Mutations in 1-actinin-4 (Actn4), an actin crosslinking protein encoded by the human ACTN4 gene, cause an autosomal dominant form of focal segmental glomerulosclerosis (FSGS). These mutations localize to the actin- binding domain (ABD) of this protein and cause a ~10-fold increase in its actin-binding activity, leading to altered dynamics and mechanics of in vitro actin cytoskeletal networks. We have developed two mouse models: an Actn4 knockout mouse and a disease-associated K255E point mutation knockin mouse. Work done in the last period of this project suggests that FSGS-associated ACTN4 mutations alter the interaction between the two calponin homology domains that comprise Actn4's ABD, exposing an otherwise hidden actin binding sequence (ABS1). Based on biochemical studies and evolutionary considerations, our working hypothesis is that this ABS1 domain of Actn4 that mediates the pathogenic increase in actin-binding in the presence of disease mutations must also have a normal role in glomerular physiology. A main goal in this application is to define how and when wild-type Actn4 adopts a conformation similar to that conferred by disease-causing mutations. Specifically, we aim to: 1. Define the regulation of the actin binding activity of Actn4. In the absence of disease-causing mutations, we hypothesize that exposure of ABS1 can be regulated by phosphorylation. We wish to define the significance of serine-159 and tyrosine-265 phosphorylation on Actn4 function. We will use forms of Actn4 that mimic phosphorylation to examine the properties of phosphorylated Actn4 and determine the dependence of these properties on ABS1. We will identify the specific kinases responsible for phosphorylation-dependent exposure of ABS1. 2. Define the relationship between Actn4-mediated in vitro cytoskeletal biophysics and the biophysical behavior of cells. Disease-causing mutations, or mutations that alter ABS1 of Actn4, change the strain hardening properties of actin networks and make these networks brittle. We will see if these alterations correlate with altered cell biomechanical properties. We will then test the effect of phosphomimetic and unphosphorylatable forms of Actn4 on these biophysical properties. We will examine the structures of the actin cytoskeleton to see if changes in vitro correlate with changes in cells. 3. Define the role of regulation of the ABD of Actn4 on the in vivo kidney. We will develop a series of mice transgenic for Actn4 BACs designed to determine the in vivo relevance of regulation of the Actn4 ABD. These will include transgenes with an inactivated ABS1 and transgenes altering phosphorylation sites. 4. Perform studies to understand the downstream effects of Actn4 mutations in regulating gene expression, based on gene expression profiling we have performed in podocytes from Actn4 KO and K255E knockin mice. We will examine the role of two specific upregulated genes, Dmpk and Gadd45b in mediating the downstream effects of Actn4 mutation and deletion.
Mutations in the human alpha-actinin-4 gene, ACTN4, cause kidney disease in humans. We are working to understand how the protein encoded by this gene works in the kidney, and how alterations in its function cause kidney disease. Better understanding how defects in this gene cause human disease will have significant and direct implications for understanding and ultimately treating common forms of renal failure and renal failure progression.
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