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Temporal Quantitative Profiling of Newly Synthesized Proteins during Aβ Accumulation.

ABSTRACT: Accumulation of aggregated amyloid beta (Aβ) in the brain is believed to impair multiple cellular pathways and play a central role in Alzheimer's disease pathology. However, how this process is regulated remains unclear. In theory, measuring protein synthesis is the most direct way to evaluate a cell's response to stimuli, but to date, there have been few reliable methods to do this. To identify the protein regulatory network during the development of Aβ deposition in AD, we applied a new proteomic technique to quantitate newly synthesized protein (NSP) changes in the cerebral cortex and hippocampus of 2-, 5-, and 9-month-old APP/PS1 AD transgenic mice. This bio-orthogonal noncanonical amino acid tagging analysis combined PALM (pulse azidohomoalanine labeling in mammals) and HILAQ (heavy isotope labeled AHA quantitation) to reveal a comprehensive dataset of NSPs prior to and post Aβ deposition, including the identification of proteins not previously associated with AD, and demonstrated that the pattern of differentially expressed NSPs is age-dependent. We also found dysregulated vesicle transportation networks including endosomal subunits, coat protein complex I (COPI), and mitochondrial respiratory chain throughout all time points and two brain regions. These results point to a pathological dysregulation of vesicle transportation which occurs prior to Aβ accumulation and the onset of AD symptoms, which may progressively impact the entire protein network and thereby drive neurodegeneration. This study illustrates key pathway regulation responses to the development of AD pathogenesis by directly measuring the changes in protein synthesis and provides unique insights into the mechanisms that underlie AD.

SUBMITTER: Ma Y

PROVIDER: S-EPMC7775911 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Adaptation to starvation is integral to the Leishmania life cycle. The parasite can survive prolonged periods of nutrient deprivation both in vitro and in vivo. The identification of parasite proteins synthesised during starvation is key to unravelling the underlying molecular mechanisms facilitating adaptation to these conditions. Additionally, as stress adaptation mechanisms in Leishmania are linked to virulence as well as infectivity, profiling of the complete repertoire of Newly Synthesised Proteins (NSPs) under starvation is important for drug target discovery. However, differential identification and quantitation of low abundance, starvation-specific NSPs from the larger background of the pre-existing parasite proteome has proven difficult, as this demands a highly selective and sensitive methodology. Herein we introduce an integrated chemical proteomics method in L. mexicana promastigotes that involves a powerful combination of the BONCAT technique and iTRAQ quantitative proteomics Mass Spectrometry (MS), which enabled temporally resolved quantitative profiling of de novo protein synthesis in the starving parasite. Uniquely, this approach integrates the high specificity of the BONCAT technique for the NSPs, with the high sensitivity and multiplexed quantitation capability of the iTRAQ proteomics MS. Proof-of-concept experiments identified over 250 starvation-responsive NSPs in the parasite. Our results show a starvation-specific increased relative abundance of several translation regulating and stress-responsive proteins in the parasite. GO analysis of the identified NSPs for Biological Process revealed translation (enrichment P value 2.47e-35) and peptide biosynthetic process (enrichment P value 4.84e-35) as extremely significantly enriched terms indicating the high specificity of the NSP towards regulation of protein synthesis. We believe that this approach will find widespread use in the study of the developmental stages of Leishmania species and in the broader field of protozoan biology.

Dataset Information

Temporal Quantitative Profiling of Newly Synthesized Proteins during Aβ Accumulation.

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