Article Search
닫기

Microbiology and Biotechnology Letters

Research Article(보문)

View PDF

Microbial Biotechnology (MB)  |  Cell Culture and Biomedical Engineering

Microbiol. Biotechnol. Lett. 2023; 51(3): 243-249

https://doi.org/10.48022/mbl.2305.05008

Received: May 22, 2023; Revised: June 26, 2023; Accepted: June 28, 2023

The Specific Binding Mechanism of the Antimicrobial Peptide CopA3 to Caspases

Ho Kim*

Division of Biohealthcare, College of Health Science, Daejin University, Pocheon 11159, Republic of Korea

Correspondence to :
Ho Kim,          hokim@daejin.ac.kr

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

Graphical Abstract


Apoptosis associated with diverse disease states is known to be highly dependent on caspase activation [15], motivating translational investigations that have led to the development of a number of caspase inhibitors [6]. 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) [25], 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 [25]. 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) [7]. 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 [1012], exerts inhibitory effects against cell apoptosis through direct binding to caspases [13]. Moreover, we found that the mechanism by which CopA3 inhibits caspases is distinct from that of many of the caspase inhibitors indicated above [13]. CopA3 binds to caspases and inhibits the proteolytic cleavage required for their activation, thereby effectively blocking the binding of upstream caspases [13]. 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 [13]. 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 [14]. The peptide corresponds to Leu22 to Lys30 in the 43-amino acid, mature coprisin peptide (VTCDVLSFEAKGIAVNHSACALHCIALRKKGGSCQNGVCVCRN-NH2), a natural peptide from the dung beetle [14]. 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 [13], we here investigated the mechanism that mediates the interaction of CopA3 with caspases and is responsible for the high selectivity of this binding.

Synthesis of CopA3, CopA3-cysteine mutant, and other antimicrobial peptides

The CopA3 peptide (LLCIAALRKK, homodimer), isolated from the Dung beetle, Copris tripartitus), and a CopA3-CA mutant, in which a cysteine in CopA3 was replaced with alanine, were synthesized by AnyGen (Korea) [15]. CopA3-deletion mutants (a leucine-deleted mutant: LLCIALRKK, a lysine-deleted mutant: LLCIALRKK, a two lysine deleted-mutant: LLCIALRKK, an arginine and two lysines-deleted mutant: LLCIALRKK, an C-terminal four amino acids-deleted CopA3 mutant: LLCIALRKK, an C-terminal five amino acids-deleted CopA3 mutant: LLCIALRKK, an C-terminal six amino acids-deleted CopA3 mutant: LLCIALRKK). These peptides were purified by reverse-phase high-performance liquid chromatography (HPLC) using a Capcell Pak C18 column (Shiseido, Japan) and eluted with a linear gradient of water-acetonitrile (0–80%) containing 0.1% trifluoroacetic acid (45% recovery). The identity of the peptide was confirmed by electrospray ionization (ESI) mass spectrometry (Platform II; Micromass, United Kingdom). The antimicrobial peptides HaA4 (IGGYCSWLRLIGGY, homodimer), isolated from the ladybug Harmonia axyridis [16], and the Lumbricusins Lum-1 (RNRRWCIDQQA), Lum-2 (QLICWRRK) and Lum-3 (QLICWRRNR), isolated as homodimers from the earthworm Lumbricus terrestris, were also purified as indicated above [1719].

Cell culture and reagents

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 [20]. 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).

Recombinant protein production and binding assay

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 [21]. 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.

Measurement of CopA3 binding to caspases in cell extracts

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 [13].

Statistical analysis

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.

