SPAAC Pulse-Chase: A Novel Click Chemistry-Based Method to Determine the Half-Life of Cellular Proteins.

Trevor M Morey,Mohammad Ali Esmaeili,Martin L Duennwald,R Jane Rylett

doi:10.3389/fcell.2021.722560

Trevor M Morey, Mohammad Ali Esmaeili + Show 2 more

Open Access

https://doi.org/10.3389/fcell.2021.722560

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Abstract

Assessing the stability and degradation of proteins is central to the study of cellular biological processes. Here, we describe a novel pulse-chase method to determine the half-life of cellular proteins that overcomes the limitations of other commonly used approaches. This method takes advantage of pulse-labeling of nascent proteins in living cells with the bioorthogonal amino acid L-azidohomoalanine (AHA) that is compatible with click chemistry-based modifications. We validate this method in both mammalian and yeast cells by assessing both over-expressed and endogenous proteins using various fluorescent and chemiluminescent click chemistry-compatible probes. Importantly, while cellular stress responses are induced to a limited extent following live-cell AHA pulse-labeling, we also show that this response does not result in changes in cell viability and growth. Moreover, this method is not compromised by the cytotoxicity evident in other commonly used protein half-life measurement methods and it does not require the use of radioactive amino acids. This new method thus presents a versatile, customizable, and valuable addition to the toolbox available to cell biologists to determine the stability of cellular proteins.

Highlights

The stability and degradation of cellular proteins are critical parameters that influence and regulate most aspects of cellular physiology and many disease-associated processes
We developed a novel pulse-chase method to determine the half-life of cellular proteins based on strain-promoted alkyne-azide cycloaddition (SPAAC) click chemistry reactions using non-radioactive labeling and detection reagents (Figure 1)
Exposure of yeast cells to 42◦C for 1 h led to an increase in these heat shock protein (HSP) (p ≤ 0.001) as compared to AHA-treated cells grown at 24◦C. These results demonstrate that our SPAAC pulse-chase method can be used successfully in yeast and, while AHA treatment did induce the heat shock response in yeast similar to some mammalian cells, this remained non-toxic and did not affect yeast viability

Summary

Introduction

The stability and degradation of cellular proteins are critical parameters that influence and regulate most aspects of cellular physiology and many disease-associated processes. Three major methods are commonly used to determine protein half-life, each of which has specific advantages and severe limitations (Eldeeb et al, 2019) These include (1) treatment of cells with cycloheximide (CHX), a fast and effective, yet highly cytotoxic, inhibitor of protein synthesis (Schneider-Poetsch et al, 2010), (2) pulse-chase experiments that involve labeling of newly synthesized proteins in living cells with radioisotopic amino acids such as 35S-methionine (Takahashi and Ono, 2003; Esposito and Kinzy, 2014), and (3) expression of a protein of interest as a GFP (green fluorescent protein) fusion, where GFP is either photoactivatable (Zhang et al, 2007) or can be permanently photobleached (Eden et al, 2011). Protocols involving either 35S-methionine pulse-chase labeling or CHX treatment require cell lysis, whereas GFP tags allow for monitoring protein half-life in living cells by microscopy This latter method requires the heterologous expression of a GFP-tagged protein, and cannot be used to study endogenous proteins. Expression of GFP alone or as a fusion protein can induce proteome changes, alter kinase and ubiquitin signaling pathways, and cause cellular toxicity (Baens et al, 2006; Coumans et al, 2014; Ansari et al, 2016)

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