Computational modeling reveals molecular details of epidermal growth factor binding

BACKGROUND: The ErbB family of receptors are dysregulated in a number of cancers, and the signaling pathway of this receptor family is a critical target for several anti-cancer drugs. Therefore a detailed understanding of the mechanisms of receptor activation is critical. However, despite a plethora of biochemical studies and recent single particle tracking experiments, the early molecular mechanisms involving epidermal growth factor (EGF) binding and EGF receptor (EGFR) dimerization are not as well understood. Herein, we describe a spatially distributed Monte Carlo based simulation framework to enable the simulation of in vivo receptor diffusion and dimerization. RESULTS: Our simulation results are in agreement with the data from single particle tracking and biochemical experiments on EGFR. Furthermore, the simulations reveal that the sequence of receptor-receptor and ligand-receptor reaction events depends on the ligand concentration, receptor density and receptor mobility. CONCLUSION: Our computer simulations reveal the mechanism of EGF binding on EGFR. Overall, we show that spatial simulation of receptor dynamics can be used to gain a mechanistic understanding of receptor activation which may in turn enable improved cancer treatments in the future

Microscopy Techniques for Investigating Interactions in Microbial Systems

Biological interactions occur on multiple length scales, ranging from molecular to population wide interactions. This work describes the study of two specific areas of biological interactions in microbial systems: intracellular protein-protein interactions and cell-to-cell interactions. The implementation of optical and atomic force microscopy and the methodologies developed during this study proved to be invaluable tools for investigating these systems. Identifying and characterizing protein interactions are fundamental steps toward understanding complex cellular networks. We have developed a unique methodology which combines an imaging-based protein interaction assay with a fluorescence recovery after photobleaching technique (FRAP). Protein interactions are readily detected by co-localization of two proteins of interest fused to green fluorescent protein (GFP) and DivIVA, a cell division protein from Bacillus subtilis. We demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions over four orders of magnitude. FRAP data was analyzed using a combination of various image processing techniques and analytical models. This combined approach made it possible to estimate cell morphology parameters such as length, diameter, the effective laser probe volume, as well as to the mobile protein concentration in vivo, the number of bound molecules at the cellular poles, and the biophysical parameter koff. Cells not only utilize molecular interactions in the intracellular environment, but also express proteins, polysaccharides and other complex molecules to mediate interactions with the surrounding extracellular environment. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting cell-to-cell interactions. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Further analyses suggest that the extracellular matrix differs between the cheA1 and the cheY1 deletion mutants, despite similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition

Partial differential equations for self-organization in cellular and developmental biology

Understanding the mechanisms governing and regulating the emergence of structure and heterogeneity within cellular systems, such as the developing embryo, represents a multiscale challenge typifying current integrative biology research, namely, explaining the macroscale behaviour of a system from microscale dynamics. This review will focus upon modelling how cell-based dynamics orchestrate the emergence of higher level structure. After surveying representative biological examples and the models used to describe them, we will assess how developments at the scale of molecular biology have impacted on current theoretical frameworks, and the new modelling opportunities that are emerging as a result. We shall restrict our survey of mathematical approaches to partial differential equations and the tools required for their analysis. We will discuss the gap between the modelling abstraction and biological reality, the challenges this presents and highlight some open problems in the field

Structure-Function Studies of Scaffolding Proteins Involved in the Formation of Neuronal Connections: AIDA1 and CASKIN2

Modular proteins serve assembly platforms and often actively regulate cellular signaling events. An intrinsic diversity of interaction modules, typical for scaffolding proteins, facilitates the organization of numerous protein partners into signaling cascades, contributing to the spatial precision, efficiency and fidelity of signal transduction. The role of complex molecular dynamics of postsynaptic density (PSD) proteins in synaptic plasticity is relatively new and yet to be fully understood. AIDA-1 is one of the most abundant members of the PSD protein family. Growing research evidence of multiple protein partnerships suggests that AIDA-1 functions as an essential PSD molecular scaffold, NMDA receptor functional mediator, and a synapse-to-nucleus messenger. The NMR structure of AIDA-1 carboxy-terminal phosphotyrosine binding domain (PTB), presented in this study, provided the structural basis for comparative analysis with the other PTB domain-containing proteins, Fe65 and X11/Mint1, that also participate in amyloid beta precursor protein (APP) processing and amyloid beta peptide (A) secretion. A combination of peptide arrays, mutagenesis and fluorescence based assays was employed to characterize the affinity and specificity of the AIDA-1 PTB domain and APP intracellular domain (AICD) interaction. Another modular protein of these studies is a pre-synaptic scaffolding protein, Caskin2. Presently, its function within the synapse is less clear compared to its more widely studied homolog, Caskin1. However, the structural differences between the two identified by our research suggest the possibility of distinct functional outcomes in the neuron. We demonstrated that Caskin2 Sterile Alpha Motif (SAM) assembles into an oligomeric architecture different from Caskin1, with the minimal repeating unit being a dimer, rather than a monomer. In invertebrates, Caskin has been functionally linked to LAR receptor tyrosine phosphatase functional pathways, implicated in axonogenesis and synaptogenesis. Using a combination of biophysical and biochemical methods, the partnership between Caskin2 and LAR Homo sapiens homologs was confirmed and characterized. These integrated structural and functional studies provide a platform for further elucidation of AIDA-1 and Caskin cellular functions

