High-throughput Analysis of in vivo Protein Stability

Abstract

Determining the half-life of proteins is critical for an understanding of virtually all cellular processes. Current methods for measuring in vivo protein stability, including large-scale approaches, are limited in their throughput or in their ability to discriminate among small differences in stability. We developed a new method, Stable-seq, which uses a simple genetic selection combined with highthroughput DNA sequencing to assess the in vivo stability of a large number of variants of a protein The regulation of protein stability is critical in order for cells to maintain proper functioning of almost every process. Thus, approaches for measuring the in vivo stability of a protein are essential for the identification of components in proteolytic pathways that affect protein turnover and for an understanding of the consequences of their activities. These approaches include traditional biochemical methods such as the Western blot, in which samples taken from time points after the inhibition of protein expression are fractionated via gel electrophoresis and the relevant protein is visualized with the use of an antibody. Another method allows one to track the degradation rate of newly synthesized proteins by metabolically labeling proteins with a radioisotope and following their radioactivity. A third method fuses a protein to a reporter enzyme like ␤-galactosidase, allowing the steady-state level of a protein to be measured via the enzymatic activity of the reporter enzyme. However, these small-scale methods are limited in the number of samples that they can analyze.Large-scale methods have been developed that allow the simultaneous quantitation of the in vivo stability of many proteins. For example, Yen et al. fused ϳ8,000 human proteins to green fluorescent protein (GFP) 1 and followed the amount of each protein over time by using fluorescence-activated cell sorting (FACS) (1). To identify proteins, the plasmids encoding the GFP fusions were isolated and PCR products derived from these plasmids were hybridized to a DNA microarray. This method was applied to identify the substrates of a ubiquitin ligase complex (2). However, this method is limited by the number of bins into which protein fusions can be sorted in the FACS analysis and, consequently, how fine changes in stability can be discriminated. Alternatively, quantitative mass spectrometry has been used to analyze the stability of native proteins (3), but this approach often requires costly labeling and extensive data analysis. Moreover, these large-scale methods generally cannot distinguish differences in in vivo stability, which are sometimes significant, that result from small changes in a protein, such as single amino acid substitutions.We present a method, Stable-seq, for measuring the in vivo stability of large numbers of variants of a protein that combines a simple genetic selection with high-throughput DNA sequencing. Stable-seq is a form of deep mutational scanning (4, 5), in which a physical association between each protein variant and the DNA that ...

High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop

“…Deep mutational scanning is an emerging technique for studying large sets of mutants by assessing the enrichment or depletion of variants after a strict selection process [44]. Different selection approaches have been designed such that a specific protein property (sensitivity to substitutions [45], thermo-stability [46], protein stability [47], etc ) can be studied. Notably, our established genetic phenotypes (Table 1) were well correlated with altered transcription elongation rates in vitro and specific transcription defects in vivo [37,41], thus providing a powerful phenotypic framework for studying TL function.…”

Variant Interpretation: Functional Assays to the Rescue

“…[35][36][37] A yeast metabolic reporter fusion assay for variant stability has already been developed. 38 A similar assay could be developed in human cells. However, measuring steady-state abundance will be difficult for proteins that oligomerize or are found in complexes.…”

Measuring the activity of protein variants on a large scale using deep mutational scanning

“…In these assays, each cell expresses a variant that is subjected to functional selection. In one example, a deep mutational scan of a yeast degron was conducted using a cell-based reporter consisting of a fusion of the degron to a metabolic enzyme 16 . The growth of cells carrying each variant depended on the intracellular concentration of the metabolic enzyme, which, in turn, depended on the stability of the degron variant fused to it.…”