Quantitative Analysis of the Effective Functional Structure in Yeast Glycolysis

Yeast glycolysis is considered the prototype of dissipative biochemical oscillators. In cellular conditions, under sinusoidal source of glucose, the activity of glycolytic enzymes can display either periodic, quasiperiodic or chaotic behavior. In order to quantify the functional connectivity for the glycolytic enzymes in dissipative conditions we have analyzed different catalytic patterns using the non-linear statistical tool of Transfer Entropy. The data were obtained by means of a yeast glycolytic model formed by three delay differential equations where the enzymatic speed functions of the irreversible stages have been explicitly considered. These enzymatic activity functions were previously modeled and tested experimentally by other different groups. In agreement with experimental conditions, the studied time series corresponded to a quasi-periodic route to chaos. The results of the analysis are three-fold: first, in addition to the classical topological structure characterized by the specific location of enzymes, substrates, products and feedback regulatory metabolites, an effective functional structure emerges in the modeled glycolytic system, which is dynamical and characterized by notable variations of the functional interactions. Second, the dynamical structure exhibits a metabolic invariant which constrains the functional attributes of the enzymes. Finally, in accordance with the classical biochemical studies, our numerical analysis reveals in a quantitative manner that the enzyme phosphofructokinase is the key-core of the metabolic system, behaving for all conditions as the main source of the effective causal flows in yeast glycolysis.Comment: Biologically improve

The Metabolic Core and Catalytic Switches Are Fundamental Elements in the Self-Regulation of the Systemic Metabolic Structure of Cells

[Background] Experimental observations and numerical studies with dissipative metabolic networks have shown that cellular enzymatic activity self-organizes spontaneously leading to the emergence of a metabolic core formed by a set of enzymatic reactions which are always active under all environmental conditions, while the rest of catalytic processes are only intermittently active. The reactions of the metabolic core are essential for biomass formation and to assure optimal metabolic performance. The on-off catalytic reactions and the metabolic core are essential elements of a Systemic Metabolic Structure which seems to be a key feature common to all cellular organisms. [Methodology/Principal Findings] In order to investigate the functional importance of the metabolic core we have studied different catalytic patterns of a dissipative metabolic network under different external conditions. The emerging biochemical data have been analysed using information-based dynamic tools, such as Pearson's correlation and Transfer Entropy (which measures effective functionality). Our results show that a functional structure of effective connectivity emerges which is dynamical and characterized by significant variations of bio-molecular information flows. [Conclusions/Significance] We have quantified essential aspects of the metabolic core functionality. The always active enzymatic reactions form a hub –with a high degree of effective connectivity- exhibiting a wide range of functional information values being able to act either as a source or as a sink of bio-molecular causal interactions. Likewise, we have found that the metabolic core is an essential part of an emergent functional structure characterized by catalytic modules and metabolic switches which allow critical transitions in enzymatic activity. Both, the metabolic core and the catalytic switches in which also intermittently-active enzymes are involved seem to be fundamental elements in the self-regulation of the Systemic Metabolic Structure.Consejo Superior de Investigaciones Cientificas (CSIC),grant 201020I026. Ministerio de Ciencia e Innovacion (MICINN). Programa Ramon y Cajal. Campus de Excelencia Internacional CEI BioTIC GENIL, grant PYR-2010-14. Junta de Andalucia, grant P09-FQM-4682

Global Self-Regulation of the Cellular Metabolic Structure

Different studies have shown that cellular enzymatic activities are able to self-organize spontaneously, forming a metabolic core of reactive processes that remain active under different growth conditions while the rest of the molecular catalytic reactions exhibit structural plasticity. This global cellular metabolic structure appears to be an intrinsic characteristic common to all cellular organisms. Recent work performed with dissipative metabolic networks has shown that the fundamental element for the spontaneous emergence of this global self-organized enzymatic structure could be the number of catalytic elements in the metabolic networks.This work was supported by the Spanish Ministry of Science and Education Grants with the projects MTM2007-62186 and MTM2005-01504 and by the Basque Government grants GIC07/151-IT-254-07 and IT-305-07. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Associative Conditioning Is a Robust Systemic Behavior in Unicellular Organisms: An Interspecies Comparison