Cysteine in CopA3 is required for direct binding of CopA3 to caspases

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 [13]. 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 in vitro binding assay using CopA3 or a CopA3-CA mutant in which alanine was substituted for the original cysteine. As expected, addition of CopA3 caused a concentration-dependent shift in the migration of GST-caspase-3 (Fig. 1A) and GST-caspase-6 (Fig. 1B) fusion proteins on SDS-PAGE gels. However, the CopA3-CA mutant did not cause a shift in the migration of either caspase, indicating no interaction (Fig. 1C), results similar to those obtained with a GST control protein (data not shown) [13]. These results suggest that CopA3 binds to caspase-3 and -6 through a disulfide bond. To confirm these results, we tested whether CopA3 binding to caspases was affected by the reducing agent, dithiothreitol (DTT) [22]. To this end, we incubated GST-caspase-3 and -6 proteins with CopA3 in the presence of 100 mM DTT. Reducing the disulfide bond with DTT completely abrogated the CopA3-induced shifts in the electrophoretic mobilities of caspase-3 (Fig. 1D) and caspase-6 (Fig. 1E). These results indicate that the binding of CopA3 to caspases is highly dependent on the cysteine in the intermediate region of CopA3, implying that CopA3 binds to caspases via a disulfide bond.

Figure 1.Role of the resident cysteine in CopA3 binding to caspases. Binding assays using different concentrations of CopA3 and 1 μg recombinant GST-caspase-3 and -6 proteins, purified using a glutathione-agarose affinity spin column. (A, B) CopA3 (0, 0.1, 0.5, 1 μg) was incubated with GST-caspase-3 (A) or GST-caspase-6 (B) in reaction buffer at 37℃ for 15 min. Reaction mixtures were then resolved by SDS-PAGE on 10% gels, after which blots were probed with the appropriate anticaspase primary antibody (1:2000 dilution). The presented results are representative of three independent experiments. (C) CopA3 (100 ng) or CopA3-CA (100 ng) was incubated with GST-caspase-3 (1 μg) or GST-caspase-6 (1 μg) in reaction buffer at 37℃ for 1 h. Reaction mixtures were then resolved by SDSPAGE on 10% gels, after which blots were probed with specific antibodies as described above. (d, e) CopA3 (100 ng) was incubated with 1 μg GST-caspase-3 (D) or GST-caspase-6 (E) in reaction buffer at 37℃ for 30 min in the presence or absence of DTT (100 mM) prior to SDS-PAGE and immunoblotting as described above.

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.

Cysteine does not uniquely confer specific binding of CopA3 to 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.

Figure 2.Cysteine does not uniquely confer the specific binding of CopA3 to caspases. (A) Antimicrobial peptides with a structure similar to that of CopA3 (D-form amino acids, disulfide bridge homodimer, <11 aa in length). (B, C) CopA3 (1 μg), HaA4 from ladybugs (1 μg), or Lumbricusins (1 μg; Lum-1, -2, and -3) from earthworms was incubated with 1 μg GST-caspase-3 (B)or GST-caspase-6 (C) at 37℃ for 15 min in a total reaction volume of 30 μl. Reaction mixtures were resolved by SDS-PAGE on 15% gels, and blots were probed with a primary antibody against the corresponding caspase. The presented results are representative of three independent experiments.

The binding specificity of CopA3 for caspases requires the N-terminal leucine and two C-terminal lysines in addition to cysteine

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.

Figure 3.Additional amino acids in CopA3 are required for specific binding to caspases. (A, B) Wild-type CopA3 (1 μg) or CopA3-deletion mutants (1 μg) were incubated with 1 μg GSTcaspase-3 (A) or GST-caspase-3 (B) at 37℃ for 15 min in a total reaction volume of 30 μl respectively. Reaction mixtures were resolved by SDS-PAGE on 10% gels, and blots were probed with a primary antibody against the corresponding caspase. The presented results are representative of three independent experiments.

The importance of leucine (L) in the CopA3 sequence is particularly intriguing, given the role of the LXXLL motif in mediating high-affinity protein-protein binding [23]. For example, a number of transcription factors possess a short helical sequence containing an LXXLL motif that is necessary for interaction with co-activators [2325]. CopA3, which has an α-helical structure, also contains a LXXXLL motif similar to LXXLL that could possibly help CopA3 bind caspases. Collectively, our findings indicate that the presence of a similar LXXLL motif and positive charges in the CopA3 structure, together with an internal cysteine residue, contribute to the selectivity and binding affinity of CopA3 for caspases.

