PDZ domains are ~90 residue-long protein domains that usually bind the C-termini of their protein targets. PDZ-containing proteins regulate diverse biological systems and frequently have multiple, distinct PDZ domains, thus acting as scaffolds/adaptors simultaneously binding multiple targets. We will study PDZK1 (four PDZ domains, PDZ1-PDZ4) to understand 1) how distinct PDZ domains in multi-PDZ domain proteins collaborate to mediate function, and 2) how a single target (SR-BI) requiring a PDZ protein in one tissue (liver) requires a different PDZ protein in other tissues (e.g., steroidogeni tissue (ST)). PDZK1 is expressed in diverse tissues, and has many targets, e.g., CFTR, prostacyclin receptor, ion channels, and the HDL receptor SR-BI. We will focus on the tissue-specific PDZ domain-mediated regulation of SR-BI, which controls plasma HDL metabolism. SR-BI influences many processes, including platelet & red blood cell structure/function, female fertility, development, synaptic plasticity & cognition, inflammation, deep vein thrombosis, endothelial physiology, infection (e.g., hepatic Hepatitis C Virus entry), and lipoprotein metabolism and associated diseases (e.g., coronary heart disease). In vivo in hepatocytes SR-BI's normal localization, abundance and function, and thus normal HDL metabolism, depend on its productive interaction with PDZK1. Normal PDZK1/SR-BI interaction requires 1) binding of SR-BI's C-terminus to either PDZ1 or PDZ3, 2) the presence of either PDZ2 or PDZ3 and 3) the presence of PDZ4, which in vitro can non-canonically bind directly to inner plasma membrane-like bilayers. PDZK1's localization to the hepatocyte plasma membrane in vivo requires PDZ4 and co-expression of SR-BI. Knock-in mice with a C-terminal deletion in SR-BI suggest a PDZK1 analog is required for SR-BI expression in ST. We will test two hypotheses (I &II): I. PDZK1's regulation of SR-BI requires two- or three-pronged binding in which 1) PDZ4 tethers PDZK1 to the membrane when SR-BI binds to PDZ1 (or PDZ3) and 2) PDZ2 or PDZ3 either binds to other target membrane proteins or plays a structural role. We will perturb PDZK1's PDZ domains individually or within the full-length protein. Perturbations will include targeted mutations or inhibition by small molecules identified using in silico screening and optimized by medicinal chemistry. The mutants & inhibitors will be used in vitro (biochemistry, biophysics) and/or in vivo (cell biology, transgenic and knock-in mice & physiology). Techniques will include analysis of purified proteins (e.g., isothermal titration calorimetry, hydrogen/deuterium exchange mass spectrometry, X-ray crystallography, neutron reflectometry) and histologic and physiologic studies with transgenic and knock-in animals (including analysis of plasma lipids and lipoproteins). Analysis of the PDZK1/SR-BI system will serve as a model for understanding in detail how other multi-PDZ proteins regulate the important biological functions of cell surface receptors. II. A PDZK1 analog(s) in ST regulates SR-BI protein expression and function (as does PDZK1 in the liver). We will isolate the analog(s) and determine the mechanism of its regulation of SR-BI.

Public Health Relevance

Many critically important processes in biology and medicine are controlled by proteins that contain one or more Lego-like blocks (called PDZ domains) that help link different molecules in a cell together so that they can function normally. We will study one such protein, called PDZK1, because it is necessary for the normal functioning of the HDL (good cholesterol) receptor (technical name: SR-BI), which is involved directly or indirectly in many normal and disease-related processes (e.g, transformation of an egg cell into an adult, blood cells that carry oxygen or form blood clots, the body's defense against infection, and the metabolism of cholesterol and its associated diseases (e.g., clogging of the arteries, heart attacks and strokes)). Thus, defining precisely how PDZK1 controls SR-BI in the body will serve as a model for understanding in general how PDZ domains regulate many important biological processes, and more specifically how we might best manipulate PDZK1's target SR-BI to influence diseases as diverse as hepatitis C virus infection and heart disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL127174-02
Application #
9198970
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Liu, Lijuan
Project Start
2016-01-01
Project End
2019-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
2
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02142