The capacity to learn new efficient systemic behavior is a fundamental issue of contemporary biology. We have recently observed, in a preliminary analysis, the emergence of conditioned behavior in some individual amoebae cells. In these experiments, cells were able to acquire new migratory patterns and remember them for long periods of their cellular cycle, forgetting them later on. Here, following a similar conceptual framework of Pavlov’s experiments, we have exhaustively studied the migration trajectories of more than 2000 individual cells belonging to three different species: Amoeba proteus, Metamoeba leningradensis, and Amoeba borokensis. Fundamentally, we have analyzed several relevant properties of conditioned cells, such as the intensity of the responses, the directionality persistence, the total distance traveled, the directionality ratio, the average speed, and the persistence times. We have observed that cells belonging to these three species can modify the systemic response to a specific stimulus by associative conditioning. Our main analysis shows that such new behavior is very robust and presents a similar structure of migration patterns in the three species, which was characterized by the presence of conditioning for long periods, remarkable straightness in their trajectories and strong directional persistence. Our experimental and quantitative results, compared with other studies on complex cellular responses in bacteria, protozoa, fungus-like organisms and metazoans that we discus here, allow us to conclude that cellular associative conditioning might be a widespread characteristic of unicellular organisms. This new systemic behavior could be essential to understand some key principles involved in increasing the cellular adaptive fitness to microenvironments.This work was supported by a grant of the University of Basque Country (UPV/EHU), GIU17/066, the Basque Government grant IT974-16, the UPV/EHU and Basque Center of Applied Mathematics, grant US18/21, and the Israel Science Foundation (536/19)Peer reviewe

On the dynamics of the adenylate energy system: homeorhesis vs homeostasis.

Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life

Global Self-Organization of the Cellular Metabolic Structure

Background: Over many years, it has been assumed that enzymes work either in an isolated way, or organized in small catalytic groups. Several studies performed using "metabolic networks models'' are helping to understand the degree of functional complexity that characterizes enzymatic dynamic systems. In a previous work, we used "dissipative metabolic networks'' (DMNs) to show that enzymes can present a self-organized global functional structure, in which several sets of enzymes are always in an active state, whereas the rest of molecular catalytic sets exhibit dynamics of on-off changing states. We suggested that this kind of global metabolic dynamics might be a genuine and universal functional configuration of the cellular metabolic structure, common to all living cells. Later, a different group has shown experimentally that this kind of functional structure does, indeed, exist in several microorganisms. Methodology/Principal Findings: Here we have analyzed around 2.500.000 different DMNs in order to investigate the underlying mechanism of this dynamic global configuration. The numerical analyses that we have performed show that this global configuration is an emergent property inherent to the cellular metabolic dynamics. Concretely, we have found that the existence of a high number of enzymatic subsystems belonging to the DMNs is the fundamental element for the spontaneous emergence of a functional reactive structure characterized by a metabolic core formed by several sets of enzymes always in an active state. Conclusions/Significance: This self-organized dynamic structure seems to be an intrinsic characteristic of metabolism, common to all living cellular organisms. To better understand cellular functionality, it will be crucial to structurally characterize these enzymatic self-organized global structures.Supported by the Spanish Ministry of Science and Education Grants MTM2005-01504, MTM2004-04665, partly with FEDER funds, and by the Basque Government, Grant IT252-07

First computational design using lambda-superstrings and in vivo validation of SARS-CoV-2 vaccine