Binding specificity of CopA3 for native endogenous caspases extracted from HT29 human colorectal adenocarcinoma cells

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.

Figure 4.The same amino acids in CopA3 are required for binding to endogenous caspases in cell extracts. (A) Extracts (10 μg) of HT29 human colorectal cells containing endogenous caspases were incubated with wild-type CopA3 (1 μg) at 37℃ for 15 min. The reaction mixture was resolved by SDS-PAGE on 15% gels, and blots were probed with antibodies (1:2000 dilution) against caspase-3 or -6. The presented results are representative of three independent experiments. (B, C) Extracts (10 μg) of HT29 cells containing endogenous caspases were incubated with wild-type CopA3 (1 μg) or CopA3-deletion mutants (1 μg) at 37℃ for 15 min. Reaction mixtures were resolved by SDS-PAGE on 15% gels, and blots were probed with antibodies (1:2000 dilution) against caspase-3 (B) or -6 (C).

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 (Copris tripartitus); CopA3-CA mutant; substitution of cysteine of CopA3 by alanine, GST protein; Glutathione S transferase protein.

This work was supported by the Daejin University Research Grants in 2023.

The author has no financial conflicts of interest to declare.

  1. Shalini S, Dorstyn L, Dawar S, Kumar S. 2015. Old, new and emerging functions of caspases. Cell Death Differ. 22: 526-539.
    Pubmed KoreaMed CrossRef
  2. Kamada S, Funahashi Y, Tsujimoto Y. 1997. Caspase-4 and caspase-5, members of the ICE/CED-3 family of cysteine proteases, are CrmA-inhibitable proteases. Cell Death Differ. 4: 473-478.
    Pubmed CrossRef
  3. Miura M, Zhu H, Rotello R, Hartwieg EA, Yuan J. 1993. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell 75: 653-660.
    Pubmed CrossRef
  4. Ray CA, Black RA, Kronheim SR, Greenstreet TA, Sleath PR, Salvesen GS, et al. 1992. Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme. Cell 69: 597-604.
    Pubmed CrossRef
  5. Zhou Q, Snipas S, Orth K, Muzio M, Dixit VM, Salvesen GS. 1997. Target protease specificity of the viral serpin CrmA. Analysis of five caspases. J. Biol. Chem. 272: 7797-7800.
    Pubmed CrossRef
  6. Ekert PG, Silke J, Vaux DL. 1999. Caspase inhibitors. Cell Death Differ. 6: 1081-1086.
    Pubmed CrossRef
  7. Rano TA, Timkey T, Peterson EP, Rotonda J, Nicholson DW, Becker JW, et al. 1997. A combinatorial approach for determining protease specificities: application to interleukin-1beta converting enzyme (ICE). Chem. Biol. 4: 149-155.
    Pubmed CrossRef
  8. Mittl PR, Di Marco S, Krebs JF, Bai X, Karanewsky DS, Priestle JP, et al. 1997. Structure of recombinant human CPP32 in complex with the tetrapeptide acetyl-Asp-Val-Ala-Asp fluoromethyl ketone. J. Biol. Chem. 272: 6539-6547.
    Pubmed CrossRef
  9. Walker NP, Talanian RV, Brady KD, Dang LC, Bump NJ, Ferenz CR, et al. 1994. Crystal structure of the cysteine protease interleukin-1 beta-converting enzyme: a (p20/p10)2 homodimer. Cell 78: 343-352.
    Pubmed CrossRef
  10. Kang JK, Hwang JS, Nam HJ, Ahn KJ, Seok H, Kim SK, et al. 2011. The insect peptide coprisin prevents Clostridium difficile-mediated acute inflammation and mucosal damage through selective antimicrobial activity. Antimicrob. Agents Chemother. 55: 4850-4857.
    Pubmed KoreaMed CrossRef
  11. Lee J, Hwang JS, Hwang IS, Cho J, Lee E, Kim Y, et al. 2012. Coprisininduced antifungal effects in Candida albicans correlate with apoptotic mechanisms. Free Radic. Biol. Med. 52: 2302-2311.
