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Decreased synthesis of ribosomal proteins in tauopathy revealed by non-canonical amino acid labelling.

ABSTRACT: Tau is a scaffolding protein that serves multiple cellular functions that are perturbed in neurodegenerative diseases, including Alzheimer's disease (AD) and frontotemporal dementia (FTD). We have recently shown that amyloid-?, the second hallmark of AD, induces de novo protein synthesis of tau. Importantly, this activation was found to be tau-dependent, raising the question of whether FTD-tau by itself affects protein synthesis. We therefore applied non-canonical amino acid labelling to visualise and identify newly synthesised proteins in the K369I tau transgenic K3 mouse model of FTD. This revealed massively decreased protein synthesis in neurons containing pathologically phosphorylated tau, a finding confirmed in P301L mutant tau transgenic rTg4510 mice. Using quantitative SWATH-MS proteomics, we identified changes in 247 proteins of the de novo proteome of K3 mice. These included decreased synthesis of the ribosomal proteins RPL23, RPLP0, RPL19 and RPS16, a finding that was validated in both K3 and rTg4510 mice. Together, our findings present a potential pathomechanism by which pathological tau interferes with cellular functions through the dysregulation of ribosomal protein synthesis.

SUBMITTER: Evans HT

PROVIDER: S-EPMC6600635 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Decreased synthesis of ribosomal proteins in tauopathy revealed by non-canonical amino acid labelling.

Evans Harrison Tudor HT Benetatos Joseph J van Roijen Marloes M Bodea Liviu-Gabriel LG Götz Jürgen J

The EMBO journal 20190522 13

Tau is a scaffolding protein that serves multiple cellular functions that are perturbed in neurodegenerative diseases, including Alzheimer's disease (AD) and frontotemporal dementia (FTD). We have recently shown that amyloid-β, the second hallmark of AD, induces de novo protein synthesis of tau. Importantly, this activation was found to be tau-dependent, raising the question of whether FTD-tau by itself affects protein synthesis. We therefore applied non-canonical amino acid labelling to visuali ...[more]

PMID: 31268600

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Project description:BACKGROUND:Assembly of the ribosome from its protein and RNA constituents must occur quickly and efficiently in order to synthesize the proteins necessary for all cellular activity. Since the early 1960's, certain characteristics of possible assembly pathways have been elucidated, yet the mechanisms that govern the precise recognition events remain unclear.We utilize a comparative analysis to investigate the amino acid composition of ribosomal proteins (r-proteins) with respect to their role in the assembly process. We compared small subunit (30S) r-protein sequences to those of other housekeeping proteins from 560 bacterial species and searched for correlations between r-protein amino acid content and factors such as assembly binding order, environmental growth temperature, protein size, and contact with ribosomal RNA (rRNA) in the 30S complex. RESULTS:We find r-proteins have a significantly high percent of positive residues, which are highly represented at rRNA contact sites. An inverse correlation between the percent of positive residues and r-protein size was identified and is mainly due to the content of Lysine residues, rather than Arginine. Nearly all r-proteins carry a net positive charge, but no statistical correlation between the net charge and the binding order was detected. Thermophilic (high-temperature) r-proteins contain increased Arginine, Isoleucine, and Tyrosine, and decreased Serine and Threonine compared to mesophilic (lower-temperature), reflecting a known distinction between thermophiles and mesophiles, possibly to account for protein thermostability. However, this difference in amino acid content does not extend to rRNA contact sites, as the proportions of thermophilic and mesophilic contact residues are not significantly different. CONCLUSIONS:Given the significantly higher level of positively charged residues in r-proteins and at contact sites, we conclude that ribosome assembly relies heavily on an electrostatic component of interaction. However, the binding order of r-proteins in assembly does not appear to depend on these electrostatics interactions. Additionally, because thermophiles and mesophiles exhibit significantly different amino acid compositions in their sequences but not in the identities of contact sites, we conclude that this electrostatic component of interaction is insensitive to temperature and is not the determining factor differentiating the temperature sensitivity of ribosome assembly.

Project description:IntroductionMapping protein synthesis and turnover during development will provide insight into functional tissue assembly; however, quantitative in vivo characterization has been hindered by a lack of tools. To address this gap, we previously demonstrated murine embryos can be labeled with the non-canonical amino acid azidohomoalanine (Aha), which enables the enrichment and identification of newly synthesized proteins. Using this technique, we now show how protein turnover varies as a function of both time and cellular compartment during murine development.MethodsPregnant C57BL/6 mice were injected with Aha or PBS (control) at different embryonic time points. Aha-labeled proteins from homogenized E12.5 and E15.5 embryos were conjugated with diazo biotin-alkyne, bound to NeutrAvidin beads, selectively released, then processed for either SDS-PAGE or LC-MS/MS. For turnover studies, embryos were harvested 0-48 h after Aha injection at E12.5, separated into different cellular fractions based on solubility, and analyzed via western blotting.ResultsWe developed an enhanced method for isolating Aha-labeled proteins from embryos that minimizes background signal from unlabeled proteins and avidin contamination. Approximately 50% of all identified proteins were found only in Aha samples. Comparing proteins present in both Aha and PBS samples, 90% were > 2-fold enriched in Aha-treated embryos. Furthermore, this method could resolve differences in the Aha-labeled proteome between developmental time points. Newly synthesized Aha-labeled proteins were observed by 3 h and peak labeling was around 6 h. Notably, extracellular matrix and cytoskeletal turnover appeared lower than the cytosolic fraction.ConclusionsThe methods developed in this work enable the identification and quantification of protein synthesis and turnover in different tissue fractions during development. This will provide insight into functional tissue assembly and ultimately inform the design of regenerative therapies that seek to promote growth and repair.

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Decreased synthesis of ribosomal proteins in tauopathy revealed by non-canonical amino acid labelling.

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Decreased synthesis of ribosomal proteins in tauopathy revealed by non-canonical amino acid labelling.

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