Gabriel Piedrafita

Allele-Specific Gene Expression Is Widespread Across the Genome and Biological Processes

Allelic specific gene expression (ASGE) appears to be an important factor in human phenotypic variability and as a consequence, for the development of complex traits and diseases. In order to study ASGE across the human genome, we have performed a study in which genotyping was coupled with an analysis of ASGE by screening 11,500 SNPs using the Mapping 10 K Array to identify differential allelic expression. We found that from the 5,133 SNPs that were suitable for analysis (heterozygous in our sample and expressed in peripheral blood mononuclear cells), 2,934 (57%) SNPs had differential allelic expression. Such SNPs were equally distributed along human chromosomes and biological processes. We validated the presence or absence of ASGE in 18 out 20 SNPs (90%) randomly selected by real time PCR in 48 human subjects. In addition, we observed that SNPs close to -but not included in- segmental duplications had increased levels of ASGE. Finally, we found that transcripts of unknown function or non-coding RNAs, also display ASGE: from a total of 2,308 intronic SNPs, 1510 (65%) SNPs underwent differential allelic expression. In summary, ASGE is a widespread mechanism in the human genome whose regulation seems to be far more complex than expected.

The widespread role of non-enzymatic reactions in cellular metabolism

Graphical abstract

A simple self-maintaining metabolic system: robustness, autocatalysis, bistability.

A living organism must not only organize itself from within; it must also maintain its organization in the face of changes in its environment and degradation of its components. We show here that a simple (M,R)-system consisting of three interlocking catalytic cycles, with every catalyst produced by the system itself, can both establish a non-trivial steady state and maintain this despite continuous loss of the catalysts by irreversible degradation. As long as at least one catalyst is present at a sufficient concentration in the initial state, the others can be produced and maintained. The system shows bistability, because if the amount of catalyst in the initial state is insufficient to reach the non-trivial steady state the system collapses to a trivial steady state in which all fluxes are zero. It is also robust, because if one catalyst is catastrophically lost when the system is in steady state it can recreate the same state. There are three elementary flux modes, but none of them is an enzyme-maintaining mode, the entire network being necessary to maintain the two catalysts.

The Impact of Non-Enzymatic Reactions and Enzyme Promiscuity on Cellular Metabolism during (Oxidative) Stress Conditions

Cellular metabolism assembles in a structurally highly conserved, but functionally dynamic system, known as the metabolic network. This network involves highly active, enzyme-catalyzed metabolic pathways that provide the building blocks for cell growth. In parallel, however, chemical reactivity of metabolites and unspecific enzyme function give rise to a number of side products that are not part of canonical metabolic pathways. It is increasingly acknowledged that these molecules are important for the evolution of metabolism, affect metabolic efficiency, and that they play a potential role in human disease—age-related disorders and cancer in particular. In this review we discuss the impact of oxidative and other cellular stressors on the formation of metabolic side products, which originate as a consequence of: (i) chemical reactivity or modification of regular metabolites; (ii) through modifications in substrate specificity of damaged enzymes; and (iii) through altered metabolic flux that protects cells in stress conditions. In particular, oxidative and heat stress conditions are causative of metabolite and enzymatic damage and thus promote the non-canonical metabolic activity of the cells through an increased repertoire of side products. On the basis of selected examples, we discuss the consequences of non-canonical metabolic reactivity on evolution, function and repair of the metabolic network.

Nonenzymatic gluconeogenesis-like formation of fructose 1,6-bisphosphate in ice

Significance It is still unknown how an early metabolism produced the sugar phosphates central for life. We provide evidence that gluconeogenesis, the anabolic counterpart to glycolysis, could have emerged nonenzymatically. We describe that the gluconeogenic carbon-bond–forming reaction has a nonenzymatic pendant that occurs in ice and that leads to the accumulation of fructose 1,6-bisphosphate as (re-)built from glycolytic catabolites. As a nonenzymatic glycolysis has been described previously, the discovery of this reaction could both help to explain the origin of the larger cellular sugar phosphates and provide a scenario in which an early metabolic system was able to escape equilibrium. The reaction further hints that the earliest anabolic enzymes could have been as simple as single amino acids. The evolutionary origins of metabolism, in particular the emergence of the sugar phosphates that constitute glycolysis, the pentose phosphate pathway, and the RNA and DNA backbone, are largely unknown. In cells, a major source of glucose and the large sugar phosphates is gluconeogenesis. This ancient anabolic pathway (re-)builds carbon bonds as cleaved in glycolysis in an aldol condensation of the unstable catabolites glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, forming the much more stable fructose 1,6-bisphosphate. We here report the discovery of a nonenzymatic counterpart to this reaction. The in-ice nonenzymatic aldol addition leads to the continuous accumulation of fructose 1,6-bisphosphate in a permanently frozen solution as followed over months. Moreover, the in-ice reaction is accelerated by simple amino acids, in particular glycine and lysine. Revealing that gluconeogenesis may be of nonenzymatic origin, our results shed light on how glucose anabolism could have emerged in early life forms. Furthermore, the amino acid acceleration of a key cellular anabolic reaction may indicate a link between prebiotic chemistry and the nature of the first metabolic enzymes.

