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001-es BibID:	BIBFORM103838
035-os BibID:	(scopus)85132050297 (cikkazonosító)e202213146
Első szerző:	Naseem, Muhammad Umair (biofizikus, molekuláris biológus)
Cím:	Cm28, a scorpion toxin having a unique primary structure, inhibits KV1.2 and KV1.3 with high affinity / Naseem Muhammad Umair, Carcamo-Noriega Edson, Beltrán-Vidal José, Borrego Jesus, Szanto Tibor G., Zamudio Fernando Z., Delgado-Prudencio Gustavo, Possani Lourival D., Panyi Gyorgy
Dátum:	2022
ISSN:	0022-1295 1540-7748
Megjegyzések:	The Cm28 in the venom of Centruroides margaritatus is a short peptide consisting of 27 amino acid residues with a mol wt of 2,820 D. Cm28 has <40% similarity with other known ?-KTx from scorpions and lacks the typical functional dyad (lysine?tyrosine) required to block KV channels. However, its unique sequence contains the three disulfide-bond traits of the ?-KTx scorpion toxin family. We propose that Cm28 is the first example of a new subfamily of ?-KTxs, registered with the systematic number ?-KTx32.1. Cm28 inhibited voltage-gated K+ channels KV1.2 and KV1.3 with Kd values of 0.96 and 1.3 nM, respectively. There was no significant shift in the conductance?voltage (G-V) relationship for any of the channels in the presence of toxin. Toxin binding kinetics showed that the association and dissociation rates are consistent with a bimolecular interaction between the peptide and the channel. Based on these, we conclude that Cm28 is not a gating modifier but rather a pore blocker. In a selectivity assay, Cm28 at 150 nM concentration (>100? Kd value for KV1.3) did not inhibit KV1.5, KV11.1, KCa1.1, and KCa3.1 K+ channels; NaV1.5 and NaV1.4 Na+ channels; or the hHV1 H+ channel but blocked ?27% of the KV1.1 current. In a biological functional assay, Cm28 strongly inhibited the expression of the activation markers interleukin-2 receptor and CD40 ligand in anti-CD3?activated human CD4+ effector memory T lymphocytes. Cm28, due to its unique structure, may serve as a template for the generation of novel peptides targeting KV1.3 in autoimmune diseases.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk KV1.2 KV1.3 scorpion toxin
Megjelenés:	Journal Of General Physiology. - 154 : 8 (2022), p. 1-18. -
További szerzők:	Carcamo-Noriega, Edson Beltrán-Vidal, José Borrego, Jesús Szántó Gábor Tibor (1980-) (vegyész) Zamudio, Fernando Z. Delgado-Prudencio, Gustavo Possani, Lourival Domingos Panyi György (1966-) (biofizikus)
Pályázati támogatás:	K143071 OTKA K142612 OTKA K132906 OTKA CONACYT 303045 Egyéb Tempus Public Foundation Egyéb
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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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
Internet cím:	Szerző által megadott URL DOI Intézményi repozitóriumban (DEA) tárolt változat
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