Modulating Protein Stability to Switch Toxic Protein Function On and Off in Living Cells.

Toxic proteins are prime targets for molecular farming (the generation of pharmacologically active or biotechnologically usable compounds in plants) and are also efficient tools for targeted cell ablation in genetics, developmental biology, and biotechnology. However, achieving conditional activity of cytotoxins and maintaining the toxin-expressing plants as stably transformed lines remain challenging. Here, we produce a switchable version of the highly cytotoxic bacterial RNase barnase by fusing the protein to a portable protein degradation cassette, the low-temperature degron cassette.

Real-time detection of N-end rule-mediated ubiquitination via fluorescently labeled substrate probes.

The N-end rule pathway has emerged as a major system for regulating protein functions by controlling their turnover in medical, animal and plant sciences as well as agriculture. Although novel functions and enzymes of the pathway have been discovered, the ubiquitination mechanism and substrate specificity of N-end rule pathway E3 ubiquitin ligases have remained elusive. Taking the first discovered bona fide plant N-end rule E3 ligase PROTEOLYSIS1 (PRT1) as a model, we used a novel tool to molecularly characterize polyubiquitination live, in real time.

Phenotypes on demand via switchable target protein degradation in multicellular organisms.

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo.

Normalized Quantitative Western Blotting Based on Standardized Fluorescent Labeling.

Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein-epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable.

An improved workflow for quantitative N-terminal charge-based fractional diagonal chromatography (ChaFRADIC) to study proteolytic events in Arabidopsis thaliana.

We applied an extended charge-based fractional diagonal chromatography (ChaFRADIC) workflow to analyze the N-terminal proteome of Arabidopsis thaliana seedlings. Using iTRAQ protein labeling and a multi-enzyme digestion approach including trypsin, GluC, and subtilisin, a total of 200 μg per enzyme, and measuring only one third of each ChaFRADIC-enriched fraction by LC-MS, we quantified a total of 2791 unique N-terminal peptides corresponding to 2249 different unique N-termini from 1270 Arabidopsis proteins. Our data indicate the power, reproducibility, and sensitivity of the applied strategy that might be applicable to quantify proteolytic events from as little as 20 μg of protein per condition across up to eight different samples.

Generic tools for conditionally altering protein abundance and phenotypes on demand.

Conditional gene expression and modulating protein stability under physiological conditions are important tools in biomedical research. They led to a thorough understanding of the roles of many proteins in living organisms. Current protocols allow for manipulating levels of DNA, mRNA, and of functional proteins.