RESUMO
Thymic egress is a crucial process for thymocyte maturation, strictly regulated by sphingosine-1-phosphate lyase (S1PL). Recently, cystathionine γ-lyase (CSE), one of the enzymes producing hydrogen sulfide (H2S), has emerged as a vital immune process regulator. However, the molecular connection between CSE, H2S and thymic egress remains largely unexplored. In this study, we investigated the regulatory function of CSE in the thymic egress of immune cells. We showed that genetic knockout of CSE or pharmacological inhibition by CSE enzyme inhibitor NSC4056 or D,L-propargylglycine (PAG) significantly enhanced the migration of mature lymphocytes and monocytes from the thymus to the peripheral blood, and this redistribution effect could be reversed by treatment with NaHS, an exogenous donor of H2S. In addition, the CSE-generated H2S significantly increased the levels of S1P in the peripheral blood, thymus and spleen of mice, suppressed the production of proinflammatory cytokines and rescued pathogen-induced sepsis in cells and in vivo. Notably, H2S or polysulfide inhibited S1PL activity in cells and an in vitro purified enzyme assay. We found that this inhibition relied on a newly identified C203XC205 redox motif adjacent to the enzyme's active site, shedding light on the biochemical mechanism of S1PL regulation. In conclusion, this study uncovers a new function and mechanism for CSE-derived H2S in thymic egress and provides a potential drug target for treating S1P-related immune diseases.
RESUMO
Horisfieldones A (1) and B (2), two dimeric diarylpropanes featuring an unprecedentedly aromatic ring-contracted framework, were isolated from Horsfieldia kingii. Their structures and absolute configurations were determined by the inspection of extensive spectroscopic data and electronic circular dichroism calculations. Molecular modeling analysis, in vitro enzyme-based bioassays, and structure-activity relationship analysis of these isolates revealed that (+)-1 (IC50 = 35.1 ± 3.9 µM, SI > 11.4) could present a new class of human DOPA decarboxylase inhibitor.
Assuntos
Dopa Descarboxilase/farmacologia , Propano/farmacologia , Dicroísmo Circular , Dopa Descarboxilase/química , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Propano/análogos & derivados , Propano/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The human ether-a-go-go-related gene potassium channel (hERG) has an unusual long turret, whose role in recognizing scorpion toxins remains controversial. Here, BmKKx2, the first specific blocker of hERG channel derived from scorpion Mesobuthus martensii, was identified and the turret role of hERG channel was re-investigated using BmKKx2 as a molecular probe. RESULTS: BmKKx2 was found to block hERG channel with an IC50 of 6.7 ± 1.7 nM and share similar functional surface with the known hERG channel inhibitor BeKm-1. The alanine-scanning mutagenesis data indicate that different residue substitutions on hERG channel by alanine decreased the affinities of toxin BmKKx2 by about 10-fold compared with that of wild-type hERG channel, which reveals that channel turrets play a secondary role in toxin binding. Different from channel turret, the pore region of hERG channel was found to exert the conserved and essential function for toxin binding because the mutant hERG-S631A channel remarkably decreased toxin BmKKx2 affinity by about 104-fold. CONCLUSIONS: Our results not only revealed that channel turrets of hERG channel formed an open conformation in scorpion toxin binding, but also enriched the diversity of structure-function relationships among the different potassium channel turrets.
RESUMO
BACKGROUND: Although the basic scorpion K(+) channel toxins (KTxs) are well-known pharmacological tools and potential drug candidates, characterization the acidic KTxs still has the great significance for their potential selectivity towards different K(+) channel subtypes. Unfortunately, research on the acidic KTxs has been ignored for several years and progressed slowly. PRINCIPAL FINDINGS: Here, we describe the identification of nine new acidic KTxs by cDNA cloning and bioinformatic analyses. Seven of these toxins belong to three new α-KTx subfamilies (α-KTx28, α-KTx29, and α-KTx30), and two are new members of the known κ-KTx2 subfamily. ImKTx104 containing three disulfide bridges, the first member of the α-KTx28 subfamily, has a low sequence homology with other known KTxs, and its NMR structure suggests ImKTx104 adopts a modified cystine-stabilized α-helix-loop-ß-sheet (CS-α/ß) fold motif that has no apparent α-helixs and ß-sheets, but still stabilized by three disulfide bridges. These newly described acidic KTxs exhibit differential pharmacological effects on potassium channels. Acidic scorpion toxin ImKTx104 was the first peptide inhibitor found to affect KCNQ1 channel, which is insensitive to the basic KTxs and is strongly associated with human cardiac abnormalities. ImKTx104 selectively inhibited KCNQ1 channel with a K(d) of 11.69 µM, but was less effective against the basic KTxs-sensitive potassium channels. In addition to the ImKTx104 toxin, HeTx204 peptide, containing a cystine-stabilized α-helix-loop-helix (CS-α/α) fold scaffold motif, blocked both Kv1.3 and KCNQ1 channels. StKTx23 toxin, with a cystine-stabilized α-helix-loop-ß-sheet (CS-α/ß) fold motif, could inhibit Kv1.3 channel, but not the KCNQ1 channel. CONCLUSIONS/SIGNIFICANCE: These findings characterize the structural and functional diversity of acidic KTxs, and could accelerate the development and clinical use of acidic KTxs as pharmacological tools and potential drugs.
Assuntos
Canais de Potássio/química , Venenos de Escorpião/química , Escorpiões/química , Sequência de Aminoácidos , Animais , Biologia Computacional , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Filogenia , Canais de Potássio/fisiologia , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nM for recombinant LmKTT-1a, 160 nM for LmKTT-1b, 124 nM for LmKTT-1c, 136 nM for BmKTT-1, 420 nM for BmKTT-2, 760 nM for BmKTT-3, and 107 nM for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ~50-80% of Kv1.3 channel currents at a concentration of 1 µM. The exception was rBmKTT-3, which had weak activity. The IC(50) values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ~129.7, 371.3, and 6.2 nM, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel-interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases.