SUMMARYAmino-terminal acetylation is ubiquitous among eukaryotic proteins and controls a myriad of biological processes. Of the N-terminal acetyltransferases (NATs) that facilitate this co-translational modification, the heterodimeric NatA complex harbors the most diversity for substrate selection and modifies the majority of all amino-terminally acetylated proteins. Here, we report the X-ray crystal structure of the 100 kDa holo-NatA complex from Schizosaccharomyces pombe in the absence and presence of a bisubstrate peptide-CoA conjugate inhibitor, as well as the structure of the uncomplexed Naa10p catalytic subunit. The NatA-Naa15p auxiliary subunit contains 13 TPR motifs and adopts a ring-like topology that wraps around the NatA-Naa10p subunit, an interaction that alters the Naa10p active site for substrate-specific acetylation. These studies have implications for understanding the mechanistic details of other NAT complexes and how regulatory subunits modulate the activity of the broader family of protein acetyltransferases.

Edited by Paul E. FraserDirect cell-to-cell transmission of proteopathic ␣-synuclein (␣-syn) aggregates is thought to underlie the progression of neurodegenerative synucleinopathies. However, the specific intracellular processes governing this transmission remain unclear because currently available model systems are limited. For example, in cell culture models of ␣-syn-seeded aggregation, it is difficult to discern intracellular from extracellular exogenously applied ␣-syn seed species. Herein, we employed fluorescently labeled ␣-syn preformed fibrils (pffs) in conjunction with the membrane-impermeable fluorescence quencher trypan blue to selectively image internalized ␣-syn seeds in cultured primary neurons and to quantitatively characterize the concentration dependence, time course, and inhibition of pff uptake. To study the long-term fates of exogenous ␣-syn pffs in neurons, we developed a pff species labeled at amino acid residue 114 with the environmentally insensitive fluorophore BODIPY or the pH-sensitive dye pHrodo red. We found that pffs are rapidly trafficked along the endolysosomal pathway, where most of the material remains for days. We also found that brief pharmacological perturbation of lysosomes shortly after the pff treatment causes aberrations in intracellular processing of pff seeds concomitant with an increased rate of inclusion formation via recruitment of endogenous ␣-syn to a relatively small number of exogenous seeds. Our results validate a quantitative assay for pff uptake in primary neurons, implicate lysosomal processing as the major fate of internalized proteopathic seeds, and suggest lysosomal integrity as a significant rate-determining step in the transmission of ␣-syn pathology. Further, lysosomal processing of transmitted seeds may represent a new therapeutic target to combat the spread of synucleinopathies.Mounting evidence implicates direct cell-to-cell transmission of misfolded amyloidogenic protein species as a central component underlying the spatiotemporal progression of pathophysiology in numerous proteinopathies (1, 2). In synucleinopathies, including Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies, and multiple system atrophy, the amyloidogenic protein ␣-synuclein (␣-syn) 3 aggregates into Lewy bodies, Lewy neurites, and glial cytoplasmic inclusions (3, 4). These intracellular proteinaceous inclusions have been widely recapitulated in a number of in vivo and cell-based model systems via introduction of pathological, insoluble ␣-syn species from either brain extracts of diseased postmortem tissue or preformed fibrils of recombinant origin (5-10). Despite strong evidence gleaned from these models implicating a causal relationship between exposure of neurons to insoluble ␣-syn aggregates and the subsequent development of inclusions bearing pathological hallmarks of synucleinopathies, the specific processes underlying cell-to-cell transmission of proteopathic seeds and resulting intracellular events leading to the development of pathological ...

No abstract

Synthesis of α-hydroxy threonine (Tah, 2R, 3S-dihydroxy-butanoic acid) (1). L-Threonine (2.2 g, 18.5 mmol), suspended in 5 ml of water at -5 °C, was treated simultaneously with a solution of 1.38 g NaNO 2 (20 mmol) in 2 ml of water and 557 µl of concentrated H 2 SO 4 (10 mmol) in 1.5 ml H 2 O. The two solutions were added slowly while stirring so that the temperature remained between 0 °C and 5 °C. The reaction turned yellow upon addition. The solution was then stirred overnight at room temperature. The reaction mixture was concentrated, the mixture treated with 3 ml of EtOH, and the salts were filtered. The solution was concentrated. The material was dry loaded onto a flash silica gel column and run in 1:1 hexanes/ethyl acetate with 1% acetic acid to give 730 mg (38 %) of hydroxythreonine Synthesis of Tah cyanomethyl ester (2R, 3S-dihydroxy-butanoate cyanomethyl ester) (2).The hydroxy acid (385 mg, 3.21 mmol) was dissolved in 5.1 ml of ClCH 2 CN (80.1 mmol) and 1.2 ml Et 3 N (8.44 mmol). Upon stirring under Ar for 30 min, the solution turned yellow. A gradient flash silica gel column from 20% to 80% ethyl acetate/hexanes was run, and the isolated product was dried on vacuum to yield 50.9 mg (10%) of hydroxythreonine cyanomethyl ester: 1 H NMR (D 2 O) δ 1.27 (d, 3H, J= 6 Hz), 4.22 (m, 1H), 4.34 (d, 1H, J= 3 Hz), 5.01 (s,

Decreasing the size of spectroscopic probes can afford higher-resolution structural information from fluorescence experiments. Therefore, we have developed p-cyanophenylalanine (Cnf) and backbone thioamides as a fluorophore/quencher pair. Through the examination of a series of thiopeptides, we have determined the working distance for this pair to be 8-30 Å. We have also carried out a proof-of-principle protein-folding experiment in which a Cnf/thioamide-labeled version of villin headpiece HP35 was thermally unfolded while the Cnf/thioamide distance was monitored by fluorescence. For a given protein, thioamide substitutions could be used to track motions with a much greater number of measurements than for current fluorescence probes, providing a dense array of data with which to model conformational changes.