Molecular Mechanisms of Dietary Fat Digestion by Pancreatic Lipases

胰脂肪酶消化膳食脂肪的分子机制

• 批准号: 8096696
• 负责人: MARK E. LOWE
• 金额: $ 32.3万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8096696
• 关键词: Active Sites; Alanine; Amino Acids; Binding; Cell membrane; Chimera organism; Cleaved cell; Colipases; Complex; Data; Data Reporting; Dietary Fats; Digestion; Elements; Emulsions; Energy-Generating Resources; Engineering; Essential Fatty Acids; Fat-Soluble Vitamin; Galactolipids; Goals; Health; Homologous Gene; Human; Investigation; Kinetics; Leukotrienes; Lipase; Lipids; Lipolysis; Measures; Mediating; Methods; Molecular; Mutate; Mutation; Nutritional Support; Obesity; Pancreas; Peptides; Phospholipase; Phospholipids; Physiological; Property; Prostaglandins; Publishing; Role; Structure; Substrate Specificity; Surface; Testing; Therapeutic; Thromboxanes; Triglycerides; Water; absorption; base; design; esterase; galactolipase; inhibitor/antagonist; interfacial; meetings; mutant; pancreatic lipase related protein 2; preference; research study; three dimensional structure

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.

描述（由申请人提供）：我们的长期目标是阐明胰腺脂肪酶消化饮食脂肪的分子机制。在此应用中，我们将重点关注影响底物特异性的脂解的步骤。脂肪酶与作用于水溶性底物的酯酶不同，该脂肪酶必须吸收底物乳液的界面，然后底物在活性位点结合。此外，胰腺脂肪酶，胰甘油三酸酯脂肪酶（PTL）的原型要求伴酶吸收到界面。重要的是，共酶吸收表面中的突变可以改变PTL-粉化酶复合物的底物偏好，强调吸收在确定底物特异性方面的重要性。因此，在活性位点中，三个不同的台阶 - - 颜色酶结合，界面吸收和底物结合都会影响底物特异性。为了研究介导PTL与底物相互作用的分子元素，我们将利用近距离PTL同源物胰腺脂肪酶相关蛋白2（PLRP2）的优势。这些脂肪酶具有高度保守的原始结构，共享一个共同的三维结构并共享常见的催化机制。但是，它们具有完全不同的酶促性能。 PTL更喜欢甘油三酸酯，而PLRP2也裂解半乳糖脂和磷脂。此外，PTL显然需要共肽酶，而PLRP2对共糖酶的需求仍然存在争议。我们的中心假设是，特定的结构结构域控制胰腺脂肪酶和底物之间相互作用的每个步骤。为了确定确定PTL和PLRP2的底物特异性涉及的域，我们将利用有关这些脂肪酶的结构和动力学特性的可用信息。我们将这种方法基于我们已发布和初步数据的多个观察结果。 1）结构研究和我们对PTL-焦糖酶复合物的已发表研究指导了影响与PTL结合的不同域的鉴定。 2）对结构数据和我们的初步研究的分析已经确定了影响这些脂肪酶对脂质乳液的吸收的PTL-焦糖酶和PLRP2中的关键残基。 3）对PTL和PLRP2的结构的比较和初步分析可以介导底物特异性差异的域。我们的特定目标的完成将提供有关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饮食脂肪至关重要，饮食脂肪提供了主要能量，必需脂肪酸，脂溶性维生素的载体，细胞膜的重要成分以及前列腺素，动物盒，动物盒和列kotrienes的前体。由于这种作用，胰腺脂肪酶在营养疗法中显着，也是肥胖和肝topo虫治疗的靶标。对介导胰腺脂肪酶功能的结构的透彻理解将增强我们对饮食脂肪消化的理解，从而导致更安全的脂肪酶抑制剂，并使我们更接近具有营养疗法特定特性的脂肪酶的合理设计。