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1.

001-es BibID:BIBFORM105430
035-os BibID:(cikkazonosító)1215 (WoS)000895364600001
Első szerző:Borrego, Jesús
Cím:Recombinant Expression in Pichia pastoris System of Three Potent Kv1.3 Channel Blockers : Vm24, Anuroctoxin, and Ts6 / Borrego Jesús, Naseem Muhammad Umair, Sehgal Al Nasar Ahmed, Panda Lipsa Rani, Shakeel Kashmala, Gaspar Attila, Nagy Cynthia, Varga Zoltan, Panyi Gyorgy
Dátum:2022
ISSN:2309-608X
Megjegyzések:The Kv1.3 channel has become a therapeutic target for the treatment of various diseases. Several Kv1.3 channel blockers have been characterized from scorpion venom; however, extensive studies require amounts of toxin that cannot be readily obtained directly from venoms. The Pichia pastoris expression system provides a cost-effective approach to overcoming the limitations of chemical synthesis and E. coli recombinant expression. In this work, we developed an efficient system for the production of three potent Kv1.3 channel blockers from different scorpion venoms: Vm24, AnTx, and Ts6. Using the Pichia system, these toxins could be obtained in sufficient quantities (Vm24 1.6 mg/L, AnTx 46 mg/L, and Ts6 7.5 mg/L) to characterize their biological activity. A comparison was made between the activity of tagged and untagged recombinant peptides. Tagged Vm24 and untagged AnTx are nearly equivalent to native toxins in blocking Kv1.3 (Kd = 4.4 pM and Kd = 0.72 nM, respectively), whereas untagged Ts6 exhibits a 53-fold increase in Kd (Kd = 29.1 nM) as compared to the native peptide. The approach described here provides a method that can be optimized for toxin production to develop more selective and effective Kv1.3 blockers with therapeutic potential.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Pichia pastoris
recombinant peptides
scorpion venoms
Kv1.3 blockers
Megjelenés:Journal of Fungi. - 8 : 11 (2022), p. 1-15. -
További szerzők:Naseem, Muhammad Umair (1993-) (biofizikus, molekuláris biológus) Sehgal, Al Nasar Ahmed Panda, Lipsa Rani Shakeel, Kashmala Gáspár Attila (1970-) (vegyész, kémikus) Nagy Cynthia (1994-) Varga Zoltán (1969-) (biofizikus, szakfordító) Panyi György (1966-) (biofizikus)
Pályázati támogatás:K143071
OTKA
K127931
OTKA
Stipendium Hungaricum Scholarship by the Tempus Public Foundation
Egyéb
Richter Gedeon Talentum Foundation
Egyéb
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Intézményi repozitóriumban (DEA) tárolt változat
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2.

001-es BibID:BIBFORM110227
035-os BibID:(cikkazonosító)6216
Első szerző:Cozzolino, Marco (biológus)
Cím:The Voltage-Gated Hv1 H⁺ Channel Is Expressed in Tumor-Infiltrating Myeloid-Derived Suppressor Cells / Cozzolino Marco, Gyöngyösi Adrienn, Korpos Eva, Gogolak Peter, Naseem Muhammad Umair, Kállai Judit, Lanyi Arpad, Panyi Gyorgy
Dátum:2023
ISSN:1422-0067
Megjegyzések:Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b+/Ly6G+) and Mo-MDSCs (CD11b+/Ly6C+) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b+/Ly6G+ PMN-MDSCs and CD11b+/Ly6C+/F4/80?/MHCII? Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H+ channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H+ selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H+ currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Myeloid-derived suppressor cell
Hv1 proton channel
tumor microenvironment
Megjelenés:International Journal Of Molecular Sciences. - 24 : 7 (2023), p. 1-24. -
További szerzők:Gyöngyösi Adrienn (1982-) (biológus) Korpos Éva Gogolák Péter (1968-) (biológus, immunológus) Naseem, Muhammad Umair (1993-) (biofizikus, molekuláris biológus) Kállai Judit (1983-) (molekuláris biológus) Lányi Árpád (1962-) (biológus, immunológus) Panyi György (1966-) (biofizikus)
Pályázati támogatás:K119417
OTKA
K131708
OTKA
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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3.

001-es BibID:BIBFORM106364
035-os BibID:(cikkazonosító)41
Első szerző:Naseem, Muhammad Umair (biofizikus, molekuláris biológus)
Cím:Characterization and Chemical Synthesis of Cm39 (α-KTx 4.8) : a Scorpion Toxin That Inhibits Voltage-Gated K+ Channel KV1.2 and Small- and Intermediate-Conductance Ca2+-Activated K+ Channels KCa2.2 and KCa3.1 / Muhammad Umair Naseem, Georgina Gurrola-Briones, Margarita R. Romero-Imbachi, Jesus Borrego, Edson Carcamo-Noriega, José Beltrán-Vidal, Fernando Z. Zamudio, Kashmala Shakeel, Lourival Domingos Possani, Gyorgy Panyi
Dátum:2023
ISSN:2072-6651
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Cm39
scorpion toxin
Centruroides margaritatus
KV1.2
KCa2.2
KCa3.1
electrophysiology
Ca2+-activated channel
Megjelenés:Toxins. - 15 : 1 (2023), p. 1-21. -
További szerzők:Gurrola-Briones, Georgina Romero-Imbachi, Margarita R. Borrego, Jesús Carcamo-Noriega, Edson Beltrán-Vidal, José Zamudio, Fernando Z. Shakeel, Kashmala Possani, Lourival Domingos Panyi György (1966-) (biofizikus)
Pályázati támogatás:K143071
OTKA
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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4.

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
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Intézményi repozitóriumban (DEA) tárolt változat
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5.

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
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Intézményi repozitóriumban (DEA) tárolt változat
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6.

001-es BibID:BIBFORM105306
035-os BibID:(scopus)85141634111
Első szerző:Wai, Dorothy C. C.
Cím:A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel Kv1.3 / Wai Dorothy C. C., Naseem Muhammad Umair, Mocsár Gábor, Babu Reddiar Sanjeevini, Pan Yijun, Csoti Agota, Hajdu Peter, Nowell Cameron, Nicolazzo Joseph A., Panyi Gyorgy, Norton Raymond S.
Dátum:2022
ISSN:1043-1802
Megjegyzések:Upregulation of the voltage-gated potassium channel KV1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of KV1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds KV1.3 with high affinity (IC50 of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5?HsTX1[R14A] retains activity against KV1.3 (IC50 ? 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5?HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5?HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5?HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5?HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5?HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Fluorescence
Imaging probes
Peptides and proteins
Potassium
Selectivity
Megjelenés:Bioconjugate Chemistry. - 33 : 11 (2022), p. 2197-2212. -
További szerzők:Naseem, Muhammad Umair (1993-) (biofizikus, molekuláris biológus) Mocsár Gábor (1981-) (biofizikus) Babu Reddiar, Sanjeevini Pan, Yijun Csóti Ágota (1989-) (biológus) Hajdu Péter (1975-) (biofizikus) Nowell, Cameron Nicolazzo, Joseph A. Panyi György (1966-) (biofizikus) Norton, Raymond S.
Pályázati támogatás:K143071
OTKA
K128525
OTKA
EFOP-3.6.1-16-2016-00022
EFOP
GINOP-2.3.2-15-2016-00015
GINOP
Stipendium Hungaricum Scholarship by the Tempus Public Foundation
Egyéb
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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