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. 2021 Sep 7:9:722560.
doi: 10.3389/fcell.2021.722560. eCollection 2021.

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

Trevor M Morey  1   2 , Mohammad Ali Esmaeili  3 , Martin L Duennwald  3 , R Jane Rylett  1   2
Affiliations

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

Trevor M Morey et al. Front Cell Dev Biol. .

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.

Keywords: SPAAC; click chemistry; mammalian cells; protein half-life; protein stability and degradation; pulse-chase analysis; yeast.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic overview of biorthogonal strain-promoted alkyne-azide cycloaddition (SPAAC) pulse-chase for fluorescent/chemiluminescent determination of protein half-life. (1) Briefly, cultured cells are live-labeled (pulsed) with L-azidohomoalanine (AHA), a bioorthogonal methionine analog that contains a reactive azide moiety, under methionine-free conditions. (2) AHA-containing media is removed and labeled cells are first washed then chased with media containing methionine for desired times (e.g., up to 24 h). (3) Cells are collected, lysed, and protein/s of interest are immunoprecipitated. (4) Immunopurified AHA-labeled proteins are reacted with a strained cyclooctyne (e.g., 4-dibenzocyclooctynol; DIBO) that is modified with either a fluorescent (e.g., tetramethylrhodamine; TAMRA) or biotin probe to form stable triazole conjugates. (5) AHA/cyclooctyne-labeled proteins are resolved on SDS-PAGE gels and either (6a) fluorescence is detected directly in-gel (e.g., labeled with TAMRA-DIBO) or, (6b) if labeled with a biotin-cyclooctyne probe, proteins are then transferred onto PVDF membranes and detected by chemiluminescence using a HRP-conjugated streptavidin. (7) Lastly, immunoblotting is completed to measure steady-state and immunoprecipitated protein levels from AHA/cyclooctyne-labeled protein samples, and (8) subsequently for downstream data analysis. Adapted with permission from Morey et al. (2016).
FIGURE 2
FIGURE 2
SPAAC pulse-chase reveals that ChAT protein half-life is reduced by mutation of an N-terminal proline-rich motif. (A) Fluorescence detection of immunoprecipitated (IP) AHA/TAMRA-labeled wild-type (WT) and P17A/P19A-ChAT from transiently transfected SN56 cells following SPAAC pulse-chase (4 h AHA pulse, 0–24 h methionine chase) with the strained cyclooctyne TAMRA-DIBO. Control unlabeled cells were incubated in media with methionine (i.e., without AHA). Fluorescent AHA/TAMRA-labeled proteins from either whole cell lysates or anti-ChAT IPs were detected in resolved SDS-PAGE gels at an Ex/Em of 555/580 nm. Anti-ChAT and anti-actin immunoblots were completed on AHA/TAMRA-labeled protein samples for downstream data analysis and as loading controls. (B) Protein half-life of P17A/P17A-ChAT (2.2 h) is reduced compared to wild-type ChAT (19.7 h). ChAT fluorescence intensities from (A) were plotted on a semi-logarithmic scale and linear regression was completed to determine ChAT protein half-life. The slopes of decay for wild-type and P17A/P19A-ChAT were significantly different: F(1,41) = 110.043, p ≤ 0.0001, n = 5. A 24 h time point was not included for the quantification of P17A/P19A-ChAT half-life as the fluorescence intensity was indistinguishable from background. (C) SN56 cells, transiently expressing either wild-type or P17A/P19A-ChAT, were treated with 100 μg/ml cycloheximide (CHX) for the time points indicated and anti-ChAT immunoblots were completed. Anti-actin immunoblots were completed as a loading control. (D) Protein half-life of P17A/P17A-ChAT (4.1 h) is reduced as compared to wild-type ChAT (44.9 h). Steady-state ChAT protein levels from (C) were plotted on a semi-logarithmic scale and linear regression completed to determine wild-type ChAT protein half-life. The slopes of decay for wild-type and P17A/P19A-ChAT were significantly different: F(1,46) = 36.802, p ≤ 0.0001, n = 5. Importantly, both methods (B,D) demonstrated that the protein half-life of P17A/P19A-ChAT is ∼10% that of wild-type ChAT. Adapted with permission from Morey et al. (2016).
FIGURE 3
FIGURE 3
Proteasome inhibition increases ChAT protein half-life. Fluorescence detection of immunoprecipitated (IP) AHA/TAMRA-labeled wild-type (WT; A) or P17A/P19A-ChAT (B) from transiently transfected SN56 cells following SPAAC pulse-chase (4 h AHA pulse, 0–24 h methionine chase) with the strained cyclooctyne TAMRA-DIBO. Cells were treated with either DMSO-control or 5 μM MG132 throughout both the 4 h AHA pulse and the 24 h chase periods (up to 28 h total). Control unlabeled cells were incubated in media with methionine (i.e., without AHA). Fluorescent AHA/TAMRA-labeled proteins from either whole cell lysates or anti-ChAT IPs were detected in resolved SDS-PAGE gels at an Ex/Em of 555/580 nm. Anti-ChAT and anti-actin immunoblots were completed on AHA/TAMRA-labeled protein samples for downstream data analysis and as loading controls. (C) Proteasome inhibition by MG132 treatment increased the protein half-life of wild-type ChAT (no protein decay) as compared to DMSO-treated control cells (22.5 h). Wild-type ChAT fluorescence intensities from (A) were plotted on a semi-logarithmic scale and linear regression was completed to determine wild-type ChAT protein half-life. The slopes of decay for DMSO- and MG132-treated cells were significantly different from each other: F(1,46) = 4.79241, p ≤ 0.05, n = 5. (D) Proteasome inhibition by MG132 treatment increased the protein half-life of mutant P17A/P19A-ChAT (16.6 h) as compared to DMSO-control (2.2 h). ChAT fluorescence intensities from (B) were plotted on a semi-logarithmic scale and linear regression was completed to determine P17A/P19A-ChAT protein half-life. The slopes of decay for DMSO- and MG132-treated cells were significantly different from each other: F(1,41) = 18.9864, p ≤ 0.0001, n = 5. A 24 h time point was not included for the quantification of P17A/P19A-ChAT half-life in DMSO-treated cells as the fluorescence intensity was indistinguishable from background.
FIGURE 4
FIGURE 4
Detection sensitivity comparison for three different strained cyclooctyne probes. HEK293 cells were live-labeled in culture for 4 h with 50 μM AHA then collected immediately without a methionine chase. Control unlabeled cells were incubated in media with methionine (i.e., without AHA). Cells were lysed and AHA-labeled protein samples from whole cell lysates were reacted with the strained cyclooctynes (A) TAMRA-DIBO, (B) 488-DBCO, or (C) Biotin-DIBO. AHA/cyclooctyne-labeled proteins were denatured in 1× Laemmli sample buffer, then serially diluted 1:1 with 1× Laemmli sample buffer a total of six times until reaching a final dilution of 1:64. Protein samples were run on SDS-PAGE gels and AHA-labeled proteins were detected in-gel at an Ex/Em of either 555/580 nm (A; TAMRA-DIBO) or 494/517 nm (B; 488-DBCO). Alternatively, AHA-labeled proteins reacted with Biotin-DIBO (C) were resolved on SDS-PAGE gels, then transferred to PVDF membranes and detected by chemiluminescence using HRP-conjugated streptavidin. Anti-actin immunoblots were completed as a loading control. Overall, similar detection sensitivity was observed between AHA-labeled protein samples reacted with TAMRA-DIBO, 488-DBCO, or Biotin-DIBO (n = 4).
FIGURE 5
FIGURE 5
Half-life determination of p53 by SPAAC pulse-chase. Detection of immunoprecipitated (IP) endogenous AHA-labeled p53 from HEK293 cells following SPAAC pulse-chase (4 h AHA pulse, 0–12 h methionine chase) with the strained cyclooctynes TAMRA-DIBO (A), 488-DBCO (B), or Biotin-DIBO (C). Control unlabeled cells were incubated in media with methionine (i.e., without AHA). AHA/cyclooctyne-labeled proteins from either whole cell lysates or anti-p53 IPs were resolved on SDS-PAGE gels and AHA-labeled proteins were detected in-gel at an Ex/Em of either 555/580 nm (A; TAMRA-DIBO) or 494/517 nm (B; 488-DBCO). Alternatively, AHA-labeled proteins reacted with Biotin-DIBO (C) were resolved on SDS-PAGE gels, then transferred to PVDF membranes and detected by chemiluminescence using a HRP-conjugated streptavidin. Anti-p53 and anti-vinculin immunoblots were completed on AHA/cyclooctyne-labeled protein samples for downstream data analysis and as loading controls. The protein half-life of AHA-labeled p53 when reacted with three different strained cyclooctynes was determined to be 10.3 h (D; TAMRA-DIBO), 12.7 h (E; 488-DBCO), or 11.0 h (F; Biotin-DIBO). Fluorescent or chemiluminescent intensities from immunoprecipitated p53 were plotted on a linear scale and linear regression analysis was completed to determine p53 protein half-life (n = 5).
FIGURE 6
FIGURE 6
SPAAC pulse-chase analysis of ChAT protein half-life in yeast. (A) Fluorescence detection of total AHA-labeled proteins from BY Δ pdr5 yeast cells following SPAAC pulse-chase (24 h AHA pulse, 0–8 h methionine chase) and labeling with the strained cyclooctyne TAMRA-DIBO. Cells were co-treated with either 50 μM MG132 or 10 μM Bafilomycin A throughout both the 24 h AHA-pulse and 8 h chase periods to inhibit the proteasome or lysosome, respectively. Control unlabeled cells were incubated in media with methionine (i.e., without AHA). (B) Fluorescence detection of immunoprecipitated (IP) AHA/TAMRA-labeled human ChAT protein from ChAT-expressing BY Δ pdr5 yeast cells following SPAAC pulse-chase with the strained cyclooctyne TAMRA-DIBO. Fluorescent AHA/TAMRA-labeled proteins from either whole cell lysates or anti-ChAT IPs were detected in resolved SDS-PAGE gels at an Ex/Em of 555/580 nm. Anti-ChAT and anti-PGK1 immunoblots were completed on AHA/TAMRA-labeled protein samples for downstream data analysis and as loading controls. (C) Proteasome inhibition by MG132 treatment increased the protein half-life of human ChAT (18.7 h) as compared to DMSO-treated control yeast cells [3.1 h; F(1,46) = 5.6432, p ≤ 0.001]. Bafilomycin A co-treatment had no significant effect on the half-life of AHA/TAMRA-labeled ChAT (2.2 h). ChAT fluorescence intensities from (B) were plotted on a semi-logarithmic scale and linear regression was completed to determine human ChAT protein half-life (n = 5).
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