Section: New Results

Stochastic Models of Biological Networks

Participants : Renaud Dessalles, Sarah Eugene, Philippe Robert, Wen Sun.

Stochastic Modelling of self-regulation in the protein production system of bacteria.

This is a collaboration with Vincent Fromion from INRA Jouy-en-Josas, which started in December 2013.

In prokaryotic cells (e.g. E. Coli. or B. Subtilis) the protein production system has to produce in a cell cycle (i.e. less than one hour) more than 106 molecules of more than 2500 kinds, each having different level of expression. The bacteria uses more than 67% of its resources to the protein production. Gene expression is a highly stochastic process: bacteria sharing the same genome, in a same environment will not produce exactly the same amount of a given protein. Some of this stochasticity can be due to the system of production itself: molecules, that take part in the production process, move freely into the cytoplasm and therefore reach any target in the cell after some random time; some of them are present in so much limited amount that none of them can be available for a certain time; the gene can be deactivated by repressors for a certain time, etc. We study the integration of several mechanisms of regulation and their performances in terms of variance and distribution. As all molecules tends to move freely into the cytoplasm, it is assumed that the encounter time between a given entity and its target is exponentially distributed.

Feedback model

We have also investigated the production of a single protein, with the transcription and the translation steps, but we also introduced a direct feedback on it: the protein tends to bind on the promoter of its own gene, blocking therefore the transcription. The protein remains on it during an exponential time until its detachment caused by thermal agitation.

The mathematical analysis aims at understanding the nature of the internal noise of the system and to quantify it. We tend to test the hypothesis usually made that such feedback permits a noise reduction of protein distribution compared to the “open loop” model. We have made the mathematical analysis of the model (using a scaling to be able to have explicit results), it appeared that reduction of variance compared to an “open loop” model is limited: the variance cannot be reduced for more than 50%.

We proposed another possible effect of the feedback loop: the return to equilibrium is faster in the case of a feedback model compared to the open loop model. Such behaviour can be beneficial for the bacteria to change of command for a new level of production of a particular protein (due, for example, to a radical change in the environment) by reducing the respond time to reach this new average. This study has been mainly performed by simulation and it has been shown that the feedback model can go 50% faster than the open loop results.

Models with Cell Cycle

Usually, classical models of protein production do not explicitly represent several aspects of the cell cycle: the volume variations, the division and the gene replication. Yet these aspects have been proposed in literature to impact the protein production. We have therefore proposed a series of “gene-centered” models (that concentrates on the production of only one type of protein) that integrates successively all the aspects of the cell cycle. The goal is to obtain a realistic representation of the expression of one particular gene during the cell cycle. When it was possible, we analytically determined the mean and the variance of the protein concentration using Marked Poisson Point Process framework.

We based our analysis on a simple model where the volume changes across the cell cycle, and where only the mechanisms of protein production (transcription and translation) are represented. The variability predicted by this model is usually assimilated to the “intrinsic noise” (i.e. directly due to the protein production mechanism itself). We then add the random segregation of compounds at division to see its effect on protein variability: at division, every mRNA and every protein has an equal chance to go to either of the two daughter cells. It appears that this division sampling of compounds can add a significant variability to protein concentration. This effect directly depends on the relative variance (Fano factor) of the protein concentration: this effect is stronger as the relative variance is low. The dependence on the relative variance can be explained by considering a simplified model. With parameters deduced from real experimental measures, we estimate that the random segregation of compounds can double the variability of the genes with the lowest relative variance.

Finally, we integrate the gene replication to the model: at some point in the cell cycle, the gene is replicated, hence doubling the transcription rate. We are able to give analytical expressions for the mean and the variance of protein concentration at any moment of the cell cycle; it allows to directly compare the variance with the previous model with division. We show that gene replication has little impact on the protein variability: an environmental state decomposition shows that the part of the variance due to gene replication represents only at most 2% of the total variability predicted by the model.

In the end, these results are compared to the real experimental measure of protein variability. It appears that the models with cell cycle presented above tend to underestimate the protein variability especially for highly expressed proteins.

Multi-protein Model

In continuation of the previous models, we propose a model that still considers the division and the gene replication but which also integrates the sharing of common resources: the different genes are in competition for the limited quantity of RNA-polymerases and ribosomes in order to produce the mRNAs and proteins. The goal is to examine if fluctuations in the availability of these macromolecules have an important impact on the protein variability, as it has been suggested in literature. As the model considers the interaction between the different protein productions, one needs to represent all the genes of the bacteria altogether: it is therefore a multi-protein model.

As this model is too complex to be studied analytically, we have developed a procedure to estimate the parameters so that they correspond to real experimental measures. We then perform simulations in order to determine the variance of each protein and compare them with the one predicted by the models with cell cycle previously presented. It appears that the common sharing of RNA-polymerases and ribosomes has a limited impact on the protein production: for most of proteins the variance increases of at most 10%.

Finally, we have investigated other possible sources of variability by presenting other simulations that integrate some specific aspects: variability in the production of RNA-polymerases and ribosomes, uncertainty in the division and DNA replication decisions, etc. None of the considered aspects seems to have a significant impact on the protein variability.

Stochastic Modelling of Protein Polymerization

This is a collaboration with Marie Doumic, Inria MAMBA team.

The first part of our work focuses on the study of the polymerization of protein. This phenomenon is involved in many neurodegenerative diseases such as Alzheimer's and Prion diseases, e.g. mad cow. In this context, it consists in the abnormal aggregation of proteins. Curves obtained by measuring the quantity of polymers formed in in vitro experiments are sigmoids: a long lag phase with almost no polymers followed by a fast consumption of all monomers. Furthermore, repeating the experiment under the same initial conditions leads to somewhat identical curves up to translation. After having proposed a simple model to explain this fluctuations, we studied a more sophisticated model, closer to the reality. We added a conformation step: before being able to polymerize, proteins have to misfold. This step is very quick and remains at equilibrium during the whole process. Nevertheless, this equilibrium depends on the polymerization which is happening on a slower time scale. The analysis of these models involves stochastic averaging principles.

We have also investigated a more detailed model of polymerisation by considering the the evolution of the number of polymers with different sizes (Xi(t)) where Xi(t) is the number of polymers of size i at time t. By assuming that the transitions rates are scaled by a large parameter N, it has been shown that, in the limit, the process (XiN(t)) is converging to the solution of Becker-Döring equations as N goes to infinity. For another model including nucleation, we have given an asymptotic description of the lag time at the first and second order. These results are obtained in particular by proving stochastic averaging theorems.

The second part concerns the study of telomeres. This work is made in collaboration with Zhou Xu, Teresa Teixeira, from IBCP in Paris.

In eukaryotic cells, at each mitosis, chromosomes are shortened, because the DNA polymerase is not able to duplicate one ending of the chromosome. To prevent loss of genetic information- which could be catastrophic for the cell-chromosomes are equipped with telomeres at their endings. These telomeres do not contain any genetic information; they are a repetition of the sequence T-T-A-G-G-G thousands times. At each mitosis, there is therefore a loss of telomere. As it has a finite length, when the telomeres are too short, the cell cannot divide anymore: they enter in replicative senescence. Our model tries to captures the two phases of the shortening of telomeres: first, the initial state of the cells, when the telomerase is still active to repair the telomeres. Second, when the telomerase is inhibited, we try to estimate the senescence threshold, when the replication of the cells stops. See [8].