Dataset Information

Probing the Roles of Calcium-Binding Sites during the Folding of Human Peptidylarginine Deiminase 4.

ABSTRACT: Our recent studies of peptidylarginine deiminase 4 (PAD4) demonstrate that its non-catalytic Ca²⁺-binding sites play a crucial role in the assembly of the correct geometry of the enzyme. Here, we examined the folding mechanism of PAD4 and the role of Ca²⁺ ions in the folding pathway. Multiple mutations were introduced into the calcium-binding sites, and these mutants were termed the Ca1_site, Ca2_site, Ca3_site, Ca4_site and Ca5_site mutants. Our data indicate that during the unfolding process, the PAD4 dimer first dissociates into monomers, and the monomers then undergo a three-state denaturation process via an intermediate state formation. In addition, Ca²⁺ ions assist in stabilizing the folding intermediate, particularly through binding to the Ca3_site and Ca4_site to ensure the correct and active conformation of PAD4. The binding of calcium ions to the Ca1_site and Ca2_site is directly involved in the catalytic action of the enzyme. Finally, this study proposes a model for the folding of PAD4. The nascent polypeptide chains of PAD4 are first folded into monomeric intermediate states, then continue to fold into monomers, and ultimately assemble into a functional and dimeric PAD4 enzyme, and cellular Ca²⁺ ions may be the critical factor governing the interchange.

SUBMITTER: Liu YL

PROVIDER: S-EPMC5445078 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Publications

Probing the Roles of Calcium-Binding Sites during the Folding of Human Peptidylarginine Deiminase 4.

Liu Yi-Liang YL Lee Chien-Yun CY Huang Yu-Ni YN Chen Hui-Yi HY Liu Guang-Yaw GY Hung Hui-Chih HC

Scientific reports 20170525 1

Our recent studies of peptidylarginine deiminase 4 (PAD4) demonstrate that its non-catalytic Ca<sup>2+</sup>-binding sites play a crucial role in the assembly of the correct geometry of the enzyme. Here, we examined the folding mechanism of PAD4 and the role of Ca<sup>2+</sup> ions in the folding pathway. Multiple mutations were introduced into the calcium-binding sites, and these mutants were termed the Ca1_site, Ca2_site, Ca3_site, Ca4_site and Ca5_site mutants. Our data indicate that during t ...[more]

PMID: 28546558

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Project description:The keystone oral pathogen Porphyromonas gingivalis is associated with severe periodontitis. Intriguingly, this bacterium is known to secrete large amounts of an enzyme that converts peptidylarginine into citrulline residues. The present study was aimed at identifying possible functions of this citrullinating enzyme, named Porphyromonas peptidylarginine deiminase (PPAD), in the periodontal environment. The results show that PPAD is detectable in the gingiva of patients with periodontitis, and that it literally neutralizes human innate immune defenses at three distinct levels, namely bacterial phagocytosis, capture in neutrophil extracellular traps (NETs), and killing by the lysozyme-derived cationic antimicrobial peptide LP9. As shown by mass spectrometry, exposure of neutrophils to PPAD-proficient bacteria reduces the levels of neutrophil proteins involved in phagocytosis and the bactericidal histone H2. Further, PPAD is shown to citrullinate the histone H3, thereby facilitating the bacterial escape from NETs. Last, PPAD is shown to citrullinate LP9, thereby restricting its antimicrobial activity. The importance of PPAD for immune evasion is corroborated in the infection model Galleria mellonella, which only possesses an innate immune system. Together, the present observations show that PPAD-catalyzed protein citrullination defuses innate immune responses in the oral cavity, and that the citrullinating enzyme of P. gingivalis represents a new type of bacterial immune evasion factor.IMPORTANCE Bacterial pathogens do not only succeed in breaking the barriers that protect humans from infection, but they also manage to evade insults from the human immune system. The importance of the present study resides in the fact that protein citrullination is shown to represent a new bacterial mechanism for immune evasion. In particular, the oral pathogen P. gingivalis employs this mechanism to defuse innate immune responses by secreting a protein-citrullinating enzyme. Of note, this finding impacts not only the global health problem of periodontitis, but it also extends to the prevalent autoimmune disease rheumatoid arthritis, which has been strongly associated with periodontitis, PPAD activity, and loss of tolerance against citrullinated proteins, such as the histone H3.

Dataset Information

Probing the Roles of Calcium-Binding Sites during the Folding of Human Peptidylarginine Deiminase 4.

Publications

Probing the Roles of Calcium-Binding Sites during the Folding of Human Peptidylarginine Deiminase 4.

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