• Ankyrin Binding Motifs: Lessons from sAnk1-Obscurin Binding

      Busby, Benjamin; Bloch, Robert J. (2009)
      My research examines the molecular details of the binding of obscurin to small ankyrin 1 (sAnk1). sAnk1 is an integral membrane protein of the sarcoplasmic reticulum, the cytosolic region of which is believed to contain ankyrin repeats, though they are not predicted by computational algorithms. Obscurin A is a large modular protein containing 6620 amino acids that binds sAnk1. Two binding sites for sAnk1 have been identified on obscurin, the first between residues 6235 and 6260 and the second between 6316 and 6436. I have compared and contrasted the binding to sAnk1 of oligopeptides and small fusion proteins that include these sequences and constructed preliminary structural models for both of these sites. I determined that residues 6316-6345 of obscurin have a higher affinity for the cytosolic region of sAnk1 than residues 6231-6260. I also used site-directed mutagenesis to identify the specific charged residues in the sequence between amino acids 6316 and 6436 that are necessary for binding, assaying binding by blot overlays and surface plasmon resonance. Using a similar approach in dual cycle mutagenic studies, I identified the complementary residues of opposite charge on sAnk1 with which they interact. Studies utilizing a molecular dynamics approach determined the most likely structures of sAnk1 and its high-affinity binding region of obscurin and refined the structure of the docked complex, selected from a larger number of possible structures by its agreement with experimental data. Molecular dynamics also confirmed the partial alpha-helical character of obscurin 6316-6345, which I determined initially with circular dichroism studies and computational modeling. I used similar methods to study the sequence of the lower affinity binding site on obscurin, between residues 6231 and 6260. I then identified short sequences in several other human proteins that are known to bind ankyrin repeat proteins. I show that these comprise at least distinct 3 types of ankyrin binding motifs and present preliminary experiments to support my predictions for the two types found in the retinoblastoma protein, Rb, and an examples of one type found in histone deacetylase 4, that resemble the motifs I have studied in obscurin. My studies show that the binding of ankyrin binding motifs to proteins with ankyrin repeats is mediated by a combination of highly specific electrostatic, and probably hydrophobic, interactions. They further suggest that the methods I have developed to identify distinct classes of ankyrin binding motifs can be used to computationally identify many other small protein domains that mediate interaction with other common protein folds.
    • Elucidating the Roles of Giant Obscurins in Breast Cancer Progression

      Shriver, Marey Raquel; Bloch, Robert J.; Kontrogianni-Konstantopoulos, Aikaterini (2014)
      Obscurins are a family of giant cytoskeletal proteins expressed in striated muscle where they play roles in structural organization and contractility. The human OBSCN undergoes alternative splicing to give rise to two giant isoforms, obscurin A (720 kDa) and obscurin B (820 kDa), composed of tandem adhesion and signaling domains. Using a panel of obscurin-specific antibodies, I reveal the presence of giant and small isoforms ranging in size from 970-50 kDa in the skin and lung of rodent tissue. Obscurins were found to exhibit nuclear, cytosolic, and membrane localization within various cell types. Interestingly, some obscurin isoforms were ubiquitously expressed, while others were tissue-specific. In addition to identifying the presence of obscurins in skin and lung tissue, I have identified robust expression of obscurins in normal mammary epithelial tissue, where they exhibit membrane localization within the ducts and lobules, compared to matched breast cancer samples which exhibit decreased expression of obscurins. Stable clones of MCF10A cells depleted of giant obscurins fail to form adhesion junctions, undergo epithelial-to-mesenchymal transition, and generate >100 μm mammospheres bearing markers of cancer-initiating cells. Additionally, obscurin-deficient MCF10A cells display altered actin organization accompanied by increased actin dynamics, resulting in increased migration and invasion. MCF10A cells that stably express the K-Ras oncogene and obscurin shRNA, but not scramble control shRNA, exhibit increased primary tumor formation and lung colonization after subcutaneous and tail vein injections, respectively. Furthermore, my studies reveal loss of giant obscurins in MCF10A cells results in the upregulation of the PI3K signaling cascade, and blockade of the catalytic subunit of the PI3K protein resulted in a decrease in mammosphere formation in anchorage independent conditions, migration of both a monolayer and single cells, and in invasion through a matrigel coated chamber. Collectively, my findings reveal that obscurins are abundantly expressed in a variety of tissues including skin, lung, and breast. Moreover, loss of giant obscurins from breast epithelial cells results in alterations within the PI3K signaling pathway, which results in a more tumorigenic phenotype.
    • Investigating the pathophysiological significance of obscurin immunoglobulin domains Ig58/59 in the heart

