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Microbial Biotechnology (MB) | Cell Culture and Biomedical Engineering
Microbiol. Biotechnol. Lett. 2023; 51(3): 243-249
Division of Biohealthcare, College of Health Science, Daejin University, Pocheon 11159, Republic of Korea
Correspondence to :
Ho Kim, email@example.com
We recently found that the insect-derived antimicrobial peptide CopA3 (LLCIALRKK) directly binds to and inhibits the proteolytic activation of caspases, which play essential roles in apoptotic processes. However, the mechanism of CopA3 binding to caspases remained unknown. Here, using recombinant GST-caspase-3 and -6 proteins, we investigated the mechanism by which CopA3 binds to caspases. We showed that replacement of cysteine in CopA3 with alanine caused a marked loss in its binding activity towards caspase-3 and -6. Exposure to DTT, a reducing agent, also diminished their interaction, suggesting that this cysteine plays an essential role in caspase binding. Experiments using deletion mutants of CopA3 showed that the last N-terminal leucine residue of CopA3 peptide is required for binding of CopA3 to caspases, and that C-terminal lysine and arginine residues also contribute to their interaction. These conclusions are supported by binding experiments employing direct addition of CopA3 deletion mutants to human colonocyte (HT29) extracts containing endogenous caspase-3 and -6 proteins. In summary, binding of CopA3 to caspases is dependent on a cysteine in the intermediate region of the CopA3 peptide and a leucine in the N-terminal region, but that both an arginine and two adjacent lysines in the C-terminal region of CopA3 also contribute. Collectively, these results provide insight into the interaction mechanism and the high selectivity of CopA3 for caspases.
Keywords: Antimicrobial peptide, caspase inhibitor, apoptosis, peptide binding
Apoptosis associated with diverse disease states is known to be highly dependent on caspase activation [1−5], motivating translational investigations that have led to the development of a number of caspase inhibitors . Caspase inhibitors are divided into two broad categories: natural product inhibitors and synthetic inhibitors. Natural product inhibitors include the cowpox virus protein CrmA (cytokine response modifier A) [2−5], the baculovirus protein P35, and the evolutionarily conserved protein IAP (inhibitor of apoptosis). CrmA, P35, and IAPs are known to bind as pseudosubstrates to the active catalytic sites of target caspases, inhibiting their catalytic activity [2−5]. Synthetic inhibitors are primarily peptides that range in size from a single O-methylaspartate residue (e.g., Boc-Asp-FMK) to tri-peptides (e.g., z-VAD-FMK) and tetrapeptides (e.g., YVAD-FMK) . Aldehyde-linked peptide inhibitors are reversible, whereas peptides linked to acylomethyl ketones and methyl ketones are irreversible [8, 9]. It has been proposed that, in the transition state, irreversible inhibitors bind via a thioester linkage to an oxyanion, with the carbonyl oxygen occupying an oxyanion hole. However, these caspase inhibitors exhibit low selectivity and are known to cause severe side effects [6, 8, 9]. Therefore, although many caspase inhibitors are available, as described above, the development of new inhibitors with distinct modes of action and caspase binding, as well as reduced side effects, remains a crucial need.
In our previous study, we showed that CopA3, a Korean dung beetle-derived antimicrobial peptide [10−12], exerts inhibitory effects against cell apoptosis through direct binding to caspases . Moreover, we found that the mechanism by which CopA3 inhibits caspases is distinct from that of many of the caspase inhibitors indicated above . CopA3 binds to caspases and inhibits the proteolytic cleavage required for their activation, thereby effectively blocking the binding of upstream caspases . For example, CopA3 binds to caspase-3, forming a complex that does not allow proteolytic cleavage of caspase-3 by the upstream caspase, caspase-8, resulting in blockade of caspase-3 activation . However, despite the scientific impact of CopA3’s ability to inhibit caspases and subsequent apoptosis, the mechanism by which CopA3 binds caspases has yet to be fully elucidated.
