Degradation-Suppressed Cocoonase for Investigating the Propeptide-Mediated Activation Mechanism

Sakata, Nana; Ogata, Ayumi; Takegawa, Mai; Murakami, Yuri; Nishimura, Misaki; Miyazawa, Mitsuhiro; Hagiwara, Tokio; Shimamoto, Shigeru; Hidaka, Yuji

doi:10.3390/molecules27228063

Cited by 3 publications

(7 citation statements)

References 25 publications

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“…The degradation that occurs during the refolding reaction of trypsin and trypsinogen complicates the folding analyses, even though investigating the folding mechanisms of serine proteases would be extremely interesting regarding the convergent enzyme evolution [25]. To address these issues, degradation-suppressed prococoonase, [K8D]-proCCN [9], was recently prepared and applied to examining the refolding reaction in this study. Importantly, significant levels of degradation products were not observed on RP-HPLC analyses, indicating that the refolding reactions of the protein occurred without degradation and that sufficient levels of intermediates had been labeled with the peptide reagent, as shown in Figure 4.…”

Section: Discussionmentioning

confidence: 99%

“…To apply the novel peptide reagent for investigating the folding mechanisms of mid-size proteins, we utilized the peptide reagent to capture the folding intermediates of prococoonase (25.1 kDa). For this purpose, a degradation-suppressed prococoonase, [K8D,K63G,K131G,K133A]-proCCN ([K8D]-proCCN ), was employed to avoid non-specific degradation during the refolding reactions [9]. To estimate the effect of the peptide moiety on separation on SDS-PAGE, N-ethylmaleimide was compared to the novel peptide reagent.…”

Section: Labeling Reaction Of the Folding Intermediates Of Bpti Using...mentioning

confidence: 99%

Section: Reduction Of the Proteinsmentioning

confidence: 99%

“…A [K8D,K63G,K131G,K133A]-proCCN, denoted as [K8D]-proCCN , was used in this study to suppress degradation during refolding reactions [9]. The oxidized form of the [K8D]-proCCN protein was prepared by a previously reported method [9]. The reduced forms of BPTI and the [K8D]-proCCN proteins were prepared by treatment with dithiothreitol as previously described, with minor modifications [8,17].…”

Section: Reduction Of the Proteinsmentioning

confidence: 99%

“…We recently found that the CCN protein is kinetically trapped in a molten globule-like state at the final stage of the folding pathway and is converted into its native conformation with the assistance of the propeptide region [8]. To further investigate the role of the propeptide region in the disulfide-coupled folding of a precursor protein, prococoonase (proCCN), we carried out refolding and auto-processing reactions [9]. However, the refolding mixtures of proCCN Molecules 2023, 28, 3494 2 of 11 contained several types of disulfide isomers that could not be completely separated by reversed-phase HPLC (RP-HPLC).…”

Section: Introductionmentioning

confidence: 99%

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A Novel Peptide Reagent for Investigating Disulfide-Coupled Folding Intermediates of Mid-Size Proteins

et al. 2023

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Investigations of protein folding have largely involved the use of disulfide-containing proteins, since the disulfide-coupled folding of proteins allows folding intermediates to be trapped and their conformations determined. However, studies of the folding mechanisms of mid-size proteins face several problems, one of which is that detecting folding intermediates is difficult. Therefore, to solve this issue, a novel peptide reagent, maleimidohexanoyl-Arg5-Tyr-NH2, was designed and applied to the detection of folding intermediates of model proteins. BPTI was chosen as a model small protein to estimate the ability of the novel reagent to detect folding intermediates. In addition, a precursor protein (prococoonase) of Bombyx mori cocoonase was used as a model mid-size protein. Cocoonase is classified as a serine protease and has a high homology with trypsin. We recently found that the propeptide sequence of prococoonase (proCCN) is important for the folding of cocoonase. However, it was difficult to study the folding pathway of proCCN since the folding intermediates could not be separated on a reversed-phase HPLC (RP-HPLC). Therefore, to separate the folding intermediates by RP-HPLC, the novel labeling reagent was used to accomplish this for proCCN. The results indicated that the peptide reagent allowed the intermediates to be captured, separated on SDS-PAGE, and analyzed by RP-HPLC without the occurrence of undesirable disulfide-exchange reactions during the labeling reactions. The peptide reagent reported herein is a practical tool for investigating the mechanisms of disulfide-coupled folding of mid-size proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Labeling Reaction Of the Folding Intermediates Of Bpti Using...mentioning

