Refolding of disulfide containing peptides in fusion with thioredoxin

Logashina, Yulia A.; Korolkova, Yuliya V.; Maleeva, Ekaterina E.; Osmakov, Dmitry I.; Козлов, С. А.; Andreev, Yaroslav A.

doi:10.1016/j.mencom.2020.03.028

Cited by 8 publications

(5 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Heterologous production of the recombinant hydrophobic APETx-like peptides is challenging, as they possess multiple disulfide bonds of which the proper closure is sensitive to expression conditions and refolding protocol. The technique, successfully applied for obtaining the recombinant APETx2 [ 43 ], was useful, but required some changes for the correct folding of Hmg1b-4. Two Met residues in the sequence prevented fusion digestion by cyanogen bromide; therefore, the construction contained thioredoxin domain, six His residues, the DDDDK sequence and the target protein ( Figure 1 ).…”

Section: Discussionmentioning

confidence: 99%

“…The folding of Trx-Hmg 1b-4 in prokaryotic cells was difficult, as with other APETx2analogous toxins [43]. To obtain a proper protein structure, the refolding with a glutathione pair was carried out for five days at a temperature of 14 • C. The target peptide was isolated after specific hydrolysis by enteropeptidase using RP-HPLC (Figure 2).…”

Section: Obtaining Recombinant Hmg 1b-4mentioning

confidence: 99%

See 1 more Smart Citation

Anxiolytic, Analgesic and Anti-Inflammatory Effects of Peptides Hmg 1b-2 and Hmg 1b-4 from the Sea Anemone Heteractis magnifica

Климович

Kalina

Kozhevnikova

et al. 2023

Toxins

Self Cite

View full text Add to dashboard Cite

Acid-sensing ion channels (ASICs) have been known as sensors of a local pH change within both physiological and pathological conditions. ASIC-targeting peptide toxins could be potent molecular tools for ASIC-manipulating in vitro, and for pathology treatment in animal test studies. Two sea anemone toxins, native Hmg 1b-2 and recombinant Hmg 1b-4, both related to APETx-like peptides, inhibited the transient current component of human ASIC3-Δ20 expressed in Xenopus laevis oocytes, but only Hmg 1b-2 inhibited the rat ASIC3 transient current. The Hmg 1b-4 action on rASIC3 as a potentiator was confirmed once again. Both peptides are non-toxic molecules for rodents. In open field and elevated plus maze tests, Hmg 1b-2 had more of an excitatory effect and Hmg 1b-4 had more of an anxiolytic effect on mouse behavior. The analgesic activity of peptides was similar and comparable to diclofenac activity in an acid-induced muscle pain model. In models of acute local inflammation induced by λ-carrageenan or complete Freund’s adjuvant, Hmg 1b-4 had more pronounced and statistically significant anti-inflammatory effects than Hmg 1b-2. It exceeded the effect of diclofenac and, at a dose of 0.1 mg/kg, reduced the volume of the paw almost to the initial volume. Our data highlight the importance of a comprehensive study of novel ASIC-targeting ligands, and in particular, peptide toxins, and present the slightly different biological activity of the two similar toxins.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Obtaining Recombinant Hmg 1b-4mentioning

confidence: 99%

Anxiolytic, Analgesic and Anti-Inflammatory Effects of Peptides Hmg 1b-2 and Hmg 1b-4 from the Sea Anemone Heteractis magnifica

Климович

Kalina

Kozhevnikova

et al. 2023

Toxins

Self Cite

View full text Add to dashboard Cite

show abstract

“…The ASIC1a/3 current can only be partly inhibited (~60% by 3 µM concentrations of APETx2). A better result was obtained for ASIC2b/3 heteromers, and the transient current of which was inhibited to approximately 36% of the control amplitude with an IC 50 of 117 nM ( Diochot et al., 2004 ; Logashina et al., 2020 ). It was shown that APETx2 potentiates the activity of rASIC1b at concentrations 30- to 100-fold higher than it inhibits rASIC3 homomers, causing an increased current’s desensitization, with no effect on the rise time.…”

Section: Natural Ligands Of Acid-sensing Ion Channelsmentioning

confidence: 91%

Animal, Herb, and Microbial Toxins for Structural and Pharmacological Study of Acid-Sensing Ion Channels

Osmakov¹,

Khasanov²,

Andreev³

et al. 2020

Front. Pharmacol.

