岡西 広樹

Proteases play crucial roles in numerous biological processes through specific protein cleavage, and their dysregulation has been implicated in various diseases. To better understand protease specificity, we developed a lauroylation-assisted proteomic identification of protease cleavage sites (PICS) workflow that labels and enriches targeted protease-generated neo-N-termini using economical reagents and standard laboratory equipment. The lauroylation enables both discrimination of the neo-N-termini in LC-MS/MS and efficient enrichment on a C18 StageTip by exploiting its hydrophobicity. Among tested acylations, we found lauroylation to be optimal for PICS and improved enrichment and fractionation conditions. We demonstrated that this method can profile specificities of multiple proteases with high sensitivity. Furthermore, we extended this concept to N-terminomics to examine proteolysis at the protein level. Protein N-terminal dimethylation is used for labeling, and tryptic internal peptides are lauroylated for removal. This approach identified over 1500 cleavages induced by etoposide, including 912 Asp-cleaved sites consistent with caspase-3 motifs and sensitive to inhibition by Z-DEVD-FMK. Additionally, 2286 protein N-termini were identified in untreated cells, including 1794 non-ORF N-termini with 665 previously annotated processing sites. These results demonstrate that our workflow provides a simple, economical, and widely applicable method for characterizing protease cleavage at both peptide and protein levels.

BACKGROUND: L-type amino acid transporter 1 (LAT1; SLC7A5), which preferentially transports large neutral amino acids (LNAAs), is highly upregulated in various cancers and represents a promising therapeutic target. The first-in-class LAT1-specific inhibitor, nanvuranlat (JPH203, KYT-0353), has exhibited potent anti-cancer effects and is under clinical evaluation. However, alterations in the amino acid availability in cancer cells underlying its pharmacological activities remain to be elucidated. METHODS: Amino acids in nanvuranlat-treated cancer cells were measured by high-performance liquid chromatography. LAT1 knockdown was performed using siRNA. To mimic LAT1 inhibition, cancer cells were incubated in culture media lacking specific LNAA(s) reduced by nanvuranlat. The consequences of these treatments were compared by cell-based assays, including analyses of amino acid contents, cell growth, amino acid-related signaling pathways, cell cycle, ATP production rate, and transcriptomes by RNA sequencing. Metabolome of nanvuranlat-treated and untreated cells was compared by mass spectrometry. The effects of nanvuranlat on amino acid composition were also examined in three-dimensional cancer cell spheroids. RESULTS: Both pharmacological and genetic inhibition of LAT1 preferentially and continuously reduced valine, isoleucine, and tryptophan in pancreatic cancer MIA PaCa-2 cells. Nanvuranlat induced similar alterations in intracellular amino acids in multiple cancer cell lines. Depletion of these amino acids from culture media selectively lowered their intracellular concentrations, recapitulating the effects of nanvuranlat on cell growth, amino acid-related mTORC1/GAAC signaling pathways, and cell cycle. Deprivation of valine or isoleucine exhibited more pronounced impacts than tryptophan in all assays. As a novel pharmacological action of nanvuranlat mediated by the reductions in valine and isoleucine, we revealed downregulation of multiple genes in the TCA cycle and respiratory chain, accompanied by a decreased mitochondrial ATP production rate. Consistently, metabolomics revealed broad decreases in the TCA cycle intermediates by LAT1 inhibition. Nanvuranlat also similarly influenced the amino acid levels in cancer cell spheroids. CONCLUSIONS: Reductions in valine and isoleucine in cancer cells primarily account for the multifaceted anti-cancer pharmacological activities of LAT1 inhibition by nanvuranlat. This study establishes the molecular basis for LAT1-targeted therapy and highlights growth-promoting processes in cancer cells that can be exploited pharmacologically by modulating the availability of specific amino acids.

In Thermus thermophilus, an aerobic Gram-negative eubacterium used as a model organism, more than half of the phosphorylation sites identified by proteomic analysis are located near the ligand-binding site, including the active site, of the enzyme in the three-dimensional structure. We investigated the effect of these phosphorylation events on the activity of six enzymes (three nucleoside monophosphate kinases, isocitrate kinase, malate dehydrogenase and inorganic pyrophosphatase) by introducing phosphomimetic mutations, Glu, into the phosphorylation sites. All phosphomimetic mutants showed severely reduced activity compared with the wild-type, particularly in the turnover number. The proteins analyzed in this study belong to different families and have various functions. This suggests that there is a widespread mechanism by which phosphorylation of amino acid residues near the active site reduces enzyme activity independent of the protein family and function.

Bacteria reduce toxic selenium oxyanions, such as selenite, to elemental selenium (Se0), forming selenium nanoparticles (SeNPs) either intracellularly or extracellularly. However, the mechanism through which extracellular SeNPs (Ex-SeNPs) are exported remains unclear. In this study, we characterized Ex-SeNPs biosynthesized by Escherichia coli during the aerobic reduction of selenite. The SeNPs appeared within 2 h of exposure, remained extracellular, and displayed a consistent spherical morphology (∼100 nm). Purified Ex-SeNPs consisted of an Se0 core enveloped by a membrane-like layer containing lipids, proteins, carbohydrates, peptidoglycan, and lipopolysaccharides. Fluorescence microscopy and gas chromatography-mass spectrometry indicated that the encapsulated membrane originates from the E. coli cell membrane. Notably, mutants deficient in the outer membrane proteins OmpC or TolA failed to excrete SeNPs, resulting in intracellular accumulation despite efficient Se0 synthesis. Our findings suggest that E. coli forms SeNPs intracellularly and exports them via an envelope-dependent process, during which the particles may become encapsulated in membrane-like structures. These findings help clarify the mechanism underlying a membrane-dependent pathway for SeNP detoxification and export that had been suggested but not directly demonstrated.

Zinc finger domains are important interaction modules for binding to nucleic acids, proteins, lipids and small molecules. Many small-sized zinc finger proteins are encoded in bacterial genomes, but most of them have not been functionally annotated. We focused on TTHA0897, ZifS, as a small zinc finger protein from the extremely thermophilic eubacterium Thermus thermophilus HB8. In vivo experiments suggested that the cellular function of ZifS is related to the growth transition of T. thermophilus from the lag to the exponential phase under nutritionally limited conditions. In vitro biochemical experiments, including electrophoretic mobility shift assay and pull-down assay, yielded no clues about molecular functions of ZifS. X-ray crystallographic analysis revealed that the dimeric ZifS globally forms a cylinder-like structure, although ZifS dimer has no overall structural similarity to other known zinc finger proteins. The zinc ion-binding manner of ZifS fitted the characteristics of the zinc ribbon fold, which are mostly found in domains from proteins involved in the transcriptional and translational machinery. The crystal structure of ZifS is the first experimental insight into the molecular structure of this protein family, revealing several conserved features that may be functionally relevant.