      Grogan, Alyssa; Kontrogianni-Konstantopoulos, Aikaterini; 0000-0002-1505-3311 (2020)
      Obscurin (720-870 kDa) is a giant cytoskeletal protein that surrounds the periphery of myofibrils at M-bands and Z-disks where it plays key structural and regulatory roles in striated muscles. Immunoglobulin domains 58/59 (Ig58/59) of obscurin mediate binding to several important modulators of muscle structure and function, including canonical titin, a smaller splice isoform of titin termed novex-3, and phospholamban (PLN). Importantly, missense mutations identified within obscurin Ig58/59 that affect binding to titins and/or PLN are linked to cardiac and skeletal myopathies in humans. To assess the pathophysiological role of Ig58/59 in vivo, we generated a constitutive deletion mouse model, Obscn-ΔIg58/59, that expresses obscurin lacking Ig58/59. Our studies revealed that young Obscn-ΔIg58/59 animals, which do not exhibit cardiac pathologies under sedentary conditions, experience tachycardia and severe arrhythmia following acute β-adrenergic stimulation and develop enlarged atria following prolonged exercise stress. Moreover, sedentary Obscn-ΔIg58/59 mice develop compensatory left ventricular hypertrophy that progresses to dilation, atrial enlargement, contractile dysfunction, and arrhythmia through aging in which males are more affected than females. Together, these findings demonstrate that deletion of obscurin Ig58/59 leads to both ventricular and atrial pathologies as a function of aging and in response to stress that manifest to different extents between sexes. We therefore aimed to decipher the cellular and molecular impact of the Ig58/59 deletion in Obscn-ΔIg58/59 ventricular and atrial myocardia, specifically focusing on males due to their enhanced disease severity. Our findings revealed that ventricular and atrial cardiomyocytes isolated from sedentary aging Obscn-ΔIg58/59 mice are enlarged and display tissue-specific alterations in Ca2+ cycling kinetics compared to controls, associated with distinct changes in the expression and phosphorylation status of major Ca2+ cycling proteins including PLN, the sarco-endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) and the ryanodine receptor 2 (RyR2). Lastly, proteomics and phospho-proteomics studies conducted on aged Obscn-ΔIg58/59 atria revealed significant changes in the levels of cytoskeletal proteins, Ca2+ regulators, and Z-disk associated complexes. Taken together, our studies demonstrate that obscurin Ig58/59 is a key regulatory module in the heart, and its deletion leads to age- and sex-dependent cardiac remodeling and arrhythmia associated with tissue-specific molecular alterations in the ventricles and atria.
    • A New Understanding of Obscurins: A Ubiquitously-Expressed Family of Cytoskeletal Regulators With Important Roles in Breast Cancer Development

      Perry, Nicole Alana; Kontrogianni-Konstantopoulos, Aikaterini (2014)
      The obscurins comprise a family of proteins that regulate the assembly, contractile function, and membrane organization of striated muscles. Expressed from the OBSCN gene, located on human chromosome 1, the giant isoforms (720-870 kDa) are modular in structure and include approximately sixty adhesion domains followed by several domains involved in signal transduction, including a RhoGEF domain and a pair of serine/threonine kinases. Although their expression and function was thought to be limited to striated muscle, whole exome sequencing of various types of tumors revealed that mutation of OBSCN could drive the development of cancer. We sought to elucidate i) the expression pattern of obscurins in normal and cancerous breast epithelial cells, ii) the signaling pathways in which obscurins participated, and iii) the expression and localization of obscurins in a broad range of tissues. We first showed that the giant isoforms of obscurin are readily expressed in normal breast, skin, and colon epithelial cells, but not in their cancerous counterparts. Abrogation of the expression of obscurin giant isoforms using shRNA in MCF10A normal breast epithelial cells resulted in increased anchorage-independent growth and reduced apoptosis in response to DNA damage or substrate detachment, properties associated with the development and metastasis of breast cancer. Furthermore, loss of giant obscurins enhances microtentacle formation and reattachment, even in the presence of the chemotherapeutic, paclitaxel. These properties can be traced to downregulated RhoA activity upon loss of the obscurin RhoGEF, and are rescued by overexpression of the obscurin RhoGEF domain. Lastly, we employed immunoblotting to demonstrate the wide range of isoforms, ranging from ~50-900 kDa, in an array of murine organs, including brain, skin, lung, liver, spleen, and kidney. Immunohistochemistry revealed obscurins to be present at diverse subcellular locations, including the nucleus and sites of cell-cell contact. Together, these results illuminate the previously unsuspected complexity of the expression and function of obscurins in non-muscle tissues, particularly their role in the development and metastasis of breast cancer.
    • Small Ankyrin-1 and Obscurin as a Model for Ankyrin Binding Motifs

      Willis, Chris; Bloch, Robert J. (2011)
      Small ankyrin-1 binds with high affinity to two regions within obscurin A. This interaction provides a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscle. Here, I show that four hydrophobic residues within the hydrophobic "hotspot" of the ankyrin-like repeats of sAnk1 are involved in binding obscurin. Alanine scanning mutagenesis of each of the four residues inhibits binding to the high affinity binding site, Obsc6316-6345, whereas two of the mutations had no effect on binding to the lower affinity site, Obsc6231-6260. Mutagenesis identified three central residues within Obsc6316-6345 that are critical for binding sAnk1, but no single residue within Obsc6231-6260 that is essential. Instead, only a triple mutant of neighboring residues of Obsc6231-6260 decreases binding. This suggests the positional importance of the hydrophobic residues within the central region of Obsc6316-6345 that have a direct role in docking to sAnk1, in contrast to a more general, hydrophobic contribution of the hydrophobic residues of Obsc6231-6260. Modeling of sAnk1 and Obsc6316-6345 are consistent with the idea that specific hydrophobic residues interact in the docked complex. Using the identified hydrophobic and previously reported charged amino acids of obscurin involved in binding, I identify two classes of ankyrin binding motifs (ABMs) The first, type 1, contains ~35% alpha-helix, between two less structured regions. The second, type 2, contains~17% alpha-helix at its amino-terminal, flanked by a disordered carboxy-terminal sequence. Using a custom matrix, I classify several known ABMs as type 1 or 2 by structural comparison and sequence alignment within the ABM-specific matrix. I use this matrix to predict ABMs in proteins that bind ankyrins. This method identified a type 1 ABM in histone deacetylase-4 and in retinoblastoma protein that bind their ligands, RFXAnk and gankyrin, respectively. I confirm this binding with in vitro assays and the ~30% helical content of retinoblastoma by circular dichroism. The method also identified a type 2 ABM in the retinoblastoma protein that binds gankyrin in vitro and has ~15% helical content. I conclude that my methods can be used to predict ABMs from primary sequence which will be applicable to the prediction of other protein-protein interactions.