confidence: 99%

Section: Reduction Of the Proteinsmentioning

confidence: 99%

Section: Reduction Of the Proteinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Novel Peptide Reagent for Investigating Disulfide-Coupled Folding Intermediates of Mid-Size Proteins

et al. 2023

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The Cell Adhesion Activity of the Joining Peptide of Proopiomelanocortin

Hiroshima,

Sakata,

Konogami

et al. 2023

Molecules

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Proopiomelanocortin (POMC) is a precursor protein of several peptide hormones, such as ACTH and β-endorphin. Almost all of the peptide hormones in POMC have been drastically investigated in terms of their biological activities. However, the biological activity of the joining peptide region (JP) in POMC is unknown. Therefore, to explore the biological activity of JP, sequence analyses of mammalian POMC were performed. We found an -Arg-Gly-Asp- (RGD) motif in several mammalian species, such as porcine, suggesting that JP has cell adhesion activity. To validate this hypothesis, the cell adhesion activities of the synthetic porcine JP peptides were examined using 293T cells. Cell adhesions were observed in a concentration-dependent manner of the JP peptides. In addition, the JP peptide competitively inhibited cell adhesion to the POMC-coated plates. Moreover, the cell adhesion activity of the joining peptide was inhibited by the addition of EDTA, indicating that the JP peptide mediates the cell adhesion activity via a receptor protein, integrin. Interestingly, a human JP peptide, which possesses an -Arg-Ser-Asp- (RSD) sequence in place of the RGD sequence, exhibited a higher ability in the cell adhesion activity than that of the porcine JP peptide, suggesting that the cell adhesion activity of the joining peptide is developed during the molecular evolution of POMC. In conclusion, our results reveal that the joining peptide in POMC plays an important role during cell adhesion and provide useful information related to signal transduction of nerve peptide hormones derived from POMC.

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Mutational Analysis of Substrate Recognition in Trypsin-like Protease Cocoonase: Protein Memory Induced by Alterations in Substrate-Binding Site

Sakata,

Shimamoto,

Murakami

et al. 2024

Molecules

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To investigate the substrate recognition mechanism of trypsin-like protease cocoonase (CCN), mutational analyses were conducted at key substrate recognition sites, Asp187 and Ser188, and their effects on substrate specificity and enzymatic activity were evaluated. Mutants with the Asp187 substitution exhibited a significant reduction in catalytic activity compared with the wild-type enzyme, whereas the Ser188 mutants displayed a comparatively minor effect on activity. This indicates that Asp187 plays a crucial role in catalytic function, whereas Ser188 serves a complementary role in substrate recognition. Interestingly, the substitution of the Asp187 to Glu or Ser caused novel substrate specificities, resulting in the recognition of Orn and His residues. In addition, when Asp187 and Ser188 were substituted with acidic residues (Glu or Asp), both the precursor proCCN and mature CCN proteins retained highly similar secondary and tertiary structures. This reveals that the structural characteristics of precursor proteins are maintained in the mature proteins, potentially influencing substrate recognition and catalytic function. These findings suggest that the pro-regions of these mutants interact much more tightly with the mature enzyme than in the wild-type CCN. These results provide fruitful insights into the structural determinants governing substrate recognition in enzyme variants.

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Degradation-Suppressed Cocoonase for Investigating the Propeptide-Mediated Activation Mechanism

Cited by 3 publications

References 25 publications

A Novel Peptide Reagent for Investigating Disulfide-Coupled Folding Intermediates of Mid-Size Proteins

A Novel Peptide Reagent for Investigating Disulfide-Coupled Folding Intermediates of Mid-Size Proteins

The Cell Adhesion Activity of the Joining Peptide of Proopiomelanocortin

Mutational Analysis of Substrate Recognition in Trypsin-like Protease Cocoonase: Protein Memory Induced by Alterations in Substrate-Binding Site

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