Masatoshi Saiki

Synergistic cooperation of PDI family members in peroxiredoxin 4-driven oxidative protein folding

The mammalian endoplasmic reticulum (ER) harbors disulfide bond-generating enzymes, including Ero1α and peroxiredoxin 4 (Prx4), and nearly 20 members of the protein disulfide isomerase family (PDIs), which together constitute a suitable environment for oxidative protein folding. Here, we clarified the Prx4 preferential recognition of two PDI family proteins, P5 and ERp46, and the mode of interaction between Prx4 and P5 thioredoxin domain. Detailed analyses of oxidative folding catalyzed by the reconstituted Prx4–PDIs pathways demonstrated that, while P5 and ERp46 are dedicated to rapid, but promiscuous, disulfide introduction, PDI is an efficient proofreader of non-native disulfides. Remarkably, the Prx4-dependent formation of native disulfide bonds was accelerated when PDI was combined with ERp46 or P5, suggesting that PDIs work synergistically to increase the rate and fidelity of oxidative protein folding. Thus, the mammalian ER seems to contain highly systematized oxidative networks for the efficient production of large quantities of secretory proteins.

Acceleration of disulfide-coupled protein folding using glutathione derivatives

Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding proteins containing disulfide bond(s) is carried out in the presence of redox reagents, such as glutathione, to permit native disulfide pairing to occur. It is well known that the formation of a disulfide bond and the correct tertiary structure of a target protein are strongly affected by the redox reagent used. However, little is known concerning the role of each amino acid residue of the redox reagent, such as glutathione. Therefore, we prepared glutathione derivatives – glutamyl‐cysteinyl‐arginine (ECR) and arginyl‐cysteinyl‐glycine (RCG) – and examined their ability to facilitate protein folding using lysozyme and prouroguanylin as model proteins. When the reduced and oxidized forms of RCG were used, folding recovery was greater than that for a typical glutathione redox system. This was particularly true when high protein concentrations were employed, whereas folding recovery using ECR was similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin revealed that the folding velocity (KRCG = 3.69 × 10−3 s−1) using reduced RCG/oxidized RCG was approximately threefold higher than that using reduced glutathione/oxidized glutathione. In addition, folding experiments using only the oxidized form of RCG or glutathione indicated that prouroguanylin was converted to the native conformation more efficiently in the case of RCG, compared with glutathione. The findings indicate that a positively charged redox molecule is preferred to accelerate disulfide‐exchange reactions and that the RCG system is effective in mediating the formation of native disulfide bonds in proteins.

Radically different thioredoxin domain arrangement of ERp46, an efficient disulfide bond introducer of the mammalian PDI family

The mammalian endoplasmic reticulum (ER) contains a diverse oxidative protein folding network in which ERp46, a member of the protein disulfide isomerase (PDI) family, serves as an efficient disulfide bond introducer together with Peroxiredoxin-4 (Prx4). We revealed a radically different molecular architecture of ERp46, in which the N-terminal two thioredoxin (Trx) domains with positively charged patches near their peptide-binding site and the C-terminal Trx are linked by unusually long loops and arranged extendedly, forming an opened V-shape. Whereas PDI catalyzes native disulfide bond formation by the cooperative action of two mutually facing redox-active sites on folding intermediates bound to the central cleft, ERp46 Trx domains are separated, act independently, and engage in rapid but promiscuous disulfide bond formation during early oxidative protein folding. Thus, multiple PDI family members likely contribute to different stages of oxidative folding and work cooperatively to ensure the efficient production of multi-disulfide proteins in the ER.

Stem-Forming Regions That Are Essential for the Amyloidogenesis of Prion Proteins

Prion diseases represent fatal neurodegenerative disorders caused by the aggregation of prion proteins. With regard to the formation of the amyloidogenic cross-β-structure, the initial mechanism in the conversion to a β-structure is critically important. To explore the core regions forming a stem of the amyloid, we designed and prepared a series of peptides comprised of two native sequences linked by a turn-inducing dipeptide moiety and examined their ability to produce amyloids. A sequence alignment of the peptides bearing the ability to form amyloid structures revealed that paired strands consisting of VNITI (residues 180-184) and VTTTT (residues 189-193) are the core regions responsible for initiating the formation of cross-β-structures and for further ordered aggregation. In addition, most of the causative mutations responsible for inherited prion diseases were found to be located in these stem-forming regions on helix H2 and their counterpart on helix H3. Moreover, the volume effect of the nonstem domain, which contains ~200 residues, was deduced to be a determinant of the nature of the association such as oligomerization, because the stem-forming domain is only a small part of a prion protein. Taken together, we conclude that the mechanism underlying the initial stage of amyloidogenesis is the exposure of a newly formed intramolecular β-sheet to a solvent through the partial transition of a native structure from an α-helix to a β-structure. Our results also demonstrate that prion diseases caused by major prion proteins except the prions of some fungi such as yeast are inherent only in mammals, as evidenced by a comparison of the corresponding sequences to the stem-forming regions among different animals.

