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001-es BibID:	BIBFORM102212
035-os BibID:	(cikkazonosító)115023 (WoS)000793623600002 (Scopus)85127510848
Első szerző:	Csóti Ágota (biológus)
Cím:	sVmKTx, a transcriptome analysis-based synthetic peptide analogue of Vm24, inhibits Kv1.3 channels of human T cells with improved selectivity / Csoti Agota, del Carmen Nájera Meza Rosby, Bogár Ferenc, Tajti Gabor, Szanto Tibor G., Varga Zoltan, Gurrola Georgina B., Tóth Gábor K., Possani Lourival D., Panyi Gyorgy
Dátum:	2022
ISSN:	0006-2952
Megjegyzések:	Kv1.3 K+ channels play a central role in the regulation of T cell activation and Ca2+ signaling under physiological and pathophysiological conditions. Peptide toxins targeting Kv1.3 have a significant therapeutic potential in the treatment of autoimmune diseases; thus, the discovery of new toxins is highly motivated. Based on the transcriptome analysis of the venom gland of V. mexicanus smithi a novel synthetic peptide, sVmKTx was generated, containing 36 amino acid residues. sVmKTx shows high sequence similarity to Vm24, a previously characterized peptide from the same species, but contains a Glu at position 32 as opposed to Lys32 in Vm24. Vm24 inhibits Kv1.3 with high affinity (Kd = 2.9 pM). However, it has limited selectivity (~1,500-fold) for Kv1.3 over hKv1.2, hKCa3.1, and mKv1.1. sVmKTx displays reduced Kv1.3 affinity (Kd = 770 pM) but increased selectivity for Kv1.3 over hKv1.2 (~9,000-fold) as compared to Vm24, other channels tested in the panel (hKCa3.1, hKv1.1, hKv1.4, hKv1.5, rKv2.1, hKv11.1, hKCa1.1, hNav1.5) were practically insensitive to the toxin at 2.5 ?M. Molecular dynamics simulations showed that introduction of a Glu instead of Lys at position 32 led to a decreased structural fluctuation of the N-terminal segment of sVmKTx, which may explain its increased selectivity for Kv1.3. sVmKTx at 100 nM concentration decreased the expression level of the Ca2+ -dependent T cell activation marker, CD40 ligand. The high affinity block of Kv1.3 and increased selectivity over the natural peptide makes sVmKTx a potential candidate for Kv1.3 blockade-mediated treatment of autoimmune diseases.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Kv1.3 Toxin-channel interaction T-cell activation Patch-clamp Ion channel selectivity Scorpion toxin
Megjelenés:	Biochemical Pharmacology. - 199 (2022), p. 1-14. -
További szerzők:	del Carmen Nájera Meza, Rosby Bogár Ferenc Tajti Gábor (1988-) (gyógyszerész, biofizikus, sejtbiológus) Szántó Gábor Tibor (1980-) (vegyész) Varga Zoltán (1969-) (biofizikus, szakfordító) Gurrola-Briones, Georgina Tóth Gábor K. Possani, Lourival Domingos Panyi György (1966-) (biofizikus)
Pályázati támogatás:	143071 OTKA K119417 OTKA K132906 OTKA EFOP-3.6.2-16-2017-00006 EFOP GINOP-2.3.2-15-2016-00044 GINOP PRONACE303045 from National Conseil of Science and Technology of Mexico Egyéb Ministry of Human Capacities, Hungary grant, TKP-2020 Egyéb
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001-es BibID:	BIBFORM098765
035-os BibID:	(cikkazonosító)733610 (scopus)85117131062 (wos)000707249000001
Első szerző:	Naseem, Muhammad Umair (biofizikus, molekuláris biológus)
Cím:	Optimization of Pichia pastoris Expression System for High-Level Production of Margatoxin / Naseem Muhammad Umair, Tajti Gabor, Gaspar Attila, Szanto Tibor G., Borrego Jesús, Panyi Gyorgy
Dátum:	2021
ISSN:	1663-9812
Megjegyzések:	Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1?Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ? 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (Kd for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.
Tárgyszavak:	Orvostudományok Gyógyszerészeti tudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Pichia pastoris patch-clamp margatoxin recombinant expression Kv1.3 blocker CD4+ TEM cells
Megjelenés:	Frontiers in Pharmacology. - 12 (2021), p. 1-18. -
További szerzők:	Tajti Gábor (1988-) (gyógyszerész, biofizikus, sejtbiológus) Gáspár Attila (1970-) (vegyész, kémikus) Szántó Gábor Tibor (1980-) (vegyész) Borrego, Jesús Panyi György (1966-) (biofizikus)
Pályázati támogatás:	K119417 OTKA EFOP-3.6.1-16-2016-00022 EFOP GINOP2.3.2-15-2016-00044 GINOP
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001-es BibID:	BIBFORM091018
Első szerző:	Tajti Gábor (gyógyszerész, biofizikus, sejtbiológus)
Cím:	Immunomagnetic separation is a suitable method for electrophysiology and ion channel pharmacology studies on T cells / Gabor Tajti, Tibor Gabor Szanto, Agota Csoti, Greta Racz, César Evaristo, Peter Hajdu, Gyorgy Panyi
Dátum:	2021
ISSN:	1933-6950 1933-6969
Megjegyzések:	Ion channels play pivotal role in the physiological and pathological function of immune cells. As immune cells represent a functionally diverse population, subtype-specific functional studies, such as single-cell electrophysiology require proper subset identification and separation. Magneticactivated cell sorting (MACS) techniques provide an alternative to fluorescence-activated cell sorting (FACS), however, the potential impact of MACS-related beads on the biophysical and pharmacological properties of the ion channels were not studied yet. We studied the aforementioned properties of the voltage-gated Kv1.3 K+ channel in activated CD4+ T-cells as well as the membrane capacitance using whole-cell patch-clamp following immunomagnetic positive separation, using the REAlease? kit. This kit allows three experimental configurations: bead-bound configuration, bead-free configuration following the removal of magnetic beads, and the labelfree configuration following removal of CD4 recognizing antibody fragments. As controls, we used FACS separation as well as immunomagnetic negative selection. The membrane capacitance and of the biophysical parameters of Kv1.3 gating, voltage-dependence of steady-state activation and inactivation kinetics of the current were not affected by the presence of MACS-related compounds on the cell surface. We found subtle differences in the activation kinetics of the Kv1.3 current that could not be explained by the presence of MACS-related compounds. Neither the equilibrium block of Kv1.3 by TEA+ or charybdotoxin (ChTx) nor the kinetics of ChTx block are affected by the presence of the magnetics beads on the cell surface. Based on our results MACS is a suitable method to separate cells for studying ion channels in non-excitable cells, such as T-lymphocytes.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Kv1.3 cd4+ T-cell magneticactivated cell sorting macs immunomagnetic separation fluorescenceactivated cell sorting facs
Megjelenés:	Channels. - 15 : 1 (2021), p. 53-66. -
További szerzők:	Szántó Gábor Tibor (1980-) (vegyész) Csóti Ágota (1989-) (biológus) Rácz Gréta Evaristo, César Hajdu Péter (1975-) (biofizikus) Panyi György (1966-) (biofizikus)
Pályázati támogatás:	OTKA K119417 Egyéb EFOP-3.6.2-16-2017-00006 EFOP GINOP-2.3.2-15-2016-00015 GINOP NKFIH K128525 Egyéb Bolyai Research Fellowship Egyéb
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