Biology subjects

Jacquet, P.

Publications and source records attributed to Jacquet, P..

4 recordsLinked to original sources

bioRxiv2026

Time-resolved functional rotation crystallography reveals protein dynamics and catalysis

Time-resolved crystallography offers a window into the transient, atomic-scale structural changes that underlie biological function. Here we introduce Time-Resolved Functional Rotation Crystallography (TR-FRX), a method that enables molecular-level studies of ligand binding and enzymatic catalysis using single protein crystals at room temperature. Unlike many state-of-the-art time-resolved methodologies, TR-FRX relies on standard rotation-based X-ray data collection and does not require serial sample delivery strategies. By dispensing nanoliter-scale droplets of ligand or substrate directly onto crystals mounted in conventional holders, TR-FRX captures real-time structural snapshots while maintaining experimental accessibility. As a proof-of-concept, we first monitor the binding of N-acetylglucosamine to hen egg-white lysozyme, demonstrating that TR-FRX can reveal ligand recognition in crystallo on sub-second timescales. We then resolve the bidirectional catalytic mechanism of a prototypical enzyme from the tricarboxylic-acid-cycle, revealing sequential cofactor and substrate binding, catalytic loop dynamics, and substrate stabilization across time points spanning from 137 milliseconds to minutes. Requiring only micrograms of protein and standard beamline infrastructure, TR-FRX provides an accessible methodology for capturing transient enzymatic states and enables 100-ms timescale studies at room temperature on single protein crystals.

biochemistry↗

bioRxiv2023

A computational model of the spatiotemporal adaptation of tumor cells metabolism in a growing spheroid

The Warburg effect, commonly depicted as an inherent metabolic trait of cancer in literature, is under intensive investigation to comprehend its origins. However, while the prolonged presence of excessive lactic acid production in tumors has been noted, it merely constitutes a fraction of the potential metabolic states cancer cells can adopt. This study aimed to elucidate the emergence of spatiotemporal diversity in tumor energy metabolism by expanding an existing model based on experimental facts. The resulting hybrid model integrates discrete formulations for individual cells and their processes, along with continuous elements for metabolism and the diffusion of crucial environmental substrates like oxygen, glucose, lactate, and the often underestimated acidity. This model enables simulation of a tumor spheroid, a standard experimental model, composed of numerous cells which can have distinct traits. By subjecting the spheroid to alterations of the environment such as cyclic hypoxia, acid shocks, or glucose deprivation, novel insights into metabolic regulation were obtained. The findings underscore the significance of the pyruvate-lactate interaction in governing tumor metabolic routes. Integrating aciditys impact into the model, revealed its pivotal role in energy pathway regulation. Consequently, the conventional portrayal of a respiration/fermentation dichotomy proves inaccurate, as cells continuously and spatially adjust the ratio of these energy production modes, in contrast to abrupt, irreversible switches. Moreover, a cooperative cellular behavior akin to the reverse Warburg effect has emerged. This implies that the Warburg effect is not universally inherent to tumor metabolism, but a contextual, transient metabolic expression. Ultimately, the dynamic cellular-environment metabolic landscape influences cells survival under external conditions, with epigenetic regulations shaping their mobility potential within this landscape. While genetic mutations within tumor cells are undoubtedly present, this study shows they are not invariably essential for extreme metabolic modes or pathological characteristics to arise. Consequently, this research paves the way for innovative perspectives on metabolism, guiding tailored therapeutic strategies that consider not just patient-specific tissue attributes but also treat tumors as intricate ecosystems beyond their genetic diversity. Author SummaryFor years, scientists have been intrigued by the peculiar energy consumption patterns of cancer cells, such as the Warburg effect characterized by excessive lactic acid production. This study aimed to decipher the underlying reasons for the varying energy behaviors observed in different parts of tumors. Using a computational model, we simulated the collaborative dynamics of cells within tumors. The results revealed compelling insights. Two molecules, pyruvate and lactate, were identified as influential players in shaping energy utilization. Remarkably, the surrounding acidity was also found to exert a significant impact. Interestingly, tumor cells display a certain flexibility in their energy production strategies, adjusting according to prevailing conditions to maintain their survival and adaptability. Interestingly, cellular cooperation challenges the Warburg effect as an omnipresent phenomenon and reveals a transient nature. Our study underscores the significance of environmental influences, shedding light on the interplay between genetic modifications and the tumor environment in shaping cellular behavior. These findings hold promise for transforming cancer comprehension and devising treatments that tailor to both patients and the distinctive characteristics of their tumors.

cancer biology↗

bioRxiv2023

Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity

Enzymatic promiscuity, the ability of enzymes to catalyze multiple, distinct chemical reactions, has been well documented and is hypothesized to be a major driver for the emergence of new enzymatic functions. Yet, the molecular mechanisms involved in the transition from one activity to another remain debated and elusive. Here, we evaluated the redesign of the active site binding cleft of the lactonase SsoPox using structure-based design and combinatorial libraries. We created variants with largely improved catalytic abilities against phosphotriesters, the best ones being > 1,000-fold better compared to the wild-type enzyme. The observed shifts in activity specificity are large, [~]1,000,000-fold and beyond, since some variants completely lost their initial activity. The selected combinations of mutations have considerably reshaped the active site cavity via side chain changes but mostly through large rearrangements of the active site loops, as revealed by a suite of crystal structures. This suggests that specific active site loop configuration is critical to the lactonase activity. Interestingly, analysis of high-resolution structures hints at the potential role of conformational sampling and its directionality in defining an enzyme activity profile.

biochemistry↗

bioRxiv2019

Why the tumor cell metabolism is not that abnormal

The cell energy metabolism is a multifactorial and evolving process that we address with a theoretical approach in order to decipher the functioning of the core system of the glycolysis-OXPHOS relationship. The model is based on some key experimental observations and well established facts. It emphasizes the role of lactate as a substrate, as well as the central role of pyruvate in the regulation of the metabolism. The simulations show how imposed environmental constraints and imposed energy requirements push the cell to adapt its metabolism to sustain its needs. The results highlight the cooperativeness of the two metabolic modes and allows to revisit the notions of metabolic switch and metabolic reprogramming. Our results thus tend to show that the Warburg effect is not an inherent characteristic of the tumor cell, but a spontaneous and transitory adaptation mechanism to a disturbed environment. This means that the tumor cell metabolism is not fundamentally different from that of a normal cell. This has implications on the way therapies are being considered. The quest to normalize the tumor acidity could be a good strategy. Author SummaryCancer cells metabolism focuses the interest of the cancer research community. Although this process is intensely studied experimentally, there exists very few theoretical models that tackle this issue. One main reason is the extraordinary complexity of the metabolism that involves many inter-related regulation networks which makes it illusory to recreate computationally this complexity. In this study we propose a simplified model of the metabolism which focuses on the interrelation of the three main energetic metabolites that are oxygen, glucose and lactate with the aim to better understand the dynamic of the core system of the glycolysis-OXPHOS relationship. However simple, the model highlights the main rules that allow the cell to dynamically adapt its metabolism to its changing environment. It moreover allows to address this impact at the tissue scale. Simulations performed in a spheroid exhibit non-trivial spatial heterogeneity of the energy metabolism. It further reveals that the metabolic features that are commonly assigned to cancer cells are not necessarily due to cell intrinsic abnormality. They can emerge spontaneously because of the disregulated over-acidic environment.

cell biology↗