Masaki Kojima

A novel GTPase, CRAG, mediates promyelocytic leukemia protein–associated nuclear body formation and degradation of expanded polyglutamine protein

Polyglutamine diseases are inherited neurodegenerative diseases caused by the expanded polyglutamine proteins (polyQs). We have identified a novel guanosine triphosphatase (GTPase) named CRAG that contains a nuclear localization signal (NLS) sequence and forms nuclear inclusions in response to stress. After ultraviolet irradiation, CRAG interacted with and induced an enlarged ring-like structure of promyelocytic leukemia protein (PML) body in a GTPase-dependent manner. Reactive oxygen species (ROS) generated by polyQ accumulation triggered the association of CRAG with polyQ and the nuclear translocation of the CRAG–polyQ complex. Furthermore, CRAG promoted the degradation of polyQ at PML/CRAG bodies through the ubiquitin–proteasome pathway. CRAG knockdown by small interfering RNA in neuronal cells consistently blocked the nuclear translocation of polyQ and enhanced polyQ-mediated cell death. We propose that CRAG is a modulator of PML function and dynamics in ROS signaling and is protectively involved in the pathogenesis of polyglutamine diseases.

Solution structure of bromelain inhibitor IV from pineapple stem: structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean.

Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain inhibitor with an 11-residue light chain and a 41-residue heavy chain by disulfide bonds and inhibits the cysteine proteinase bromelain competitively. The structure of BI-VI in aqueous solution was determined using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Its three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded antiparallel beta-sheet. Unexpectedly, BI-VI was found to share a similar folding and disulfide bond connectivities not with cystatin superfamily inhibitors which inhibit the same cysteine proteinases but with the Bowman-Birk trypsin/chymotrypsin inhibitor from soybean (BBI-I). BBI-I is a 71-residue inhibitor which has two independent inhibitory sites toward the serine proteinases trypsin and chymotrypsin. These structural similarities with BBI-I suggest that they have evolved from a common ancestor and differentiated in function during a course of molecular evolution.

Structure of full-length class I chitinase from rice revealed by X-ray crystallography and small-angle X-ray scattering.

The rice class I chitinase OsChia1b, also referred to as RCC2 or Cht‐2, is composed of an N‐terminal chitin‐binding domain (ChBD) and a C‐terminal catalytic domain (CatD), which are connected by a proline‐ and threonine‐rich linker peptide. Because of the ability to inhibit fungal growth, the OsChia1b gene has been used to produce transgenic plants with enhanced disease resistance. As an initial step toward elucidating the mechanism of hydrolytic action and antifungal activity, the full‐length structure of OsChia1b was analyzed by X‐ray crystallography and small‐angle X‐ray scattering (SAXS). We determined the crystal structure of full‐length OsChia1b at 2.00‐Å resolution, but there are two possibilities for a biological molecule with and without interdomain contacts. The SAXS data showed an extended structure of OsChia1b in solution compared to that in the crystal form. This extension could be caused by the conformational flexibility of the linker. A docking simulation of ChBD with tri‐N‐acetylchitotriose exhibited a similar binding mode to the one observed in the crystal structure of a two‐domain plant lectin complexed with a chitooligosaccharide. A hypothetical model based on the binding mode suggested that ChBD is unsuitable for binding to crystalline α‐chitin, which is a major component of fungal cell walls because of its collisions with the chitin chains on the flat surface of α‐chitin. This model also indicates the difference in the binding specificity of plant and bacterial ChBDs of GH19 chitinases, which contribute to antifungal activity. Proteins 2010.

Characterization of fibrillation process of α-synuclein at the initial stage

alpha-Synuclein is the major component of the filamentous Lewy bodies and Lewy-related neurites, neuropathological hallmarks of Parkinsons disease. Although numerous studies on alpha-synuclein fibrillation have been reported, the molecular mechanisms of aggregation and fibrillation at the initial stage are still unclear. In the present study, structural properties and propensities to form fibrils of alpha-synuclein at the initial stage were investigated using 2D (1)H-(15)N NMR spectroscopy, electron microscope, and small angle X-ray scattering (SAXS). Observation of the 2D (1)H-(15)N HSQC spectra indicated significant attenuation of many cross peak intensities in the regions of KTKEGV-type repeats and the non-Abeta component of Alzheimers disease amyloid (NAC), suggesting that these regions contributed fibril formation. Oligomerization comprising heptamer was successfully monitored at the initial stage using the time-dependent SAXS measurements.

