A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 “clients”. We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

Deciphering Translation - a Genome-Wide Analysis of Translation Limitations and Competitions in Saccharomyces cerevisiae

Vassily Hatzimanikatis, Julien Racle

Protein synthesis is one of the central elements in every living cell. For this process, the mRNAs coding for genes are simultaneously competing for the same translation machinery, i.e. for the same ribosomes and the same protein building blocks, the amino acids. But the ultimate determinants of cellular functions are the proteins, it is therefore of crucial importance to understand well this translation process. Nowadays, it is possible to measure the changes in the levels of all mRNAs and proteins in the cell, under different environmental and genetic perturbations. The large amount of data from transcriptomic and proteomic studies requires development of efficient systems-wide mathematical and computational frameworks to analyze and interpret the data. For this purpose, we developed a genome-wide, mechanistic, mathematical model for translation in S. cerevisiae. This model includes information on the translation state of each mRNA (i.e. polysome size), and mRNA length, and can be used to estimate genome-scale kinetics of the protein synthesis machinery. It is then used to quantify the rate limiting steps of the translation process and gain insights into the mechanism of regulation of expression between different proteins. This framework allows us therefore to quantify the effect of a change in the total number of ribosomes, or also the effect of varying the level of a single mRNA on the various proteins. This information is then used to determine functional couplings in the yeast genome. We find that while 95% of the mRNA species are initiation and elongation limited, approximately 5% are elongation limited and have higher specific protein synthesis rates. We also observed that the mRNAs with ribosomal coverage greater than 0.8 display a decrease in the specific protein production rate with increasing coverage, and can be acting as sinks for system resources like ribosomes. It was also possible to determine which mRNA species have the most / least influence on the other, and we found that the most influential species are linked to proteins which form structural constituents of ribosomes, proteins related to translation elongation factor activity or key enzymes of the central carbon metabolism. Thus a perturbation to these key mRNA species has the maximum impact on protein synthesis from other mRNA species. Our results thus have implications in the understanding of the translation machinery response to perturbations and of the global systems response.

2010

Deciphering translation - a genome-wide analysis of translation in Escherichia coli

Vassily Hatzimanikatis, Julien Racle

Protein synthesis is one of the central elements in every living cell. For this process, mRNAs coding for genes are simultaneously competing for the same translation machinery (ribosomes and amino acids). But the ultimate determinants of cellular functions are the proteins, a good understanding of this process is therefore of crucial importance. For this purpose, we developed a genome-wide, mechanistic, mathematical model of translation, taking into account individual codon kinetics. It is used to quantify the rate limiting steps of translation and gain insight into the mechanism of regulation of expression between different proteins. We observe for each gene in a first low-occupancy phase a nearly linear increase in protein synthesis rate with amount of bound ribosomes to the mRNA, followed by a plateau of maximum synthesis rate. A further increase in the ribosomal coverage of the mRNA leads to a decrease in protein production rate, and might act as sinks for system resources like ribosomes. Native E. coli gene sequences are also compared to randomly obtained synonymous sequences, coding the same protein, and it is found some genes of higher significance for the cell have been better optimized for protein synthesis rate than other.

2010

A computational framework for the design of optimal protein synthesis

Vassily Hatzimanikatis, Jan Overney, Julien Racle

Despite the establishment of design principles to optimize codon choice for heterologous expression vector design, the relationship between codon sequence and final protein yield remains poorly understood. In this work, we present a computational framework for the identification of a set of mutant codon sequences for optimized heterologous protein production, which uses a codon-sequence mechanistic model of protein synthesis. Through a sensitivity analysis on the optimal steady state configuration of protein synthesis we are able to identify the set of codons, that are the most rate limiting with respect to steady state protein synthesis rate, and we replace them with synonymous codons recognized by charged tRNAs more efficient for translation, so that the resulting codon-elongation rate is higher. Repeating this procedure, we iteratively optimize the codon sequence for higher protein synthesis rate taking into account multiple constraints of various types. We determine a small set of optimized synonymous codon sequences that are very close to each other in sequence space, but they have an impact on properties such as ribosomal utilization or secondary structure. This limited number of sequences can then be offered for further experimental study. Overall, the proposed method is very valuable in understanding the effects of the different properties of mRNA sequences on the final protein yield in heterologous protein production and it can find applications in synthetic biology and biotechnology.

Wiley-Blackwell2012

Deciphering Translation - a Genome-Wide Analysis of Translation Limitations and Competitions in Saccharomyces cerevisiae

Vassily Hatzimanikatis, Julien Racle

2010