Neli Panova

Bioluminescence Methodology for the Detection of Protein–Protein Interactions Within the Voltage-Gated Sodium Channel Macromolecular Complex

Protein-protein interactions are critical molecular determinants of ion channel function and emerging targets for pharmacological interventions. Yet, current methodologies for the rapid detection of ion channel macromolecular complexes are still lacking. In this study we have adapted a split-luciferase complementation assay (LCA) for detecting the assembly of the voltage-gated Na+ (Nav) channel C-tail and the intracellular fibroblast growth factor 14 (FGF14), a functionally relevant component of the Nav channelosome that controls gating and targeting of Nav channels through direct interaction with the channel C-tail. In the LCA, two complementary N-terminus and C-terminus fragments of the firefly luciferase were fused, respectively, to a chimera of the CD4 transmembrane segment and the C-tail of Nav1.6 channel (CD4-Nav1.6-NLuc) or FGF14 (CLuc-FGF14). Co-expression of CLuc-FGF14 and CD4-Nav1.6-NLuc in live cells led to a robust assembly of the FGF14:Nav1.6 C-tail complex, which was attenuated by introducing single-point mutations at the predicted FGF14:Nav channel interface. To evaluate the dynamic regulation of the FGF14:Nav1.6 C-tail complex by signaling pathways, we investigated the effect of kinase inhibitors on the complex formation. Through a platform of counter screenings, we show that the p38/MAPK inhibitor, PD169316, and the IκB kinase inhibitor, BAY 11-7082, reduce the FGF14:Nav1.6 C-tail complementation, highlighting a potential role of the p38MAPK and the IκB/NFκB pathways in controlling neuronal excitability through protein-protein interactions. We envision the methodology presented here as a new valuable tool to allow functional evaluations of protein-channel complexes toward probe development and drug discovery targeting ion channels implicated in human disorders.

Prenatal Diagnosis of a Fetus with Unbalanced Translocation (4;13)(p16;q32) with Overlapping Features of Patau and Wolf-Hirschhorn Syndromes

Wolf-Hirschhorn syndrome (WHS) and Patau syndrome are two of the most severe conditions resulting from chromosome abnormalities. WHS is caused by a deletion of 4p16, while Patau syndrome is caused by trisomy for some or all regions of chromosome 13. Though the etiologies of these syndromes differ, they share several features including pre- and postnatal growth retardation, microcephaly, cleft lip and palate, and cardiac anomalies. We present here a female fetus with deletion of 4p16 → pter and duplication of 13q32 → qter due to unbalanced segregation of t(4;13)(p16;q32) in the father. She displayed overlapping features of both of these syndromes on ultrasound. To the best of our knowledge, this is the first report of a fetus with both partial trisomy 13 and deletion of 4p16, the critical region for WHS.

Identifying a Kinase Network Regulating FGF14:Nav1.6 Complex Assembly Using Split-Luciferase Complementation

Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA) is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14). Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC) as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3) and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the normal and diseased brain.

Modulation of the FGF14:FGF14 homodimer interaction through short peptide fragments.

Fibroblast growth factor 14 (FGF14) is a member of the intracellular FGF (iFGFs) family and a functionally relevant component of the neuronal voltage-gated Na(+) (Nav) channel complex. Through a monomeric interaction with the intracellular C-terminus of neuronal Nav channels, FGF14 modulates Na(+) currents in an Nav isoform-specific manner serving as a fine-tuning regulator of excitability. Previous studies based on the highly homologous FGF13 homodimer crystal structure have proposed a conserved protein:protein interaction (PPI) interface common to both Nav channel binding and iFGF homodimer formation. This interface could provide a novel target for drug design against neuronal Nav channels. Here, we provide the first in-cell reconstitution of the FGF14:FGF14 protein complex and measure the dimer interaction using the split-luciferase complementation assay (LCA). Based on the FGF14 dimer structure generated in silico, we designed short peptide fragments against the FGF14 dimer interface. One of these fragments, FLPK aligns with the pocket defined by the β12-strand and β8-β9 loop, reducing the FGF14:FGF14 dimer interaction by 25% as measured by LCA. We further compared the relative interaction strength of FGF14 wild type homodimers with FGF14 hetero- and homodimers carrying double N mutations at the Y153 and V155 residues, located at the β8-β9 loop. The Y153N/V155N double mutation counteracts the FLPK effect by increasing the strength of the dimer interaction. These data suggest that the β12 strand of FGF14 might serve as scaffold for drug design against neuronal FGF14 dimers and Nav channels.