Viability Conditions for a Compartmentalized Protometabolic System: A Semi-Empirical Approach

In this work we attempt to find out the extent to which realistic prebiotic compartments, such as fatty acid vesicles, would constrain the chemical network dynamics that could have sustained a minimal form of metabolism. We combine experimental and simulation results to establish the conditions under which a reaction network with a catalytically closed organization (more specifically, an ()-system) would overcome the potential problem of self-suffocation that arises from the limited accessibility of nutrients to its internal reaction domain. The relationship between the permeability of the membrane, the lifetime of the key catalysts and their efficiency (reaction rate enhancement) turns out to be critical. In particular, we show how permeability values constrain the characteristic time scale of the bounded protometabolic processes. From this concrete and illustrative example we finally extend the discussion to a wider evolutionary context.

Stochastic simulations of mixed-lipid compartments: from self-assembling vesicles to self-producing protocells.

The computational platform ENVIRONMENT, developed to simulate stochastically reaction systems in varying compartmentalized conditions [Mavelli and Ruiz-Mirazo: Philos Trans R Soc Lond B Biol Sci 362:1789-1802, 2007; Physical Biology 7(3): 036002, 2010], is here applied to study the dynamic properties and stability of model protocells that start producing their own lipid molecules (e.g., phospholipids), which get inserted in previously self-assembled vesicles, made of precursor amphiphiles (e.g., fatty acids). Attention is mainly focused on the changes that this may provoke in the permeability of the compartment, as well as in its eventual osmotic robustness.

Permeability-driven selection in a semi-empirical protocell model: the roots of prebiotic systems evolution

The origin-of-life problem has been traditionally conceived as the chemical challenge to find the type of molecule and free-solution reaction dynamics that could have started Darwinian evolution. Different autocatalytic and ‘self-replicative’ molecular species have been extensively investigated, together with plausible synthetic pathways that might have led, abiotically, to such a minimalist scenario. However, in addition to molecular kinetics or molecular evolutionary dynamics, other physical and chemical constraints (like compartmentalization, differential diffusion, selective transport, osmotic forces, energetic couplings) could have been crucial for the cohesion, functional integration, and intrinsic stability/robustness of intermediate systems between chemistry and biology. These less acknowledged mechanisms of interaction and molecular control might have made the initial pathways to prebiotic systems evolution more intricate, but were surely essential for sustaining far-from-equilibrium chemical dynamics, given their functional relevance in all modern cells. Here we explore a protocellular scenario in which some of those additional constraints/mechanisms are addressed, demonstrating their ‘system-level’ implications. In particular, an experimental study on the permeability of prebiotic vesicle membranes composed of binary lipid mixtures allows us to construct a semi-empirical model where protocells are able to reproduce and undergo an evolutionary process based on their coupling with an internal chemistry that supports lipid synthesis.

Identifying ultrasensitive HGF dose-response functions in a 3D mammalian system for synthetic morphogenesis.

Nonlinear responses to signals are widespread natural phenomena that affect various cellular processes. Nonlinearity can be a desirable characteristic for engineering living organisms because it can lead to more switch-like responses, similar to those underlying the wiring in electronics. Steeper functions are described as ultrasensitive, and can be applied in synthetic biology by using various techniques including receptor decoys, multiple co-operative binding sites, and sequential positive feedbacks. Here, we explore the inherent non-linearity of a biological signaling system to identify functions that can potentially be exploited using cell genome engineering. For this, we performed genome-wide transcription profiling to identify genes with ultrasensitive response functions to Hepatocyte Growth Factor (HGF). We identified 3,527 genes that react to increasing concentrations of HGF, in Madin-Darby canine kidney (MDCK) cells, grown as cysts in 3D collagen cell culture. By fitting a generic Hill function to the dose-responses of these genes we obtained a measure of the ultrasensitivity of HGF-responsive genes, identifying a subset with higher apparent Hill coefficients (e.g. MMP1, TIMP1, SNORD75, SNORD86 and ERRFI1). The regulatory regions of these genes are potential candidates for future engineering of synthetic mammalian gene circuits requiring nonlinear responses to HGF signalling.

On the transition from prebiotic to proto-biological membranes: from 'self-assembly' to 'self-production'

A model is here presented to analyse how vesicles may turn into protocells that synthesize their own lipid components and the consequences that this may have on the properties of the resulting membrane (in particular, on its permeability), as well as on the overall stability of the system.