Isolation of the LBRC and characterization of its protein components

Northwestern University At Chicago, Evanston IL

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Abstract

DESCRIPTION (provided by applicant): The movement of leukocytes across the endothelial cell layer of blood vessels (referred to as transendothelial migration, TEM) is critical to mounting an effective inflammatory response, but is also associated with the pathology of such diseases at atherosclerosis, multiple sclerosis, and arthritis. Although the process of TEM is only beginning to be understood, it is known to be facilitated by the proteins PECAM and CD99. During TEM, these molecules are recycled to the plasma membrane from a novel compartment, the lateral border recycling compartment (LBRC). The targeted recycling of the LBRC is required for TEM, and confirms the importance of this compartment in inflammation. However, the LBRC is poorly characterized and only a few of its resident proteins have been identified. The long-term goal of this proposal is to understand how the LBRC regulates TEM so that new targets could be identified for anti-inflammatory therapies. Toward this end, we will identify and characterize the molecular components of the LBRC so that we may better understand its function in TEM. Specifically, we will isolate the LBRC it using subcellular fractionation and biochemical techniques to identify its proteins. Candidate proteins will be assayed to confirm their localization in the LBRC and also to determine whether they are critical for TEM. As a parallel approach and to better characterize the known components of the LBRC that are necessary for TEM, namely PECAM and CD99, we will identify novel PECAM and CD99 interacting proteins and also determine the molecular components required for CD99 localization to the LBRC. Relevance: The movement of white blood cells across the lining of the blood vessel is critical to inflammation and requires the function of a recently identified intracellular compartment referred to as the Lateral Border Recycling Compartment (LBRC). The identification and characterization of the protein constituents of the LBRC will provide new targets and strategies for the development of drugs and therapies to control unwanted inflammation in diseases such as atherosclerosis, multiple sclerosis, and arthritis.