Genetics of Glomerular Disease and Immune Cell Activation
Summary: Andrey Shaw is searching for genetic mutations that cause chronic kidney disease by interfering with the kidney's ability to filter proteins from the blood. His work is also focused on understanding biochemical mechanisms that underlie the recognition of antigen by lymphocytes and on understanding the mechanism of activation of the critical Ras-MAP kinase pathway.
The work in my lab is focused in three general areas: the genetics of glomerular disease and the role of CD2AP, signal transduction in the immune system, and the role of KSR1 in activation of the mitogen-activated protein (MAP) kinase pathway.
The Podocyte and Glomerular Disease
The podocyte is one of three kinds of cells that make up the glomerulus, a collection of capillaries that constitutes the primary blood filtration apparatus. The podocyte has a fascinating morphology, with multiple long foot processes that cover the entire outside surface of the capillary. The exact function of the podocyte is unknown, but it has been implicated as a component of the filtration barrier.
Our interest in podocyte biology began with studies of knockout mice lacking a gene that we had cloned called CD2-associated protein, or CD2AP. These mice leak protein into their urine and then die by six weeks of age. Analysis of renal expression of CD2AP revealed that CD2AP is expressed almost exclusively in the kidney in the podocyte. Since CD2AP appears critical for podocyte function, studies of CD2AP-deficient animals have the potential to reveal the function of the podocyte.
For more than 10 years, we have been trying to understand the exact function of CD2AP. Recently, human genetic studies have implicated CD2AP in Alzheimer's disease, providing further urgency to our effort. Using a variety of approaches, we have implicated CD2AP in actin cytoskeletal regulation, in endocytosis, and in membrane protein recycling. Using a variety of imaging methods, we have shown that CD2AP localizes in clathrin-coated pits as well as in recycling tubular endosomes. An understanding of the function of CD2AP may provide novel insights into its role in human disease.
Based on our data showing that mutations in CD2AP can lead to human glomerular diseases such as focal segmental glomerulosclerosis (FSGS), our current strategy is to use human genetics to define the epistatic network of genes involving CD2AP. Because FSGS is a disease of podocytes, we used bioinformatics to identify only those genes expressed in podocytes. We then selected a subset of 3,000 genes that we believe to be the likeliest epistatic candidates. Our goal is to sequence a set of kidney-specific genes in about 1,000 FSGS patients and use statistical methods to analyze the pattern of rare variants in patients versus controls to assemble a list of potential FSGS disease genes. We expect that these genes will be epistatic with CD2AP.
To test our candidate list of genes, we have engineered a mouse embryonic stem (ES) cell that is genetically susceptible to FSGS, allows for inducible expression of an inhibitory RNA (RNAi), and allows for efficient and genome-site-specific targeting of transgene constructs. To test gene candidates rapidly and efficiently, we have also taken advantage of new technologies that allow us to use our ES cells to generate mice that are close to 100 percent pure. We believe that our strategy of harnessing the power of human genetics and coupling this with state-of-the-art mouse genetics will play an important role in the interpretation of human genome sequencing.
T Cell Activation
In our second major area of work, we are focused on signal transduction events mediated by antigen recognition. In the early years of my laboratory, we focused on the biochemical processes that are stimulated by immune cell receptors. In the second phase of our work, recognizing that antigenic molecules are rare and lymphocyte recognition is highly sensitive and discriminatory, we focused on events occurring on the outside of the lymphocyte. Our immunological synapse hypothesis proposed that the sensitivity and specificity of T cell recognition is mediated by changes in the arrangement of receptors in the contact area between the T cell and the antigen-presenting cell. We have used a variety of methods, including mathematical modeling, to understand the role of the immunological synapse in the kinetics of T cell signaling.
Recent intravital imaging approaches have shown that signaling in vivo is much more dynamic than expected. This has led us to try and develop tools to measure signaling in live cells in vivo. Our strategy is to develop fluorescent reporters that will allow for signaling to be assessed by two-photon microscopy. We have developed a mouse that inducibly expresses a FRET (fluorescence resonance energy transfer) reporter for calcium levels that allows us to measure changes in calcium in vivo. We have also knocked in GFP (green fluorescent protein) so that it is fused to ERK2 (a member of the MAP family of kinases), allowing us to follow ERK2 activation by its intracellular localization. Here we take advantage of the fact that activated ERK2 translocates to the cell nucleus. Because cytokine secretion is an important property of activated immune cells, we have been generating reporter mice that will allow us to use two-photon microscopy to visualize the expression of a wide variety of different cytokines. Imaging by two-photon microscopy is challenging, as our reporters need to be bright enough to visualize in intact tissue.
The Role of KSR1 in the Activation of the ERK MAP Kinase Pathway
Kinase suppressor of Ras (KSR) was originally identified in fly and worm as a protein important in the Ras/ERK signaling pathway. Although it is highly related to the protein kinase RAF, the long-time inability of scientists to measure KSR kinase activity suggested that KSR might be a pseudokinase. Because KSR binds to all three components of the ERK MAP kinase pathway—RAF, MEK, and ERK—we originally proposed that KSR functions as a scaffold for the pathway, facilitating activation and suppressing cross-talk.
To prove that KSR is not a kinase, we generated a mutant that could not bind to ATP. Surprisingly, we found that this mutation impairs the function of KSR, suggesting that KSR kinase activity might be important for its function. We are actively trying to understand the potential role of KSR kinase activity. Since KSR is also required for the ability of mutated Ras to transform cells, our data also suggest that KSR is a potential cancer drug target. We are testing whether targeting the kinase activity of KSR could be useful in inhibiting the growth of cancer cells.
This work was supported in part by grants from the National Institute of Diabetes and Digestive and Kidney Diseases; the National Institute of Allergy and Infectious Diseases; the Siteman Cancer Center, Washington University; and the Rheumatic Diseases Core Center, Washington University.
As of September 20, 2012