    Pubmed CrossRef
  12. Lee J, Lee D, Choi H, Kim HH, Kim H, Hwang JS, et al. 2014. Structureactivity relationships of the intramolecular disulfide bonds in coprisin, a defensin from the dung beetle. BMB Rep. 47: 625-630.
    Pubmed KoreaMed CrossRef
  13. Kim YH, Hwang JS, Yoon IN, Lee JH, Lee J, Park KC, et al. 2021. The insect peptide CopA3 blocks programmed cell death by directly binding caspases and inhibiting their proteolytic activation. Biochem. Biophys. Res. Commun. 547: 82-88.
    Pubmed CrossRef
  14. Hwang JS, Lee J, Kim YJ, Bang HS, Yun EY, Kim SR, et al. 2009. Isolation and characterization of a defensin-like peptide (Coprisin) from the Dung Beetle, Copris tripartitus. Int. J. Pept. 2009: 136284.
    Pubmed KoreaMed CrossRef
  15. Yoon IN, Hwang JS, Lee JH, Kim H. 2019. The antimicrobial peptide CopA3 inhibits Clostridium difficile toxin a-induced viability loss and apoptosis in neural cells. J. Microbiol. Biotechnol. 29: 30-36.
    Pubmed CrossRef
  16. Kim IW, Lee JH, Kwon YN, Yun EY, Nam SH, Ahn MY, et al. 2013. Anticancer activity of a synthetic peptide derived from harmoniasin, an antibacterial peptide from the ladybug Harmonia axyridis. Int. J. Oncol. 43: 622-628.
    Pubmed CrossRef
  17. Kim DH, Lee IH, Nam ST, Hong J, Zhang P, Lu LF, et al. 2015. Antimicrobial peptide, lumbricusin, ameliorates motor dysfunction and dopaminergic neurodegeneration in a mouse model of Parkinson's disease. J. Microbiol. Biotechnol. 25: 1640-1647.
    Pubmed CrossRef
  18. Seo M, Lee JH, Baek M, Kim MA, Ahn MY, Kim SH, et al. 2017. A novel role for earthworm peptide Lumbricusin as a regulator of neuroinflammation. Biochem. Biophys. Res. Commun. 490: 1004-1010.
    Pubmed CrossRef
  19. Kim DH, Lee IH, Nam ST, Hong J, Zhang P, Hwang JS, et al. 2014. Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin. Biochem. Biophys. Res. Commun. 448: 292-297.
    Pubmed CrossRef
  20. Kim H, Kokkotou E, Na X, Rhee SH, Moyer MP, Pothoulakis C, et al. 2005. Clostridium difficile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-activated protein kinase. Gastroenterology 129: 1875-1888.
    Pubmed CrossRef
  21. Arbach H, Butler C, McMenimen KA. 2017. Chaperone activity of human small heat shock protein-GST fusion proteins. Cell Stress Chaperones 22: 503-515.
    Pubmed KoreaMed CrossRef
  22. Nadkarni DV. 2020. Conjugations to endogenous cysteine residues. Methods Mol. Biol. 2078: 37-49.
    Pubmed CrossRef
  23. Martinez-Zapien D, Ruiz FX, Poirson J, Mitschler A, Ramirez J, Forster A, et al. 2015. Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53. Nature 529: 541-545.
    Pubmed KoreaMed CrossRef
  24. Hu M, Luo Q, Alitongbieke G, Chong S, Xu C, Xie L, et al. 1996. Celastrol-induced Nur77 interaction with TRAF2 alleviates inflammation by promoting mitochondrial ubiquitination and autophagy. Mol. Cell. 66: 141-153.
    Pubmed KoreaMed CrossRef
  25. Adamidou T, Arvaniti KO, Glykos NM. 2011. Folding simulations of a nuclear receptor box-containing peptide demonstrate the structural persistence of the LxxLL motif even in the absence of its cognate receptor. J. Phys. Chem B. 122: 106-116.
    Pubmed CrossRef

Starts of Metrics

Share this article on :

Related articles in MBL

Most Searched Keywords ?

What is Most Searched Keywords?

  • It is most registrated keyword in articles at this journal during for 2 years.