Computer simulations of lysosomal and osteoclast microphysiology

描述(由申请人提供)：细胞在特定的隔室中创造和严格调节化学环境的能力对许多基本的生物过程至关重要。溶酶体和骨吸收破骨细胞的胞外室(ECC)必须保持内部的酸性pH，约为4.5，才能有效地破坏大分子和骨基质。这些细胞室紧密相连，因为ECC来自溶酶体；它们都使用一组共同的膜蛋白来实现低腔pH。溶酶体酸化过程中的功能障碍可导致溶酶体储存疾病；破骨细胞的异常骨吸收与包括骨质疏松症在内的许多骨骼疾病有关，骨质疏松症在美国影响着1000万人，并导致显著的发病率和死亡率。尽管破骨细胞和溶酶体的正常功能对人类健康有着深远的影响，但对于这些系统中的pH是如何调节的，还没有统一的理解。近年来，我们对参与溶酶体和ECC酸化的蛋白质及其生物物理性质的了解有了戏剧性的增长。这一过程中的几个关键蛋白质包括V-ATPase质子泵、ClC-7氯逆向转运体和质子泄漏通道。这项提议的目标是建立溶酶体pH调节(目标1)和破骨细胞ECC(目标2)的综合数学模型。我们将首先在大范围的环境条件下分别校准每个相关传输器和离子通道的离子通量。这些性能表面随后将被用作构建基于常微分方程(ODE)的溶酶体和ECC中的离子调节模型的输入。ODE模型将根据实验数据进行校准。我们还将创建两个细胞隔间的3D模型，并使用蒙特卡罗模拟进行酸化研究。我们预计，我们的研究将有助于解决该领域关于质子泵浦所需的反离子通量的身份的争议，这些模型将指导新的实验。此外，我们的模型将揭示抑制ECC酸化的潜在药物靶点，这可能导致新的骨质疏松症治疗方法。

DESCRIPTION (provided by applicant): The ability of cells to create and tightly regulate the chemical environment in specialized compartments is critical to many fundamental biological processes. Lysosomes and the extracellular compartment (ECC) of bone resorbing osteoclasts must maintain an acidic interior pH, around 4.5, to effectively destroy macromolecules and bone matrix, respectively. These cellular compartments are intimately linked, since the ECC is derived from lysosomes; and they both use a common set of membrane proteins to achieve a low lumenal pH. Dysfunction in the acidification process in lysosomes can lead to lysosomal storage diseases; and aberrant bone resorption by osteoclasts is associated with a number of bone diseases including osteoporosis, which affects 10 million people in the United States and causes significant morbidity and mortality. Despite the profound influence that the proper functioning of osteoclasts and lysosomes has on human health, there is no unified understanding of how pH is regulated in these systems. In recent years, there has been a dramatic increase in our knowledge of the proteins involved in acidification of lysosomes and ECC and their biophysical properties. Several critical proteins in this process include the V-ATPase proton pump, ClC-7 chloride antiporter, and the proton leak channel. The goal of this proposal is to create comprehensive mathematical models of pH regulation in lysosomes (Aim 1) and the ECC of osteoclasts (Aim 2). We will start by individually calibrating the ionic flux of each relevant transporter and ion channel over a large range of environmental conditions. These performance surfaces will then be used as inputs in the construction of ordinary differential equation (ODE) based models of ion regulation in lysosomes and the ECC. The ODE models will be calibrated against experimental data. We will also create 3D models of both cellular compartments, and carry out acidification studies using Monte Carlo simulations. We expect that our studies will help resolve a controversy in the field regarding the identity of the counter-ion flux required for proton pumping, and the models will guide new experiments. Additionally, our models will reveal potential drug targets for inhibiting ECC acidification that could lead to new osteoporosis treatments.