CK2 activity is required for the interaction of FGF14 with voltage-gated sodium channels and neuronal excitability

Recent data shows that fibroblast growth factor 14 (FGF14) binds to and controls the function of the voltage‐gated sodium (Nav) channel with phenotypic outcomes on neuronal excitability. Mutations in the FGF14 gene in humans have been associated with brain disorders that are partially recapitulated in Fgf14–/– mice. Thus, signaling pathways that modulate the FGF14:Nav channel interaction may be important therapeutic targets. Bioluminescence‐based screening of small molecule modulators of the FGF14:Nav1.6 complex identified 4,5,6,7‐tetrabromobenzotriazole (TBB), a potent casein kinase 2 (CK2) inhibitor, as a strong suppressor of FGF14:Nav1.6 interaction. Inhibition of CK2 through TBB reduces the interaction of FGF14 with Nav1.6 and Nav1.2 channels. Mass spectrometry confirmed direct phosphorylation of FGF14 by CK2 at S228 and S230, and mutation to alanine at these sites modified FGF14 modulation of Nav1.6‐mediated currents. In 1 d in vitro hippocampal neurons, TBB induced a reduction in FGF14 expression, a decrease in transient Na+ current amplitude, and a hyperpolarizing shift in the voltage dependence of Nav channel steady‐state inactivation. In mature neurons, TBB reduces the axodendritic polarity of FGF14. In cornu ammonis area 1 hippocampal slices from wild‐type mice, TBB impairs neuronal excitability by increasing action potential threshold and lowering firing frequency. Importantly, these changes in excitability are recapitulated in Fgf14–/– mice, and deletion of Fgf14 occludes TBB‐dependent phenotypes observed in wild‐type mice. These results suggest that a CK2‐FGF14 axis may regulate Nav channels and neuronal excitability.—Hsu, W.‐C. J., Scala, F., Nenov, M. N., Wildburger, N. C., Elferink, H., Singh, A. K., Chesson, C. B., Buzhdygan, T., Sohail, M., Shavkunov, A. S., Panova, N. I., Nilsson, C. L., Rudra, J. S., Lichti, C. F., Laezza, F. CK2 activity is required for the interaction of FGF14 with voltage‐gated sodium channels and neuronal excitability. FASEB J. 30, 2171–2186 (2016). www.fasebj.org

Role of chromosome 1 pericentric heterochromatin (1q) in pathogenesis of myelodysplastic syndromes: report of 2 new cases.

Chromosome 1 pericentromeric heterochromatin (1q) has been shown to play an important role in the pathogenesis of non-Hodgkin lymphoma and multiple myeloma. Myelodysplastic syndrome (MDS) results from marrow failure in two or more cell lineages. Although trisomy 1q has been reported in MDS, it is usually present with additional common abnormalities such as trisomy 8, monosomy 5 or monosomy 7, leading to speculation that 1q abnormalities are mostly secondary events representing clonal evolution. We report two cases of MDS in which consistent involvement of 1q heterochromatin is seen as the primary clonal abnormality. Both patients presented with fatigue and pancytopenia. Based on the published reports and our cases, we propose that the 1q heterochromatin plays a vital role in the pathophysiology of MDS. Abnormalities involving 1q result in aberrant heterochromatin/euchromatin junctions, leading to gene dosage abnormalities. Further studies of 1q abnormalities in MDS might provide specific insights as to the exact role of the excess 1q heterochromatin in the etiology of MDS.

Thermal property and assessment of biocompatibility of poly(lactic-co-glycolic) acid/graphene nanocomposites

Polymer-matrix nanocomposites based on Poly(lactic-co-glycolic) acid (PLGA) and Graphene platelets (GNPs) were studied. GNPs, nanomaterials with a 2D flat surface, were chosen with or without chemical modification in PLGA/GNP nanocomposites and their microstructure, thermal property, and their compatibility as scaffolds for cell growth were investigated. PLGA/GNP nanocomposites (0, 1, and 5 wt. % of GNPs) were prepared using a solution based technique. Transmission electron microscopy, X-ray diffraction, Differential scanning calorimeter, and Thermogravimetric analyzer were used to analyze morphology and thermal properties. This work demonstrated the role of GNPs flat surface to provide a favorable platform resulting in an enhanced PLGA crystallization. Functionalized GNPs suppress both the thermal stability and the crystallization of PLGA. Finally, to determine the potential usefulness of these scaffolds for biomedical applications, mammalian cells were cultured on various PLGA/GNP nanocomposites (0, 1, and 5 wt. % GNPs). 1 wt. % PLGA/GNP nanocomposites showed better biocompatibility for cell growth with/without graphenes functionalization compared to pure PLGA and 5 wt. % PLGA/GNP. The function of GNPs in PLGA/GNPs (1 wt. %) composites is to provide a stage for PLGA crystallization where cell growth is favored. These results provide strong evidence for a new class of materials that could be important for biomedical applications.

