Molecular dynamics simulations of binding, unfolding, and global conformational changes of signaling and adhesion molecules

Molecular dynamics (MD) simulations were used to investigate the structural basis for the functions of three proteins: Fc(gamma) receptor III (CD16), von Willebrand factor (VWF), and integrin. CD16, a heavily glycosylated protein expressed on human immune cells, plays a crucial role in immune defense by linking antibody-antigen complexes with cellular effector functions. Glycosylation of CD16 decreases its affinity for IgG. MD simulations were run for CD16-IgG Fc complexes with or without an N-glycan on CD16. The two simulated complexes show different conformations. Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) approach was used to calculate the binding free energy of the CD16-IgG Fc complexes. The calculated binding free energy helped to identify critical residues. VWF, a multimeric multidomain glycoprotein, initiates platelet adhesion at the sites of vascular injury. A specific VWF metalloprotease, A Disintegrin And Metalloprotease with ThromboSpondin motifs member 13 (ADAMTS-13), cleaves the Tyr1605-Met1606 bond in the VWF A2 domain to generate the full spectrum of plasma VWF species. Shear stress or denaturants assist VWF cleavage by ADAMTS-13 due to the unfolding of A2. MD was used to simulate the unfolding processes of A2 under force or high temperature. The beta-strands of A2 were pulled out sequentially by force, during which the cleavage site changed in steps from the fully buried state to the fully exposed state. Thermal unfolding follows a very different pathway. Integrins are adhesion molecules mediating cell-cell, cell-extracellular matrix, and cell-pathogen interactions. Experiments suggest that integrins can undergo a large-scale change from a bent to an extended conformation, associating with a transition from low to high affinity states, i.e., integrin activation. Steered MD was utilized to simulate the bent-to-extended conformational transition in time of aVb3 integrin. The integrin was observed to change smoothly from the bent to the extended conformation. One major energy barrier was overcome, corresponding to the disruption of the interactions at Hybrid/EGF4/bTD interfaces. A partially extended conformation tends to bend back while a fully extended conformation is stabilized by the coordination of Asp457 with Ca2+ at alpha-genu. Unbending with separated legs overcomes more energy barriers.Ph.D.Committee Chair: Zhu, Cheng; Committee Member: Harvey, Stephen; Committee Member: Hud, Nicholas; Committee Member: Zamir, Evan; Committee Member: Zhu, Tin

Integrins as biomechanical sensors of the microenvironment

Integrins, and integrin-mediated adhesions, have long been recognized to provide the main molecular link attaching cells to the extracellular matrix (ECM) and to serve as bidirectional hubs transmitting signals between cells and their environment. Recent evidence has shown that their combined biochemical and mechanical properties also allow integrins to sense, respond to and interact with ECM of differing properties with exquisite specificity. Here, we review this work first by providing an overview of how integrin function is regulated from both a biochemical and a mechanical perspective, affecting integrin cell-surface availability, binding properties, activation or clustering. Then, we address how this biomechanical regulation allows integrins to respond to different ECM physicochemical properties and signals, such as rigidity, composition and spatial distribution. Finally, we discuss the importance of this sensing for major cell functions by taking cell migration and cancer as examples

Recent Advances and Prospects in the Research of Nascent Adhesions

Nascent adhesions are submicron transient structures promoting the early adhesion of cells to the extracellular matrix. Nascent adhesions typically consist of several tens of integrins, and serve as platforms for the recruitment and activation of proteins to build mature focal adhesions. They are also associated with early stage signalling and the mechanoresponse. Despite their crucial role in sampling the local extracellular matrix, very little is known about the mechanism of their formation. Consequently, there is a strong scientific activity focused on elucidating the physical and biochemical foundation of their development and function. Precisely the results of this effort will be summarized in this article.Comment: 38 pages, 2 figures, review articl

Chemomechanical regulation of integrin activation and cellular processes in acidic extracellular pH