The antimicrobial peptide CopA3 is a D-type dimer with the amino acid sequence LLCIALRKK . The peptide corresponds to Leu22 to Lys30 in the 43-amino acid, mature coprisin peptide (VTCDVLSFEAKGIAVNHSACALHCIALRKKGGSCQNGVCVCRN-NH2), a natural peptide from the dung beetle . NMR experiments have revealed that the Leu22−Lys30 region adopts an α-helix structure. During development of CopA3, the histidine in the N-terminal region of this sequence in coprisin was replaced with a leucine because the partial hydrophobicity it introduces is known to potentially affect antimicrobial activity. The antimicrobial peptide CopA3 shares several features with defensin, including its short length (9-mer peptide), amphipathic α-helical structure, positively charged amino acids, and an internal cysteine. Structurally, CopA3 is composed of two leucines in the N-terminal region that contribute to increased hydrophobicity, two hydrophilic lysines in the C-terminal region, and a cysteine in the intermediate region that forms a cysteine-disulfide bridge that is crucial for the fundamental peptide architecture.
Based on our previous finding that CopA3 directly binds to caspases and inhibits their proteolytic activation , we here investigated the mechanism that mediates the interaction of CopA3 with caspases and is responsible for the high selectivity of this binding.
The CopA3 peptide (LLCIAALRKK, homodimer), isolated from the Dung beetle,
Human colorectal adenocarcinoma HT29 cells were grown in McCoy's 5A medium (Invitrogen, USA) supplemented with 10% FBS (fetal bovine serum), 1% penicillin (Gibco, USA) and 1% streptomycin (Invitrogen), and cultured at 37℃ in a humidified incubator containing 5% CO2 . The polyclonal antibodies against caspase-3, and -6 were purchased from Cell Signaling Technology (USA). The antibody against β-actin, the DTT (dithiothreitol) were purchased from Sigma-Aldrich (USA).
The plasmids pGST-caspase-3 and pGST-caspase-6 were constructed by cloning into the commercial vector pGEX4T1 (Pharmacia, Sweden). GST-chimeric proteins were purified to homogeneity by glutathione-agarose affinity chromatography (Sigma-Aldrich) according to the manufacturer’s instructions . GST-caspase-3 (1 μg) and GST-caspase-6 (1 μg) were incubated with CopA3 (100 ng) in reaction buffer (50 Mm HEPES, 50 mM NaCl, 0.1% CHAPS, 10 mM EDTA, 5% glycerol, 10 mM DTT) at 37℃ for 30 min. After 1 h, the mixtures were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 15% gels, after which blots were incubated with antibodies against caspase-3 or -6.
Crude cell extracts were prepared from HT29 cells and divided into aliquots. Aliquots (20 μg) containing inactive forms of caspase-3 and caspase-6 were incubated with CopA3 (1 μg) in reaction buffer (50 mM HEPES, 50 mM NaCl, 0.1% CHAPS, 10 mM EDTA, 5% glycerol, 10 mM DTT) at 37℃ for 30 min. After 1 h, the mixtures were resolved by SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) on 15% gels, after which blots were incubated with antibodies against caspase-3 or -6 .
The results are presented as mean values ± SEM. Data were analyzed using the SIGMA-STAT professional statistics software package (Jandel Scientific Software, USA). Analyses of variance with protected t-tests were used for intergroup comparisons.
We previously reported that CopA3 binds to caspases and that this causes a distinct shift in the migration of caspases on SDS-PAGE gels, reflecting the increased molecular weight of the CopA3-caspase complex . Thus, immunoblot analysis can detect direct binding of CopA3 to caspases, making this a facile experimental approach for evaluating caspase-CopA3 interactions. Using this approach, we investigated how CopA3 binds to caspases, first testing the role of the cysteine in CopA3. To this end, we prepared recombinant GSTcaspase-3 and GST-caspase-6 fusion proteins and conducted
Given that one or more cysteines in the caspase protein are binding sites for CopA3, these findings collectively suggest three possible concepts: (1) CopA3 binds to caspases at multiple sites, preventing the substrate caspase from entering the initiator caspase’s active site; (2) multiple CopA3 peptides bind to downstream caspases, hindering the enzymatic activity of the upstream caspase; and (3) binding of multiple CopA3 peptides to caspase alters the 3-dimensional structure of the substrate caspase such that it blocks binding of upstream caspases to substrate caspases.