Partial trisomy and partial monosomy resulting from a reciprocal segregating in a large family

Partial trisomy 7p with partial monosomy 9p is a rare disorder with only 3 cases reported. Both these abnormalities i.e., partial trisomy 7p and partial monosomy 9p result in distinct clinical phenotypes. However, patients with combined 7p trisomy/9p monosomy present with a phenotype consistent with trisomy 7p. We present a fourth case of trisomy 7p/monosomy 9p with long term follow-up and document the medical complications associated with this disorder. Long term follow-up of patients with chromosome abnormalities provides a unique opportunity to document the medical history and complications associated with such abnormalities.

Contents Vol. 17, 2002

1 A.W. Liley Medal 2001 Awarded to Prof. Wolfgang Holzgreve Chervenak, F.A. (New York, N.Y.) 3 Prenatal and Perinatal Aspects of a Giant Fetal Cervicothoracal Lymphangioma Axt-Fliedner, R.; Hendrik, H.J.; Schwaiger, C.; Ertan, A.K.; Friedrich, M.; Schmidt, W. (Homburg/Saar) 8 Gelatin Sponge Embolization. A Method for the Management of Iatrogenic Preterm Premature Rupture of the Membranes O’Brien, J.M.; Milligan, D.A.; Barton, J.R. (Lexington, Ky.) 11 Diagnostic and Therapeutic Dilemma with Large Prenatally Detected Cystic Adrenal Masses de Luca, J.L.; Rousseau, T.; Durand, C.; Sagot, P.; Sapin, E. (Dijon) 17 The Influence of Smoking and Parity on Serum Markers for Down’s Syndrome Screening Tišlarić, D. (Zagreb); Brajenović-Milić, B.; Ristić, S. (Rijeka); Latin, V. (Zagreb); ZZ uvić-Butorac, M.; BacZ ić, J. (Rijeka); Petek, M. (Zagreb); Kapović, M. (Rijeka) 22 Erythropoietin as Treatment for Late Hyporegenerative Anemia in Neonates with Rh Hemolytic Disease after in utero Exchange Transfusion Nicaise, C.; Gire, C.; Casha, P.; d’Ercole, C.; Chau, C.; Palix, C. (Marseille) 25 Alpha-Fetoprotein Values in Amniotic Fluid Obtained during Early Amniocentesis (11–13 Weeks) Nwebube, N.I.; Lockitch, G.; Halstead, C. (Vancouver); Johnson, J. (Toronto); Wilson, R.D. (Vancouver) 29 Prenatal Diagnosis of Arthrogryposis multiplex congenita with Increased Nuchal Translucency but without Any Underlying Fetal Neurogenic or Myogenic Pathology Madazl}, R.; Tüysüz, B.; Aksoy, F.; Barbaros, M.; UludagE , S.; Ocak, V. (Istanbul) 34 Prenatal Diagnosis of Lissencephaly (Type II) by Ultrasound and Fast Magnetic Resonance Imaging Kojima, K.; Suzuki, Y.; Seki, K.; Yamamoto, T.; Sato, T.; Tanaka, T.; Suzumori, K. (Nagoya) 37 Expression of C-Type Natriuretic Peptide in Human Placenta and Myometrium in Normal Pregnancies and Pregnancies Complicated by Intrauterine Growth Retardation. Preliminary Results Stepan, H.; Faber, R.; Stegemann, S. (Leipzig); Schultheiss, H.-P.; Walther, T. (Berlin) 42 Early Transvaginal Biometry of Fetal Orbits: A Cross-Sectional Study Guariglia, L.; Rosati, P. (Rome) 48 Uterine Artery Doppler Velocimetry in Patients with Idiopathic Hydramnios Hershkovitz, R.; Sheiner, E.; Furman, B.; Smolin, A.; Hallak, M.; Mazor, M. (Beer-Sheva) 52 First-Trimester Ductus venosus Velocimetry in Relation to Nuchal Translucency Thickness and Fetal Karyotype Zoppi, M.A.; Putzolu, M.; Ibba, R.M.; Floris, M.; Monni, G. (Cagliari) 58 MR Autopsy in Fetuses Huisman, T.A.G.M.; Wisser, J.; Stallmach, T.; Krestin, G.P.; Huch, R.; Kubik-Huch, R.A. (Zürich) No. 2