Recognition of the N-Terminal Histone H2A and H3 Peptides by Peptidylarginine Deiminase IV

Peptidylarginine deiminase IV (PAD4) catalyzes the conversion of an Arg residue to a citrulline residue in various proteins. In particular, citrullination of histone subunits, such as H2A and H3, by PAD4 is thought to be related to rheumatoid arthritis. However, the details of the citrullination mechanism of histone H2A and H3 are not yet well known. Moreover, the effects of N-terminal acetylation on histone subunits with respect to PAD4 recognition have not yet been studied. To further study the mechanism of PAD4 recognition of histone H2A and H3 subunits, a series of the N-terminal peptides was chemically synthesized and the citrullination sites were identified using MALDI-TOF/MS. N-terminal acetylation of histone H2A was not significant with respect to PAD4 recognition in vitro, but the acetylation of H3 peptide had a significant effect on PAD4 recognition in vitro, resulting in predominant citrullination at the Arg2 residue.

Structural stability of amyloid fibrils depends on the existence of the peripheral sequence near the core cross-β region

Amyloid fibrils are fibrous protein assemblies with distinctive cross‐β structures. For amyloidosis, there are disease‐associated mutations outside of the cross‐β structures. Thus, it is necessary to elucidate the role of peripheral sequences outside the cross‐β structure. Amyloid fibrils are generally 10 nm in width; however, the amyloid fibrils of truncated barnase M1 peptides missing the C‐terminal sequence outside the cross‐β structure are 20 nm in width. In this study, we performed comparative analysis of the structural stability of amyloids formed by the respective peptides. We found that the C‐terminal amino acids dramatically affect the conformational instability in the presence of a denaturing reagent.

Fiber formation of a synthetic spider peptide derived from Nephila clavata

Dragline silk is a high‐performance biopolymer with exceptional mechanical properties. Artificial spider dragline silk is currently prepared by a recombinant technique or chemical synthesis. However, the recombinant process is costly and large‐sized synthetic peptides are needed for fiber formation. In addition, the silk fibers that are produced are much weaker than a fiber derived from a native spider. In this study, a small peptide was chemically synthesized and examined for its ability to participate in fiber formation. A short synthetic peptide derived from Nephila clavata was prepared by a solid‐phase peptide method, based on a prediction using the hydrophobic parameter of each individual amino acid residue. After purification of the spider peptide, fiber formation was examined under several conditions. Fiber formation proceeded in the acidic pH range, and larger fibers were produced when organic solvents such as trifluoroethanol and acetonitrile were used at an acidic pH. Circular dichroism measurements of the spider peptide indicate that the peptide has a β‐sheet structure and that the formation of a β‐sheet structure is required for the spider peptide to undergo fiber formation.

Gly Scan of Prouroguanylin to Investigate Essential Amino Acid Residues for the Intra-Molecular Chaperone Function

Peptide hormones are often produced in the form of the precursor proteins with prepro-sequences in vivo. The pre-region acts as a signal peptide to carry the precursor protein to endoplasmic reticulum. However, little is known concerning the role of pro-sequences in peptide hormones.Uroguanylin, an endogenous ligand of guanylyl cyclase C, is also produced via the processing of the precursor protein, prouroguanylin. We previously reported that the pro-peptide region in prouroguanylin functions as an intra-molecular chaperone in the correct folding of the mature peptide, uroguanylin. Furthermore, folding analyses of the N-terminal mutants of prouroguanylin suggested that the Ile3 residue in prouroguanylin plays an important role in the chaperone function of pro-peptide region.Recently, the NMR structural analysis of proguanylin, related protein to prouroguanylin, suggested that the N-terminal region of pro-peptide interacts with the mature region to stabilize the tertiary structure of the mature peptide. However, the role of each amino acid residue in pro-peptide region remains to be studied. Therefore, we further performed the sited-directed mutagenesis for prouroguanylin to estimate the role of each amino acid residue in the intra-molecular chaperone function.For this purpose, Gly scanning was employed to estimate the role of side chains of each amino acid residue for the intra-molecular chaperone function. Each N-terminal amino acid residue of the pro-peptide region was sequentially mutated to a Gly residue. Mutants were prepared using the T7-promoter expression system in E. coli cells. After the purification of the reduced form of mutants by RP HPLC, the refolding reactions were carried out in the presence of glutathione. The folding reaction was monitored by RP-HPLC and the Circular Dichroism measurement. Folding products were analyzed by MALDI-TOF/MS. The results will be discussed in this paper.