Structural Characterization of the Interactions between Palladin and α-Actinin

The interaction between α-actinin and palladin, two actin-crosslinking proteins, is essential for proper bidirectional targeting of these proteins. As a first step toward understanding the role of this complex in organizing cytoskeletal actin, we have characterized binding interactions between the EF hand domain of α-actinin (Act-EF34) and peptides derived from palladin, and generated a NMR-derived structural model for the Act-EF34/palladin peptide complex. The critical binding site residues are similar to an actinin binding motif previously suggested for the complex between Act-EF34 and titin Z-repeats. The structure-based model of the Act-EF34/palladin peptide complex expands our understanding of binding specificity between the scaffold protein α-actinin and various ligands, which appears to require an α-helical motif containing four hydrophobic residues, common to many α–actinin ligands. We also provide evidence that the Family-X mutation in palladin, associated with a highly penetrant form of pancreatic cancer, does not interfere with α-actinin binding

Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections

Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility

NMR structure of a fungal virulence factor reveals structural homology with mammalian saposin B

The fungal protein CBP (calcium binding protein) is a known virulence factor with an unknown virulence mechanism. The protein was identified based on its ability to bind calcium and its prevalence as Histoplasma capsulatum’s most abundant secreted protein. However, CBP has no sequence homology with other calcium binding proteins and contains no known calcium-binding motifs. Here, the NMR structure of CBP reveals a highly intertwined homodimer and represents the first atomic level NMR model of any fungal virulence factor. Each CBP monomer is comprised of four α-helices that adopt the saposin fold, characteristic of a protein family that binds to membranes and lipids. This structural homology suggests that CBP functions as a lipid-binding protein, potentially interacting with host glycolipids in the phagolysosome of host cells

Myopalladin promotes muscle growth through modulation of the serum response factor pathway

Myopalladin (MYPN) is a striated muscle-specific, immunoglobulin-containing protein located in the Z-line and I-band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss-of-function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe

Structure and Function of Palladin's Actin Binding Domain

Here we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct F-actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and crosslinking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin crosslinking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo

Expression profiling of in vivo ductal carcinoma in situ progression models identified B cell lymphoma-9 as a molecular driver of breast cancer invasion

Abstract Introduction There are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of patients. We profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using in vivo DCIS progression models. These studies identified B cell lymphoma-9 (BCL9) as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However BCL9 role in breast cancer had not been previously recognized. Methods Microarray and RNA sequencing were utilized to characterize the sequential changes in mRNA expression during DCIS invasive transition. BCL9-shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, epithelial-mesenchymal transition (EMT) and canonical Wnt-signaling. Immunofluorescence of 28 patient samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. The cancer genome atlas data were analyzed to assess the status of BCL9 gene alterations in breast cancers. Results Analysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: Estrogen receptor (ER) and progesterone receptor (PR) negative, high nuclear grade, and high human epidermal growth factor receptor2 (HER2). In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of EMT. Analysis of the TCGA data showed BCL9 to be altered in 26 % of breast cancers. This is a significant alteration when compared to HER2 (ERBB2) gene (19 %) and estrogen receptor (ESR1) gene (8 %). A significantly higher proportion of basal like invasive breast cancers compared to luminal breast cancers showed BCL9 amplification. Conclusion BCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC

Palladin's Ig4 Mutation: Exploring the link with pancreatic cancer

Click on the DOI link to access the article (may not be free).Palladin is a recently discovered protein that is expressed in human cells and plays a key role in cytoskeletal dynamics by directly binding and bundling filaments of actin. These processes provide an important function in cell motility and are made possible by the Ig3 and Ig4 domains of palladin. Since cancer survival is often dependent upon migration of cancerous cells to other parts of the body, palladin has been implicated as playing a critical role in cancer metastasis. In addition, a mutation from a conserved tryptophan to cysteine in palladin's Ig4 domain has recently been linked to a form of pancreatic cancer. This mutation is called “PaTu2,” and understanding how it affects palladin is the focus of this research project. We began by isolating the wild type and mutant Ig4 and Ig3-4 domains of palladin. Obtaining the PaTu2 mutant protein required a different approach due to the protein remaining insoluble within inclusion bodies in E. coli bacteria after cell lysis. This was amended by incorporating a maltose-binding protein tag to increase solubility so that the protein could undergo affinity purification. Next, we determined whether the mutation affected palladin's ability to bind and bundle actin filaments by conducting cosedimentation assays, with initial results showing no significant difference when compared to the wild-type. Future directions of the project include using nuclear magnetic resonance and circular dichroism spectroscopy to see if the mutation affects the structure and stability of palladin. Such analysis will provide the necessary data that lead towards a greater understanding the cause of metastatic pancreatic cancer

Palladin Nucleates Actin Assembly and Regulates Cytoskeleton Architecture

Click on the DOI link to access the article (may not be free).The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. Palladin localizes to actin-rich protrusions and has a well-documented effect on metastasis of invasive cancers. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal immunoglobulin-like domain of palladin (Ig3) that is directly responsible for actin binding and bundling also potently nucleates the formation of actin filaments in vitro