Modeling of supramolecular biopolymers: Leading the <i>in silico</i> revolution of tissue engineering and nanomedicine

Abstract The field of tissue engineering is poised to be positively influenced by the advent of supramolecular biopolymers, because of their promising tailorability coming from the bottom-up approach used for their development, absence of toxic byproducts from their gelation reaction and intrinsic better mimicry of extracellular matrix nanotopography and mechanical properties. However, a deep understanding of the phenomena ruling their properties at the meso- and macroscales is still missing. In silico approaches are increasingly helping to shine a light on questions still of out of reach for almost all empirical methods. In this review, we will present the most significant and updated efforts on molecular modeling of SBP properties, and their interactions with the living counterparts, at all scales. In detail, the currently available molecular mechanic approaches will be discussed, paying attention to the pros and cons related to their representability and transferability. We will also give detailed insights for choosing different biomolecular modeling strategies at various scales. This is a systematic overview of tools and approaches yielding to advances at atomistic, molecular, and supramolecular levels, with a holistic perspective demonstrating the urgent need for theories and models connecting biomaterial design and their biological effect in vivo

Modulación del TLR4 : estudios de reconocimiento molecular y diseño de fármacos por modelado molecular

Tesis de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Química Orgánica y Farmacéutica, leída el 04/05/2018The heterodimeric complex, formed by Toll-Like Receptor 4 (TLR4) and its accessory protein Myeloid Differentiation factor 2 (MD-2) is responsible of activating the innate immune system when sensing the presence of particular pathogen-associated molecular patterns (PAMPs) from bacteria. The outer membrane of Gram-negative bacteria is primarily populated by lipopolysaccharides (LPS) which are essential for their growth and survival. These LPSs are specifically recognized by the TLR4/MD-2 complex as follows: an LPS binds to MD-2 inside a deep molecular hydrophobic pocket causing molecular rearrangements of the receptorial complex resulting in the dimerization of another TLR4/MD-2 unit. TLR4 ectodomains dimerization event brings together the TLR4 intercellular domains initiating the activation of innate immune system signaling pathways. Interestingly, this activation is not only modulated by naturally occurring LPSs from many different Gram-negative bacteria but also by non-naturally occurring glycolipids and other non-LPS like molecules...El complejo heterodimérico, formado por el receptor Toll-like 4 (Toll-like receptor 4, TLR4) y su proteína accesoria, el Myeloid Differentiation factor 2 (MD-2), es responsable de activar la respuesta del sistema inmune innato cuando detecta la presencia de patrones moleculares asociados a patógenos (pathogen associated molecular patterns, PAMPs), que provienen de bacterias y virus. En concreto, la membrana externa de bacterias Gram-negativas está poblada principalmente por lipopolisacáridos (lipopolisaccharides, LPS), compuestos que son esenciales para su crecimiento y supervivencia. Estos LPS son reconocidos de forma específica por el complejo TLR4/MD-2 de la siguiente manera: una molécula de LPS se une a la proteína MD-2 dentro de un profundo bolsillo hidrofóbico dando lugar al reordenamiento molecular del complejo resultando en la dimerización de otra unidad de TLR4/MD-2. El evento de dimerización de los ectodominios del TLR4 hace que se acercan los dominios intracelulares que inician la activación de las vías de señalización del sistema inmune innato. Curiosamente, esta activación no sólo está modulada por LPS naturales de muchas bacterias Gram-negativas distintas, sino también por glicolípidos no naturales y otras moléculas de estructura química diferente a los LPS...Fac. de FarmaciaTRUEunpu

The intersection of theory and application in elucidating pattern formation in developmental biology

We discuss theoretical and experimental approaches to three distinct developmental systems that illustrate how theory can influence experimental work and vice-versa. The chosen systems - Drosophila melanogaster, bacterial pattern formation, and pigmentation patterns - illustrate the fundamental physical processes of signaling, growth and cell division, and cell movement involved in pattern formation and development. These systems exemplify the current state of theoretical and experimental understanding of how these processes produce the observed patterns, and illustrate how theoretical and experimental approaches can interact to lead to a better understanding of development. As John Bonner said long ago 'We have arrived at the stage where models are useful to suggest experiments, and the facts of the experiments in turn lead to new and improved models that suggest new experiments. By this rocking back and forth between the reality of experimental facts and the dream world of hypotheses, we can move slowly toward a satisfactory solution of the major problems of developmental biology.' © EDP Sciences, 2009

ATM in focus:a damage sensor and cancer target

The ability of a cell to conserve and maintain its native DNA sequence is fundamental for the survival and normal functioning of the whole organism and protection from cancer development. Here we review recently obtained results and current topics concerning the role of the ataxia-telangiectasia mutated (ATM) protein kinase as a damage sensor and its potential as therapeutic target for treating cancer. This monograph discusses DNA repair mechanisms activated after DNA double-strand breaks (DSBs), i.e. non-homologous end joining, homologous recombination and single strand annealing and the role of ATM in the above types of repair. In addition to DNA repair, ATM participates in a diverse set of physiological processes involving metabolic regulation, oxidative stress, transcriptional modulation, protein degradation and cell proliferation. Full understanding of the complexity of ATM functions and the design of therapeutics that modulate its activity to combat diseases such as cancer necessitates parallel theoretical and experimental efforts. This could be best addressed by employing a systems biology approach, involving mathematical modelling of cell signalling pathways