendrin expression (C-KO) and analyzed the expression and cellular localization of CaBP1 and caldendrin in the mouse brain. Immunoperoxidase labeling with antibodies recognizing both CaBP1 and caldendrin was absent in the brain of C-KO mice, but was intense in multiple brain regions of wild-type mice. By western blot, the antibodies detected two proteins that were absent in the C-KO mouse and consistent in size with caldendrin variants originating from alternative translation initiation sites. By quantitative PCR, caldendrin transcript levels were far greater than those for CaBP1 particularly in the cerebral cortex and hippocampus. In the frontal cortex but not in the hippocampus, caldendrin expression increased steadily from birth. By double-label immunofluorescence, CaBP1/caldendrin was localized in principal neurons and parvalbumin-positive interneurons. In the cerebellum, CaBP1/caldendrin antibodies labeled interneurons in the molecular layer and in basket cell terminals surrounding the soma and axon initial segment of Purkinje neurons, but immunolabeling was absent in Purkinje neurons. We conclude that CaBP1/ caldendrin is localized both pre-and postsynaptically where it may regulate Ca 2+ signaling and excitability in select groups of excitatory and inhibitory neurons.

Nature Neuroscience, 2002

Calcium entry into cells through voltage-gated Ca 2+ channels initiates a wide range of cellular ... more Calcium entry into cells through voltage-gated Ca 2+ channels initiates a wide range of cellular processes including protein phosphorylation, gene expression and neurotransmitter release 1. Neuronal Ca 2+ channels consist of a pore-forming α 1 subunit and auxiliary β, α 2 δ and sometimes γ subunits 2 , and their function depends considerably on interactions with additional regulatory factors. For example, the activation of G-protein-coupled receptors by neurotransmitters inhibits Ca v 2.1 and Ca v 2.2 channels, which mediate P/Q-type and Ntype Ca 2+ currents, respectively, through the binding of G-protein βγ subunits to distinct sites on the Ca 2+ channel α 1 subunit 3-5. These channels are also inhibited by direct interactions with synaptic SNARE (soluble NSF attachment protein receptor proteins)-a process that may optimize coupling between Ca 2+ entry and synaptic vesicle fusion 6-8. Characterizing the functional interactions between Ca 2+ channels and other signaling molecules is therefore crucial to understanding how many Ca 2+-dependent processes in neurons are regulated. We have shown previously that the prominent Ca 2+ sensor CaM binds to a CaM-binding site (CBD) in the carboxy-terminal domain of the α 1 2.1 subunit and mediates the dual feedback regulation of Ca v 2.1 channels by Ca 2+ ions 9,10. A second site, located amino-terminal to the CBD, is analogous to the IQ domain that mediates Ca 2+ /CaM-dependent inactivation of Ca v 1 (L-type) channels 11-13. The IQ domain of α 1 2.1 interacts with CaM in vitro and also contributes to the regulation of Ca v 2.1 channels by CaM 14,15. Ca 2+ /CaM mediates both facilitation and enhanced inactivation of Ca v 2.1 channels in transfected cells during trains of depolarizations 10,15. Presynaptic Ca v 2.1 channels in the brain undergo similar forms of Ca 2+

MGG Molecular & General Genetics, 1992

A genomic library of Mycobacterium bovis BCG has been constructed by cloning DNA partially digest... more A genomic library of Mycobacterium bovis BCG has been constructed by cloning DNA partially digested with Sau3A into the Escherichia coli expression vector pAS 1. The gene coding for ornithine carbamoyltransferase (EC.2.1.3.3; OTCase), hereafter referred to as argF, was isolated from the library by complementation of a double argF-argI mutant of E. coli and its sequence was determined. The translation initiation codon used, GTG, was identified by comparing the amino acid sequence deduced from the gene with the N-terminal sequence of the corresponding purified protein. On this basis, the M. boris BCG OTCase monomer consists of 307 amino acid residues and displays about 44% identity with other OTCases, the most closely related homologue being the anabolic enzyme of Pseudomonas aeruginosa. The native enzyme has an estimated molecular mass of 110 kDa, suggesting a trimeric structure as is the case for most of the anabolic OTCases known from various organisms.

Molecular and Biochemical Parasitology, 1993

The DNA coding for the circumsporozoite protein of Plasmodium falciparum (CSP; aa 1-412) has been... more The DNA coding for the circumsporozoite protein of Plasmodium falciparum (CSP; aa 1-412) has been placed under the control of the mycobacterial promoter derived from the gene encoding the 64-kDa antigen of Mycobacterium bovis-BCG. This expression cassette was cloned into pJRD184, an Escherichia coli multicloning site vector, together with the kanamycin resistance gene from Tn903 and the attachment site and integrase gene from the temperate mycobacteriophage FRAT1. One of the resulting plasmids, pNIV2173, introduced by electroporation into both Mycobacterium smegmatis and M. bovis-BCG, integrated at a specific site in the genome of each recipient. Recombinants expressed immunoreactive polypeptides, ranging in size from 62 to 43 kDa, at a level of about 1% of total soluble proteins. Part of this material was present in the culture medium indicating that mycobacterial recombinants were able to secrete the CSP. The M. smegmatis and M. bovis-BCG recombinants, transformed with pNIV2173 and grown in absence of antibiotic, were followed for more than 400 and 50 generations respectively. Over this time span, neither DNA rearrangement nor loss of expression was observed. Inoculation of the recombinant BCG to mice did not induce humoral response to CSP nor proliferative response to CSP Th2R CD4+ T lymphocyte epitope.

The Journal of Clinical Endocrinology & Metabolism, 1997

We previously reported that human natural autoantibodies enriched in antiestrogen receptor Ig (Ig... more We previously reported that human natural autoantibodies enriched in antiestrogen receptor Ig (IgGs) display estrogenic activity in MCF-7 mammary carcinoma cells. In this study, we investigated IgGs' mechanism of action. We showed that: 1) IgGs Fab fragments (which contain only one antigen binding site) induced an estrogenic response in MCF-7 cells, producing estrogen receptor (ER) down-regulation and an increase in progesterone receptor concentration; 2) IgGs specifically inhibited MCF-7 cell surface labeling with fluorescent estradiol (E 2)-BSA conjugates; 3) this inhibition of E 2-BSA binding to membrane estrogen binding sites was largely caused by natural anti-E 2-BSA antibodies (Ab) selectively associated with the natural anti-ER Ab within IgGs;