Coronavirus disease 2019 (COVID-19) is the greatest threat to global health at the present time, and considerable public and private effort is being devoted to fighting this recently emerged disease. Despite the undoubted advances in the development of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uncertainty remains about their future efficacy and the duration of the immunity induced. It is therefore prudent to continue designing and testing vaccines against this pathogen. In this article we computationally designed two candidate vaccines, one monopeptide and one multipeptide, using a technique involving optimizing lambda-superstrings, which was introduced and developed by our research group. We tested the monopeptide vaccine, thus establishing a proof of concept for the validity of the technique. We synthesized a peptide of 22 amino acids in length, corresponding to one of the candidate vaccines, and prepared a dendritic cell (DC) vaccine vector loaded with the 22 amino acids SARS-CoV-2 peptide (positions 50-71) contained in the NTD domain (DC-CoVPSA) of the Spike protein. Next, we tested the immunogenicity, the type of immune response elicited, and the cytokine profile induced by the vaccine, using a non-related bacterial peptide as negative control. Our results indicated that the CoVPSA peptide of the Spike protein elicits noticeable immunogenicity in vivo using a DC vaccine vector and remarkable cellular and humoral immune responses. This DC vaccine vector loaded with the NTD peptide of the Spike protein elicited a predominant Th1-Th17 cytokine profile, indicative of an effective anti-viral response. Finally, we performed a proof of concept experiment in humans that included the following groups: asymptomatic non-active COVID-19 patients, vaccinated volunteers, and control donors that tested negative for SARS-CoV-2. The positive control was the current receptor binding domain epitope of COVID-19 RNA-vaccines. We successfully developed a vaccine candidate technique involving optimizing lambda-superstrings and provided proof of concept in human subjects. We conclude that it is a valid method to decipher the best epitopes of the Spike protein of SARS-CoV-2 to prepare peptide-based vaccines for different vector platforms, including DC vaccines.Luis Martínez and Iker Malaina were supported by the Basque Government, grants IT974-16 and KK-2018/00090 and by the UPV/EHU and Basque Center of Applied Mathematics, grants US18/21 and US21/27. Carmen Alvarez-Dominguez was funded by the Instituto de Salud Carlos III, grants DTS18-00022 and PI19-01580, co-funded in part with European FEDER funds “A new way of making Europe”, the Instituto de Investigación Marqués de Valdecilla, grant INNVAL20/01, and the COST European action ENOVA CA-16231. David Salcines-Cuevas was supported by a predoctoral contract for the BioHealth research program of the Cantabria government. Hector Teran-Navarro salary was supported by the Instituto de Investigación Marqués de Valdecilla, grant INNVAL19/26. Andrea Zeoli was an Erasmus student from the University of Milan “La Statale” (Milan, Italy) performing a stay at IDIVAL.Peer reviewe

Stability of SARS-CoV-2 spike antigens against mutations

Modern health care needs preventive vaccines and therapeutic treatments with stability against pathogen mutations to cope with current and future viral infections. At the beginning of the COVID-19 pandemic, our analytic and predictive tool identified a set of eight short SARS-CoV-2 S-spike protein epitopes that had the potential to persistently avoid mutation. Here a combination of genetic, Systems Biology and protein structure analyses confirm the stability of our identified epitopes against viral mutations. Remarkably, this research spans the whole period of the pandemic, during which 93.9% of the eight peptides remained invariable in the globally predominant 43 circulating variants, including Omicron. Likewise, the selected epitopes are conserved in 97% of all 1,514 known SARS-CoV-2 lineages. Finally, experimental analyses performed with these short peptides showed their specific immunoreactivity. This work opens a new perspective on the design of next-generation vaccines and antibody therapies that will remain reliable against future pathogen mutations.Dr. Lozano-Perez acknowledges the European Commission ERDF/FEDER Operational Program 'Murcia' CCI No. 2007ES161PO001 (Project No. 14-20/20). Miodrag Grbic acknowledges support from the NSERC Discovery grant (Canada). This work also has received funding from the Department of Education of the Basque Government via the Consolidated Research Group MATH MODE (IT1456-22). Besides, Ildefonso Martinez De la Fuente and Iker Malaina were supported by the UPV/EHU and Basque Center of Applied Mathematics, grant US21/27N