The mechanism of β-N-methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation

β-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS–purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

The biosynthetic implications of late-stage condensation domain selectivity during glycopeptide antibiotic biosynthesis

The condensation domain synthesising the last peptide bond in glycopeptide antibiotic biosynthesis has a preference for linear peptide substrates, with effective peptide formation linked to the rate of amino acid activation by the preceding adenylation domain.

Class III PI-3-kinase activates phospholipase D in an amino acid–sensing mTORC1 pathway

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex, mTORC1, regulates cell growth in response to mitogenic signals and amino acid availability. Phospholipase D (PLD) and its product, phosphatidic acid, have been established as mediators of mitogenic activation of mTORC1. In this study, we identify a novel role for PLD1 in an amino acid–sensing pathway. We find that amino acids activate PLD1 and that PLD1 is indispensable for amino acid activation of mTORC1. Activation of PLD1 by amino acids requires the class III phosphatidylinositol 3-kinase hVps34, which stimulates PLD1 activity through a functional interaction between phosphatidylinositol 3-phosphate and the Phox homology (PX) domain of PLD1. Furthermore, amino acids stimulate PLD1 translocation to the lysosomal region where mTORC1 activation occurs in an hVps34-dependent manner, and this translocation is necessary for mTORC1 activation. The PX domain is required for PLD1 translocation, mTORC1 activation, and cell size regulation. Finally, we show that the hVps34-PLD1 pathway acts independently of, and in parallel to, the Rag pathway in regulating amino acid activation of mTORC1.