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student submitted PDF version of thesis.Includes bibliographical references (p. 162-176).It is well established that extracellular pH (pHe) becomes acidic in several important physiological and pathological contexts, including the tumor and wound microenvironments. Although it is known that acidic pHe can have profound effects on cell adhesion and migration processes integral to tumor progression and wound healing, the molecular mechanisms underlying the cellular responses to acidic pHe are largely unknown. Transmembrane integrin receptors form a physical linkage between cells and the extracellular matrix, and are thus capable of modulating cell adhesion and migration in response to extracellular conditions. In this thesis, computational and experimental approaches are used to investigate the role of acidic extracellular pH in regulating activation and binding of integrin [alpha]v[beta]3, and to characterize the consequences for downstream subcellular- and cellular-scale processes. Molecular dynamics simulations demonstrate that opening of the integrin [alpha]v[beta]3 headpiece occurs more frequently in acidic pHe than in normal pHe, and that this increased headpiece opening can be partially attributed to protonation of ASP[beta]127 in acidic pHe. These computational data indicate that acidic pHe can promote activation of integrin [alpha]v[beta]3. This is consistent with flow cytometry and atomic force microscope-enabled molecular force spectroscopy experiments, which demonstrate that there are more activated [alpha]v[beta]3 receptors on live [alpha]v[beta]3 CHO-B2 cell surfaces at acidic pHe than at normal pHe 7.4. Put together, these atomistic- and molecular-level data suggest a novel mechanism of outside-in integrin activation regulation by acidic extracellular pH. Next, the consequences of acid-induced integrin activation for subcellular- and cellular-scale processes are investigated. Kymography experiments show that [alpha]v[beta]3 CHO-B2 cell membrane protrusion lifetime is increased and protrusion velocity is decreased for cells in pHe 6.5, compared to cells in pHe 7.4. Furthermore, [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 form more actin-integrin adhesion complexes than cells in pHe 7.4, and acidic extracellular pH results in increased cell area and decreased cell circularity. Cell migration measurements demonstrate that [alpha]v[beta]3 CHO-B2 cells in pHe 6.5 migrate slower than cells in pHe 7.4, and that the fibronectin ligand density required for peak migration speed is lower for cells in pHe 6.5. Together, these data show that acidic pHe affects subcellular- and cellular-scale processes in a manner that is consistent with increased integrin activation in this condition. Finally, the migration behavior of [alpha]v[beta]3 CHO-B2 cells, bovine retinal microvascular endothelial cells, and NIH-3T3 fibroblasts in an extracellular pH gradient is investigated. Results demonstrate that NIH-3T3 fibroblasts do not exhibit directional preferences in the pHe gradient, but that [alpha]v[beta]3 CHO-B2 cells and bovine retinal microvascular endothelial cells migrate preferentially toward the acidic end of the gradient. These data suggest that acidic extracellular pH may serve as a cue that directs migration of angiogenic endothelial cells to poorly vascularized regions of tumors and wounds. Overall, this thesis research results in multiscale, in-depth understanding of extracellular pH as a critical regulator of cell function, with associated implications for tumor growth, wound healing, and the role of proton pumps in cell migration.by Ranjani Krishnan Paradise.Ph.D

The use of miR-92a inhibitor to enhance endothelial progenitor cell-mediated regeneration of injured arteries