Sec22b-dependent assembly of endoplasmic reticulum Q-SNARE proteins

SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins involved in membrane fusion usually contain a conserved alpha-helix (SNARE motif) that is flanked by a C-terminal transmembrane domain. They can be classified into Q-SNARE and R-SNARE based on the structural property of their motifs. Assembly of four SNARE motifs (Qa, b, c and R) is supposed to trigger membrane fusion. We have previously shown that ER (endoplasmic reticulum)-localized syntaxin 18 (Qa) forms a complex with BNIP1 (Qb), p31/Use1 (Qc), Sec22b (R) and several peripheral membrane proteins. In the present study, we examined the interaction of syntaxin 18 with other SNAREs using pulldown assays and CD spectroscopy. We found that the association of syntaxin 18 with Sec22b induces an increase in alpha-helicity of their SNARE motifs, which results in the formation of high-affinity binding sites for BNIP1 and p31. This R-SNARE-dependent Q-SNARE assembly is quite different from the assembly mechanisms of SNAREs localized in organelles other than the ER. The implication of the mechanism of ER SNARE assembly is discussed in the context of the physiological roles of the syntaxin 18 complex.

Identification of a glutamine residue essential for catalytic activity of aspergilloglutamic peptidase by site-directed mutagenesis

Aspergilloglutamic peptidase (AGP, formerly called aspergillopepsin II) from Aspergillus niger var. macrosporus is a unique acid protease recently classified to the peptidase family G1. Our previous study using site‐directed mutagenesis on the glutamic and aspartic acid residues of AGP conserved among the G1 family suggested that Glu219 and Asp123 (numbering in the preproform) are important for catalytic activity. However, the Asn mutant of Asp123 retained weak but significant activity and therefore it was unclear whether it is an active site residue. In this study, we performed site‐directed mutagenesis on all the other hydrophilic residues including Gln, Asn, Ser, Thr, and Tyr, conserved in this family to screen other residues that might be essential for catalytic function, and found that mutations of only Gln133 resulted in almost complete loss of enzymatic activity without change in the native conformation of the enzyme. Meanwhile, the 3D structure of scytalidoglutamic peptidase, a homologue from Scytalidium lignicolum, has been reported, indicating that Glu136 and Gln53 (the counterparts of Glu219 and Gln133 in AGP) form a catalytic dyad. Therefore, the results obtained in this and our previous studies provide with complementary evidence for the definitive conclusion on the catalytic function of the Glu/Gln dyad in glutamic peptidases.

Structural and enzymatic characterization of physarolisin (formerly physaropepsin) proves that it is a unique serine-carboxyl proteinase.

Previously, we purified and partially characterized physarolisin, a lysosomal acid proteinase from Physarum polycephalum, which had been suggested to be concerned with the morphological changes of the mold. In this study, a cDNA for the enzyme was cloned and sequenced, and the structural and enzymatic features were investigated. The enzyme shows a sequence similarity to the serine-carboxyl proteinase family (MEROPS S53). Indeed, diisopropylfluorophosphate (DFP) was shown to strongly inhibit the activity of the enzyme. However, the enzyme possesses several unique features distinct from the other members of the family, such as the two-chain structure and inhibition by diazoacetyl-D,L-norleucine methyl ester (DAN). The sites and mode of processing of the precursor to the mature enzyme were deduced, and the major DAN-reactive residue in the enzyme was identified to be Asp529. These features were suggested to be due to the unique local tertiary structure of the enzyme by molecular modeling. We now propose the name physarolisin for the enzyme.

