赤木 謙一

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysis. Beyond this normal function, GAPDH acts as a moonlighting protein, interacting with nonglycolytic molecules to fulfill additional roles, such as apoptosis induction. However, the three-dimensional (3D) structural details underlying these interactions remain unclear, likely due to their dynamic and transient nature. To address this issue, we investigated the structural properties of human and porcine GAPDH using a combination of biophysical techniques, including nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, gel filtration chromatography, and thermal shift assays, with a particular focus on their 3D structures. Our results revealed that although GAPDH becomes unstable upon nicotinamide adenine dinucleotide (NAD+) depletion (apo state), its oligomeric structure as a tetramer remains preserved regardless of temperature. In contrast, the presence of adenosine triphosphate (ATP) promotes dimerization at low temperatures, as previously reported. Furthermore, our NMR data suggest that ATP binding exposes the dimer interface and increases the flexibility of side chains in this region. These findings indicate that GAPDH maintains a stable tetrameric structure in the presence of NAD+ but becomes structurally unstable and likely more susceptible to oxidation upon NAD+ depletion. Additionally, our analyses showed that partial nitrosylation of GAPDH subunits does not induce significant tertiary structural changes. However, significant structural alterations were observed when all four subunits were nitrosylated, although the possibility remains that residues other than the active site residue, Cys152, may have been oxidized. We propose that NAD+ depletion, along with oxidation or nitrosylation─most likely at Cys152─destabilizes the GAPDH conformation, and that subsequent ATP binding promotes dimerization. This subunit dissociation may serve as a structural basis for GAPDH's interactions with other molecules and its moonlighting functions.

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock considering that vancomycin is the last resort for controlling methicillin-resistantStaphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, which is typically considered too large for NMR studies, we successfully assigned the main chain using an amino acid-selective unlabeling method. Because we found that the Zn²⁺-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by mutating the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

An olfactory receptor mimetic peptide-modified graphene field-effect transistor (gFET) is a promising solution to overcome the principal challenge of low specificity graphene-based sensors for volatile organic compound (VOC) sensing. Herein, peptides mimicking a fruit fly olfactory receptor, OR19a, were designed by a high-throughput analysis method that combines a peptide array and gas chromatography for the sensitive and selective gFET detection of the signature citrus VOC, limonene. The peptide probe was bifunctionalized via linkage of a graphene-binding peptide to facilitate one-step self-assembly on the sensor surface. The limonene-specific peptide probe successfully achieved highly sensitive and selective detection of limonene by gFET, with a detection range of 8-1000 pM, while achieving facile sensor functionalization. Taken together, our target-specific peptide selection and functionalization strategy of a gFET sensor demonstrates advancement of a precise VOC detection system.

T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing β-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity.