A myopathy-linked desmin mutation perturbs striated muscle actin filament architecture.

Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85724, USA.
Molecular biology of the cell (Impact Factor: 5.98). 12/2008; 20(3):834-45. DOI: 10.1091/mbc.E08-07-0753
Source: PubMed

ABSTRACT Desmin interacts with nebulin establishing a direct link between the intermediate filament network and sarcomeres at the Z-discs. Here, we examined a desmin mutation, E245D, that is located within the coil IB (nebulin-binding) region of desmin and that has been reported to cause human cardiomyopathy and skeletal muscle atrophy. We show that the coil IB region of desmin binds to C-terminal nebulin (modules 160-164) with high affinity, whereas binding of this desmin region containing the E245D mutation appears to enhance its interaction with nebulin in solid-phase binding assays. Expression of the desmin-E245D mutant in myocytes displaces endogenous desmin and C-terminal nebulin from the Z-discs with a concomitant increase in the formation of intracellular aggregates, reminiscent of a major histological hallmark of desmin-related myopathies. Actin filament architecture was strikingly perturbed in myocytes expressing the desmin-E245D mutant because most sarcomeres contained elongated or shorter actin filaments. Our findings reveal a novel role for desmin intermediate filaments in modulating actin filament lengths and organization. Collectively, these data suggest that the desmin E245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, providing new insights into the potential molecular consequences of expression of certain disease-associated desmin mutations.

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Available from: Gloria M Conover, Dec 13, 2013
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    ABSTRACT: Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. Cataloged from PDF version of thesis. Includes bibliographical references (p. 137-154). Because there is little knowledge in the areas of stereocilia development, maintenance, and function in the hearing system, I decided to pursue a proteomics-based approach to discover proteins that play a role in stereocilia function. I employed a modified "twist-off" technique to isolate hair bundle proteins, and I developed a method to purify proteins and to process them for analysis using multi-dimensional protein identification technology (MudPIT). The MudPIT analysis yielded a substantial list of proteins. I verified the presence of 21 out of 34 (62%) existing proteins known to be present in stereocilia. This provided strong evidence that my proteomics approach was efficient in identifying hair bundle proteins. Next, I selected three proteins and localized them to murine cochlear stereocilia. StarD10, a putative phospholipid binding protein, was detectable along the shaft of stereocilia. Nebulin, a putative F-actin regulator, was located toward the base of stereocilia. Finally, twinfilin 2, a putative modulator of actin polymerization, was found at the tips of stereocilia. In order to determine the function of twinfilin 2, I localized the protein predominately to the tips of shorter stereocilia where it is up-regulated during the final phase of elongation. When overexpressed, I found that twinfilin 2 causes a shortening of microvilli in LLC-PK1/CL4 cells and in native cochlear stereocilia. The main result of this thesis was determining the sub-cellular localization of three interesting proteins and functionally characterizing one protein. My thesis also confirmed the proteomics screen I developed as an efficient method for identifying proteins in stereocilia. by Anthony Wei Peng. Ph.D.
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    ABSTRACT: The striated muscle sarcomere is a force generating and transducing unit as well as an important sensor of extracellular cues and a coordinator of cellular signals. The borders of individual sarcomeres are formed by the Z-disks. The Z-disk component myotilin interacts with Z-disk core structural proteins and with regulators of signaling cascades. Missense mutations in the gene encoding myotilin cause dominantly inherited muscle disorders, myotilinopathies, by an unknown mechanism. In this thesis the functions of myotilin were further characterized to clarify the molecular biological basis and the pathogenetic mechanisms of inherited muscle disorders, mainly caused by mutated myotilin. Myotilin has an important function in the assembly and maintenance of the Z-disks probably through its actin-organizing properties. Our results show that the Ig-domains of myotilin are needed for both binding and bundling actin and define the Ig domains as actin-binding modules. The disease-causing mutations appear not to change the interplay between actin and myotilin. Interactions between Z-disk proteins regulate muscle functions and disruption of these interactions results in muscle disorders. Mutations in Z-disk components myotilin, ZASP/Cypher and FATZ-2 (calsarcin-1/myozenin-2) are associated with myopathies. We showed that proteins from the myotilin and FATZ families interact via a novel and unique type of class III PDZ binding motif with the PDZ domains of ZASP and other Enigma family members and that the interactions can be modulated by phosphorylation. The morphological findings typical of myotilinopathies include Z-disk alterations and aggregation of dense filamentous material. The causes and mechanisms of protein aggregation in myotilinopathy patients are unknown, but impaired degradation might explain in part the abnormal protein accumulation. We showed that myotilin is degraded by the calcium-dependent, non-lysosomal cysteine protease calpain and by the proteasome pathway, and that wild type and mutant myotilin differ in their sensitivity to degradation. These studies identify the first functional difference between mutated and wild type myotilin. Furthermore, if degradation of myotilin is disturbed, it accumulates in cells in a manner resembling that seen in myotilinopathy patients. Based on the results, we propose a model where mutant myotilin escapes proteolytic breakdown and forms protein aggregates, leading to disruption of myofibrils and muscular dystrophy. In conclusion, the main results of this study demonstrate that myotilin is a Z-disk structural protein interacting with several Z-disk components. The turnover of myotilin is regulated by calpain and the ubiquitin proteasome system and mutations in myotilin seem to affect the degradation of myotilin, leading to protein accumulations in cells. These findings are important for understanding myotilin-linked muscle diseases and designing treatments for these disorders. Lihaksen rakennetta ja toimintaa säätelevät useat valkuaisaineet, joiden avulla aktiini- ja myosiinisäikeiden tuottama supistusvoima synkronoidaan ja siirretään tukikudoksiin. Myotiliini on lihaksen rakenneproteiini ja osa lihaksen supistusyksikköä, sarkomeeriä. Myotiliinin perityt pistemutaatiot aikaansaavat luuranko- ja sydänlihaksen häiriöitä, eli myotilinopatioita. Vaikka myotilinopatian perinnöllinen tausta tunnetaan, on vielä selvittämättä, kuinka myotiliinin muutokset johtavat lihasten surkastumiseen. Solutasolla tiedämme, että myotilinopatiapotilailla on vakavia sarkomeerin rakennehäiriöitä, ja että viallisen myotiliinin ilmentäminen lihassoluissa johtaa sarkomeerirakenteen hajoamiseen. Tässä tutkimuksessa selvitettiin myotiliinin toimintamekanismeja. Myotiliinin päätehtävänä lienee aktiinisäikeiden yhteenliittäminen sarkomeerin Z-levyssä. Osoitimme, että myotiliinin immunoglobuliinin (Ig) kaltaisia rakenneyksiköitä eli domeeneja tarvitaan aktiinin säätelyyn. Nämä Ig domeenit ovat yhtenäisiä rakenneosia muiden myotiliiniperheen jäsenten kanssa. Tauteja aiheuttavat geenivirheet eivät kuitenkaan vaikuttane myotiliinin ja aktiinin väliseen vuorovaikutukseen. Lihasmassaa säädellään tarpeen mukaan ja esimerkiksi fyysinen harjoittelu kasvattaa lihaksia. Rasitusvastetta ja siihen liittyvää geenien luentaa säätelevät Z-levyn rakenneproteiinit. Häiriö proteiinien, kuten myotiliinin, vuorovaikutuksissa voi johtaa lihastauteihin. Selvitimme sydän- ja lihastaudeissa mutatoituneiden myotiliinin- ja FATZ-perheen proteiinien vuorovaikutusta Z-levyssä sijaitsevien Enigma proteiiniperheen jäsenten kanssa. Osoitimme, että myotiliini- ja FATZ-perheiden jäsenet sitovat Enigma proteiinien PDZ-domeenia uuden ja ainutlaatuisen luokka III:n PDZ:tä sitovan motiivin avulla. Näitä proteiinien vuorovaikutuksia voidaan säädellä fosforylaation avulla. Vuorovaikutukset ovat osa mekanismia, jolla lihassolun sisäinen viestintä tapahtuu. Myotilinopatioille tyypillisiä lihaksen rakennevirheitä ovat Z-levyn muutokset ja tiheän säiemäisen materiaalin keräytyminen. Kertymien syytä ja mekanismia ei tunneta, mutta yksi syy tähän voisi olla proteiinien puutteellinen hajoaminen. Osoitimme, että kalpaiini-proteaasi pilkkoo myotiliinin lihassoluissa ja että myotiliini hajoaa pienempiin rakenneosiin proteasomireitin välityksellä. Määritimme myös ensimmäisen toiminnallisen eron normaalin ja mutatoituneen myotiliinin välillä, sillä myotilinopatioissa mutatoitunut myotiliini hajoaa huomattavasti normaalia hitaammin. Lisäksi näytimme, että mikäli myotiliinin hajoamista estetään kemikaaleilla, seurauksena on proteiinikertymiä, jotka muistuttavat potilasnäytteissä nähtäviä muutoksia. Näiden tulosten perusteella ehdotamme taudin syntymekanismiksi mallia, jossa mutatoitunut myotiliini ei hajoa normaalisti vaan muodostaa kertymiä, jotka johtavat lihaksen rakenneosien hajoamiseen ja lihastautiin. Tulokset edesauttavat myotiliiniin liittyvien lihassairauksien ymmärtämistä ja lihassairauksien hoitoon tarkoitettujen lääkkeiden kehittämistä.