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide IDENTIFICATION OF CRITICAL SEQUENCES AND RESIDUES IN THE PROPEPTIDE

Aspergilloglutamic peptidase (formerly called aspergillopepsin II) is an acid endopeptidase produced by Aspergillus niger var. macrosporus, with a novel catalytic dyad of a glutamic acid and a glutamine residue, thus belonging to a novel peptidase family G1. The mature enzyme is generated from its precursor by removal of the putative 41-residue propeptide and an 11-residue intervening peptide through autocatalytic activation. In the present study, the propeptide (Ala1–Asn41) and a series of its truncated peptides were chemically synthesized, and their effects on the enzyme activity and thermal stability were examined to identify the sequences and residues in the propeptide most critical to the inhibition and thermal stabilization. The synthetic propeptide was shown to be a potent competitive inhibitor of the enzyme (Ki = 27 nm at pH 4.0). Various shorter propeptide fragments derived from the central region of the propeptide had significant inhibitory effect, whereas their Ala scan-substituted peptides, especially R19A and H20A, showed only weak inhibition. Substitution of the Pro23-Pro24 sequence near His20 with an Ala-Ala sequence changed the peptide Lys18–Tyr25 to a substrate with His20 as the P1 residue. Furthermore, the propeptide was shown to be able to significantly protect the enzyme from thermal denaturation (ΔTm = ∼19 °C at pH 5.6). The protective potencies of the propeptide as well as truncated propeptides and their Ala scan-substituted peptides are parallel with their inhibitory potencies. These results indicate that the central part, and especially Arg19 and His20 therein, of the propeptide is most critical to the inhibition and thermal stabilization and that His20 interacts with the enzyme at or near the S1 site in a nonproductive fashion.

Thermal unfolding of ribonuclease T1 studied by multi- dimensional NMR spectroscopy

Abstract Thermal unfolding of ribonculease (RNase) T1 was studied by 1H nuclear Overhauser enhancement spectroscopy (NOESY) and 1H-15N heteronuclear single-quantum coherence (HSQC) NMR spectroscopy at various temperatures. Native RNase T1 is a single-chain molecule of 104 amino acid residues, and has a single α-helix and two β-sheets, A and B, which consist of two and five strands, respectively. Singular value decomposition analysis based on temperature-dependent HSQC spectra revealed that the thermal unfolding of RNase T1 can be described by a two-state transition model. The midpoint temperature and the change in enthalpy were determined as 54.0°C and 696 kJ/mol, respectively, which are consistent with results obtained by other methods. To analyze the transition profile in more detail, we investigated local structural changes using temperature-dependent NOE intensities. The results indicate that the helical region starts to unfold at lower temperature than some β-strands (B3, B4, and B5 in β-sheet B). These β-strands correspond to the hydrophobic cluster region, which had been expected to be a folding core. This was confirmed by structure calculations using the residual NOEs observed at 56°C. Thus, the two-state transition of RNase T1 appears to involve locally different conformational changes.

Nuclear magnetic resonance studies on the pKa values and interaction of ionizable groups in bromelain inhibitor VI from pineapple stem.

Abstract Bromelain inhibitor VI (BI-VI), a cysteine proteinase inhibitor from pineapple stem, is a unique doublechain molecule composed of two distinct domains A and B. In order to clarify the molecular mechanism of the proteinase-inhibitor interaction, we investigated the electrostatic properties of this inhibitor. The inhibitory activity toward bromelain was revealed to be maximal at pH 3–4 and the gross conformation to be stable over a wide range of pH. Based on these results, pH titration experiments were performed on the proton resonances of BI-VI in the pH range of 1.5–9.9, and p values (pKaKexp) were determined for all carboxyl groups and α-amino groups. The pKexp were also compared with theoretical values calculated from the NMR-derived structures of BI-VI. The electrostatic surface potential map constructed using the pKexp values revealed that BI-VI possesses continuous negatively charged and scattered positively charged regions on the molecular surface and both regions appear to serve for docking properly with a basic target enzyme. Furthermore, it was suggested that the ionic interaction of the inhibitor with the target enzyme is primarily important for the inhibition, which seems to involve some carboxyl groups in the inhibitor and a thiol group in the proteinase.