Molecular Mechanisms of Dietary Fat Digestion by Pancreatic Lipases

描述（由申请人提供）：我们的长期目标是阐明胰脂肪酶消化膳食脂肪的分子机制。在本应用中，我们将重点关注影响底物特异性的脂肪分解步骤。脂肪酶与作用于水溶性底物的酯酶的不同之处在于，在底物与活性位点结合之前，脂肪酶必须吸附到底物乳液的界面上。此外，胰腺脂肪酶的原型胰腺甘油三酯脂肪酶（PTL）需要胶原酶吸收到界面上。重要的是，胶原酶吸收表面的突变可以改变ptl -胶原酶复合物对底物的偏好，这强调了吸收在确定底物特异性方面的重要性。因此，三个不同的步骤——胶原酶结合、界面吸收和活性位点的底物结合——都影响底物特异性。为了研究介导PTL与底物相互作用的分子元件，我们将利用PTL的密切同源物胰腺脂肪酶相关蛋白2 （PLRP2）。这些脂肪酶具有高度保守的一级结构，具有共同的三维结构和催化机制；然而，它们具有完全不同的酶性质。PTL偏爱甘油三酯，而PLRP2也裂解半乳糖脂和磷脂。此外，PTL明确需要colipase，而PLRP2对colipase的要求仍然存在争议。我们的中心假设是特定的结构域控制胰腺脂肪酶和底物之间相互作用的每一步。为了确定参与确定PTL和PLRP2底物特异性的结构域，我们将利用这些脂肪酶的结构和动力学性质的现有信息。我们基于对已发表和初步数据的多次观察得出了这种方法。1)结构研究和我们发表的关于PTL-colipase复合物的研究指导了对影响colipase与PTL结合的不同结构域的识别。2)通过结构数据分析和我们的初步研究，确定了PTL-colipase和PLRP2中影响这些脂肪酶对脂质乳吸收的关键残基。3)比较和初步分析PTL和PLRP2鉴定结构域介导底物特异性差异的结构。完成我们的特定目标将提供胰腺脂肪酶与底物相互作用的每个步骤的分子细节。最终，我们将能够合理地设计脂肪酶的酶特性，以满足特定的治疗或工业需求。公共卫生相关性：胰腺脂肪酶对于有效消化膳食脂肪至关重要，脂肪是能量、必需脂肪酸、脂溶性维生素的主要来源，也是细胞膜的重要组成部分和前列腺素、血栓烷和白三烯的前体。由于这一作用，胰脂肪酶在营养治疗中占有突出地位，并作为治疗肥胖和肝纤维化的靶点。对介导胰腺脂肪酶功能的结构的深入了解将增强我们对膳食脂肪消化的理解，产生更安全的脂肪酶抑制剂，并使我们更接近合理设计具有特定性质的脂肪酶用于营养治疗。

DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the molecular mechanisms of dietary fat digestion by pancreatic lipases. In this application, we will focus on the steps in lipolysis that influence substrate specificity. Lipases differ from esterases that act on water soluble substrates in that lipases must absorb to the interface of the substrate emulsion before substrate binds in the active site. In addition, the archetype of pancreatic lipases, pancreatic triglyceride lipase (PTL), requires colipase to absorb to interfaces. Importantly, mutations in the absorption surface of colipase can change the substrate preference of the PTL-colipase complex underscoring the importance of absorption in determining substrate specificity. Thus, three distinct steps--colipase binding, interfacial absorption and substrate binding in the active site--all influence substrate specificity. To investigate the molecular elements that mediate the interaction of PTL with substrate, we will take advantage of a close PTL homologue, pancreatic lipase related protein 2 (PLRP2). These lipases have highly conserved primary structures, share a common three-dimensional structure and share common catalytic machinery; yet, they have quite different enzymatic properties. PTL prefers triglycerides whereas PLRP2 also cleaves galactolipids and phospholipids. Furthermore, PTL clearly requires colipase whereas the PLRP2 requirement for colipase remains controversial. Our central hypothesis is that specific structural domains govern each step of the interaction between pancreatic lipases and substrate. To identify domains involved in determining the substrate specificity of PTL and PLRP2, we will utilize available information on structure and kinetic properties of these lipases. We base this approach on multiple observations from our published and preliminary data. 1) Structural studies and our published studies of the PTL-colipase complex have guided the identification of distinct domains that influence colipase binding to PTL. 2) Analysis of structural data and our preliminary studies have identified critical residues in PTL-colipase and PLRP2 that influence the absorption of these lipases to lipid emulsions. 3) Comparison and preliminary analysis of the structures of PTL and PLRP2 identified domains that can mediate the differences in substrate specificity. Completion of our Specific Aims will provide molecular details about each step in the interaction of pancreatic lipases with substrate. Ultimately, we will be able to rationally engineer the enzymatic properties of lipases to meet specific therapeutic or industrial needs. PUBLIC HEALTH RELEVANCE: Pancreatic lipases are critical for the efficient digestion of dietary fats, which provide a major source of energy, essential fatty acids, a vehicle for fat-soluble vitamins, important components of cell membranes and precursors for prostaglandins, thromboxanes, and leukotrienes. Because of this role pancreatic lipases figure prominently in nutritional therapy and as targets for therapy of obesity and hepatosteatosis. A thorough understanding of the structures that mediate the functions of pancreatic lipases will enhance our understanding of dietary fat digestion, result in safer lipase inhibitors and bring us closer to the rational design of lipases with specific properties for nutritional therapy.