Restenosis, a pathological condition characterised by neointima formation and lumen narrowing, can occur in some patients submitted to percutaneous coronary intervention. Reducing its incidence remains an important medical issue. Circulating endothelial precursor cells (EPCs) home to the vascular injury site and contribute to re-endothelialisation and neointima attenuation. However, the engraftment and repair capacity of EPCs from patients with cardiovascular disease are typically impaired. Priming strategies to increase EPCs’ engraftment may improve post-injury outcomes. Antiangiogenic miR-92a is upregulated in EPCs of cardiovascular patients, contributing to their reduced regenerative capacity. It was hypothesized that miR-92a antagonism in EPCs could result in a more favourable angiogenesis profile, with the rationale of developing a future functional priming strategy before cell transplantation which could lead to increased engrafting/thriving and accelerated re-endothelialisation on injured segments, hence, contributing towards post-PCI restenosis prevention. The aims of the work were: 1) to differentiate and characterise CD34+ -derived late-outgrowth EPCs from an enriched progenitor human source; 2) to characterise target gene expression and demonstrate in vitro the functional priming following the treatment of EPCs with miR-92a inhibitor and relate it to the ensueing integrin α5 subunit (ITGA5) derepression. A human EPC culture was obtained following differentiation of cord blood CD34+ cells. miR-92a inhibitor treatment using oligofectamine in CD34+ -derived late-outgrowth EPCs revealed proangiogenic,-migratory,-proliferative, and -adhesive effects in vitro, which was accompanied by the derepression of integrin α5 (ITGA5). Remarkably, siRNA ITGA5 abrogated the enhanced matrix adhesion in primed EPCs, highlighting the role of the miR-92a downstream target in EPC engraftment. Preliminary intraluminal transplantation results suggested enhanced engraftment capacity of primed EPCs in the rat carotid balloon angioplasty model

Experimental and Computational simulation of strain in medium sized arteries at macro- and micro-level.

Cardiovascular disease remains the leading cause of mortality in Western world. Current treatments for vascular disease include vascular grafting and stenting. Owing to the limitations of current treatments, over the past few years a significant research effort has been directed towards the development of tissue engineered blood vessels (TEBV). However, a TEBV that matches the biological and biomechanical functionality of natural blood vessels has yet to be developed. One of the strategies employed in the development of TEBVs involves the use of decellularised or synthetic scaffolds, which are seeded with the patient’s own cells and physically conditioned in bioreactors, with a view to developing blood-vessel-equivalent functionality prior to implantation. Along this line, the physical conditioning in bioreactors needs to replicate the in vivo haemodynamic stimulation, in order to guide normal cellular function and appropriate graft remodelling and regeneration. However, the in vitro set up, with the cells seeded on to a decellularised or synthetic scaffold and subjected to pulsatile flow in a bioreactor, does not represent a physiological scenario. Even if the bioreactor is able to simulate physiological haemodynamic conditions at the macroscale, the stimulus that would be transferred to the microscale and sensed by the cells to regulate their function is likely to be different from the micro-stimulus sensed by the cells in vivo in a native blood vessel. Therefore, in order to appropriately guide cellular function in vitro it would be necessary to assess the level of micro-stimulus sensed by the native cells in the native blood vessel in vivo, with a view to simulating this micro-stimulus in artificial bioreactor environments for conditioning the cells that are seeded onto scaffolds with non-native histoarhitectures. However, this micro-stimulus that vascular cells are exposed to in vivo cannot be assessed experimentally. The advances in computing and software resources have enabled the use of computational modelling for conducting such assessments. The aim of this project was to develop computational models for assessing the stress and strain fields on the vascular tissue and cells at the macro- and micro-scales, which will assist the bioreactor conditioning towards the development of tissue engineered vascular grafts. The 3D macro-scale simulations involved fluid-structure interaction (FSI) analysis with main focus on the strain on the vascular wall, while the 2D micro-scale simulation involved finite element analysis (FEA), focused on the local strain variation. All simulations were based on relatively physiological structures after experimental assessment. The simulations were also compared against experimental findings for strain. Macro strain resulted in approximately 11% for FSI against 19% for experimental pressure test. However, the limitations of the experimental procedure overestimated the performed dilation. Moreover, the 2D FEA simulations performed under different material properties, as an attempt to approach more physiological conditions, and under uniaxial strain only. The variation in material properties indicated inhomogeneicity, as expected, and also seemed to replicate the local strain spread when compared to experimental findings. However, further investigation is needed, which will involve the development of 3D FEA models, more physiological material properties and biaxial stretching. Under these circumstances, more information may be extracted and eventually applied to the bioreactor conditioning. Nevertheless, the novel methodology developed in this project for the study of the strain at the micro-level allows the further investigation on the tissue micro-environment