Self Cite

View full text Add to dashboard Cite

Acid-sensing ion channels (ASICs) are of the most sensitive molecular sensors of extracellular pH change in mammals. Six isoforms of these channels are widely represented in membranes of neuronal and non-neuronal cells, where these molecules are involved in different important regulatory functions, such as synaptic plasticity, learning, memory, and nociception, as well as in various pathological states. Structural and functional studies of both wild-type and mutant ASICs are essential for human care and medicine for the efficient treatment of socially significant diseases and ensure a comfortable standard of life. Ligands of ASICs serve as indispensable tools for these studies. Such bioactive compounds can be synthesized artificially. However, to date, the search for such molecules has been most effective amongst natural sources, such as animal venoms or plants and microbial extracts. In this review, we provide a detailed and comprehensive structural and functional description of natural compounds acting on ASICs, as well as the latest information on structural aspects of their interaction with the channels. Many of the examples provided in the review demonstrate the undoubted fundamental and practical successes of using natural toxins. Without toxins, it would not be possible to obtain data on the mechanisms of ASICs’ functioning, provide detailed study of their pharmacological properties, or assess the contribution of the channels to development of different pathologies. The selectivity to different isoforms and variety in the channel modulation mode allow for the appraisal of prospective candidates for the development of new drugs.

show abstract

“…Sevanol was obtained by chemical synthesis [ 18 ]. Peptides APETx2 and mambalgin-2 were produced with heterologous expression in Escherichia coli [ 17 , 19 ]. Lyophilized compounds were dissolved in fresh buffers directly prior to experiments in extracellular solution.…”

Section: Methodsmentioning

confidence: 99%

Retinoic Acid-Differentiated Neuroblastoma SH-SY5Y Is an Accessible In Vitro Model to Study Native Human Acid-Sensing Ion Channels 1a (ASIC1a)

Kalinovskii

Osmakov

Koshelev

et al. 2022

Biology

Self Cite

View full text Add to dashboard Cite

Human neuroblastoma SH-SY5Y is a prominent neurobiological tool used for studying neuropathophysiological processes. We investigated acid-sensing (ASIC) and transient receptor potential vanilloid-1 (TRPV1) and ankyrin-1 (TRPA1) ion channels present in untreated and differentiated neuroblastoma SH-SY5Y to propose a new means for their study in neuronal-like cells. Using a quantitative real-time PCR and a whole-cell patch-clamp technique, ion channel expression profiles, functionality, and the pharmacological actions of their ligands were characterized. A low-level expression of ASIC1a and ASIC2 was detected in untreated cells. The treatment with 10 μM of retinoic acid (RA) for 6 days resulted in neuronal differentiation that was accompanied by a remarkable increase in ASIC1a expression, while ASIC2 expression remained almost unaltered. In response to acid stimuli, differentiated cells showed prominent ASIC-like currents. Detailed kinetic and pharmacological characterization suggests that homomeric ASIC1a is a dominant isoform among the present ASIC channels. RA-treatment also reduced the expression of TRPV1 and TRPA1, and minor electrophysiological responses to their agonists were found in untreated cells. Neuroblastoma SH-SY5Y treated with RA can serve as a model system to study the effects of different ligands on native human ASIC1a in neuronal-like cells. This approach can improve the characterization of modulators for the development of new neuroprotective and analgesic drugs.

show abstract

Refolding of disulfide containing peptides in fusion with thioredoxin

Cited by 8 publications

References 26 publications

Anxiolytic, Analgesic and Anti-Inflammatory Effects of Peptides Hmg 1b-2 and Hmg 1b-4 from the Sea Anemone Heteractis magnifica

Anxiolytic, Analgesic and Anti-Inflammatory Effects of Peptides Hmg 1b-2 and Hmg 1b-4 from the Sea Anemone Heteractis magnifica

Animal, Herb, and Microbial Toxins for Structural and Pharmacological Study of Acid-Sensing Ion Channels

Retinoic Acid-Differentiated Neuroblastoma SH-SY5Y Is an Accessible In Vitro Model to Study Native Human Acid-Sensing Ion Channels 1a (ASIC1a)

Contact Info

Product

Resources

About