We found that a cysteine in the CopA3 peptide is critical for CopA3 binding to caspases, but this does not clarify how CopA3 binds selectively to caspases because many intracellular proteins contain cysteine. To further investigate the specificity of CopA3-caspase binding, we tested whether other structurally similar, cysteinecontaining antimicrobial peptides, namely HaA4, Lum-1, Lum-2 and Lum-3, bind to caspases and subsequently affect their electrophoretic mobility (Fig. 2A). To this end, we incubated CopA3 or the indicated antimicrobial peptides with recombinant GST-caspase-3 or GSTcaspase-6 fusion proteins for 30 h and examined their direct binding by immunoblot analysis. As shown in Fig. 2B, addition of CopA3 caused a shift in the migration of GST-caspase-3 fusion protein, as expected. However, none of the peptides HaA4, Lum-1, Lum-2 and Lum-3, affected caspase-3 migration on SDS-PAGE gels despite having similar sequences that include a cysteine residue (Fig. 2A). Similar results were obtained in binding assays using GST-caspase-6 fusion protein (Fig. 2C). Collectively, these results suggest that cysteine does not uniquely confer the specific binding of CopA3 for caspases and that additional CopA3 amino acids are required.
Next, we further determined which amino acids in CopA3 are necessary for specific interactions with caspases. To explore this, we prepared CopA3-deletion mutants and performed immunoblot analysis-based binding assays. As shown in Fig. 3A, a leucine-deleted CopA3 mutant (LLCIALRKK) lost the ability to cause a shift in the electrophoretic mobility of GST-caspase-3, despite the presence of a cysteine in the sequence. CopA3 with a deletion of the C-terminal lysine residue (LLCIALRKK) showed a slight reduction in the ability to promote a shift in the electrophoretic mobility of GSTcaspase-3, a reduction that was additively increased by deletion of the second C-terminal lysine residues in CopA3 (LLCIALRKK). Deletion of the arginine in the Cterminal region together with the two adjacent lysines in CopA3 (LLCIALRKK) caused complete inhibition of CopA3-caspase-3 binding, as evidenced by the absence of an electrophoretic mobility shift in GST-caspase-3. Stepwise deletion of additional C-terminal residues (lysine and alanine) caused no further reduction. Similar results were obtained for the GST-caspase-6 fusion protein (Fig. 3B). Taken together, our results suggest that the internal cysteine residue together with the Nterminal leucine on CopA3 are absolutely required for binding to caspases, and that the C-terminal lysine residues, and to a lesser extent the adjacent arginine residue, further contribute to the selective, high-affinity binding of CopA3 to caspases and not to other intracellular proteins.
The importance of leucine (L) in the CopA3 sequence is particularly intriguing, given the role of the L
Finally, to confirm that the changes in binding activity of CopA3-deletion mutants for recombinant caspases also applies to native forms of caspases, we performed binding assays in which each CopA3-deletion mutant was incubated with extracts of HT29 cells, which may contain both activated, cleaved forms of caspases and inactive proforms of caspases. As expected, direct addition of wild-type CopA3 to cell extracts markedly enhanced the shift in the migration of predicted caspase-3 and -6 bands (Fig. 4A). The fact that a low concentration of added CopA3 caused a robust shift in the electrophoretic mobility of caspases in whole-cell extracts containing many intracellular proteins clearly suggests that CopA3 strongly and selectively binds to caspases. Next, we incubated CopA3 (1 μg) or the prepared CopA3-deletion mutants (1 μg) with HT29 cell extracts (10 μg) for 30 min and assessed binding. Consistent with results shown in Fig. 3, addition of the leucine-deleted CopA3 mutant (LLCIALRKK) caused a marked reduction in the electrophoretic mobility shift of caspase-3 (Fig. 4B) and -6 (Fig. 4C). The C-terminal lysine-deleted CopA3 mutant (LLCIALRKK) also strongly reduced caspase-3 migration, but this inhibition was incomplete (Figs. 4B and 4C). Complete inhibition was observed upon deletion of the C-terminal arginine and two adjacent lysines in CopA3 (LLCIALRKK) (Figs. 4B and C), results that are consistent with findings presented in Fig. 3.
Collectively, these results suggest that CopA3 represents a new concept in specific caspase inhibition. Most caspase inhibitors bind as pseudosubstrates to the active catalytic sites of caspases, inhibiting their catalytic activity against their downstream caspase substrates. In contrast, our previous study, taken together with the present study, reveal a distinct mechanism for CopA3, showing that it binds to caspases, preventing subsequent activating proteolytic cleavage by upstream caspases. This represents a completely different paradigm for caspase inhibition compared with previously developed caspase inhibitors.
CopA3, a peptide isolated from the dung beetles (
This work was supported by the Daejin University Research Grants in 2023.
The author has no financial conflicts of interest to declare.
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