Characterization of desmin disease mutants and their association with αB-crystallin [alpha-B-crystallin] in desminopathy [Elektronische Ressource] / Sarika Sharma

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Characterization of Desmin Disease Mutants and their Association with B-Crystallin in Desminopathy Sarika Sharma Doctoral Thesis German Cancer Research Centre, University Hospital of Heidelberg Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by: Sarika Sharma, MSc Biotechnology Born in: Patna, India Oral-examination: October 2010  Characterization of Desmin Disease Mutants and their Association with B-Crystallin in Desminopathy Referees: Prof. Dr. Harald Herrmann-Lerdon PD Dr. Karsten Rippe Acknowledgments To Tatjana, Dr. Norbert Mücke, Michaela and Dr. Karsten Richter, for readily sharing their technical expertise and immensely helping me in the field of protein biochemical methods, analytical ultracentrifugation analyses, cell culture and microscopy techniques, respectively. To past and present B065 colleagues (Doro, Michaela, Thorsten, Helga, Julia, Tatjana, Tanja, Ulrike, Pavle, Moni Z., Stephi, Moni M., Katharina, Petra, Bettina, Rolf), as well as to members of B060 and B140, for providing a congenial working atmosphere “on the same floor” that made lab-life “fun”. To Pavle and Dr.

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Published 01 January 2010
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Characterization of Desmin Disease
Mutants and their Association with
B-Crystallin in Desminopathy















Sarika Sharma
Doctoral Thesis
German Cancer Research Centre, University Hospital of Heidelberg


Dissertation


submitted to the
Combined Faculties for the Natural Sciences
and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences















Presented by: Sarika Sharma, MSc Biotechnology
Born in: Patna, India
Oral-examination: October 2010  
Characterization of Desmin Disease
Mutants and their Association with
B-Crystallin in Desminopathy





















Referees: Prof. Dr. Harald Herrmann-Lerdon
PD Dr. Karsten Rippe
Acknowledgments




To Tatjana, Dr. Norbert Mücke, Michaela and Dr. Karsten Richter, for readily sharing their
technical expertise and immensely helping me in the field of protein biochemical methods,
analytical ultracentrifugation analyses, cell culture and microscopy techniques, respectively.
To past and present B065 colleagues (Doro, Michaela, Thorsten, Helga, Julia, Tatjana, Tanja,
Ulrike, Pavle, Moni Z., Stephi, Moni M., Katharina, Petra, Bettina, Rolf), as well as to members of
B060 and B140, for providing a congenial working atmosphere “on the same floor” that made
lab-life “fun”.
To Pavle and Dr. Gloria Conover, for taking out their “precious” time to go through my thesis, as
well as for those fruitful discussions that lead to the generation of novel ideas.
To Prof. Roy Quinlan and his lab-members, for cooperating closely on the “desmin- B-crystallin”
project, as well as for their generous supply of the B-crystallin protein.
To Prof. Rüdiger Hell, Prof. Peter Lichter and Dr. Karsten Rippe, for readily accepting to be my
examiners.
To Prof. Harald Bär, for closely supervising my thesis through all these years, and for
encouraging me to give talks at various conferences, and also for his genuine feedback on
various manuscripts.
To Prof. Harald Herrmann, for readily accepting me as a doctoral student in his lab, for
encouraging me to participate actively in meetings and seminars, and above all for his constant
support such that I could finish my thesis in a “decent” time-frame.
To Jochen, Irene and Alfred, for all their kindness and compassion.
To Frank
To Mummy & Papa





Abstract




Mutations in intermediate filaments (IFs) and associated proteins have been shown to cause a
number of diseases in humans, ranging from blistering skin diseases to premature aging, as well
as from cataract to cardiomyopathies. Desminopathy, a disease caused by dysfunctional
mutations in type III muscle-specific IF protein desmin, constitutes a distinct sub-group of
myofibrillar myopathies (MFM) manifesting as skeletal and / or cardiac myopathy. Mutations in
the chaperone B-crystallin, that supposedly maintains cytoskeletal integrity, have also been
identified to cause MFM. Intracytoplasmic aggregates in desminopathy uniformly comprise
aberrantly folded desmin that, among other proteins, recruits B-crystallin. Currently, the
molecular basis of sequential events that lead to such aggregates in the myocyte of patients
harboring desmin mutations is not well understood. Thus, to unravel the molecular basis of
desminopathy, we have investigated the interdependence between filament alterations arising
from desmin mutations and their functional consequences in terms of interaction with the small
heat shock protein B-crystallin.
We have systematically characterized various mutants spanning the non- -helical amino-
terminal “head”, central -helical “rod” and non- -helical carboxy-terminal “tail” domain of
desmin. We show by in vitro characterization of the five head mutants, that two mutant variants
residing in the conserved nonapeptide motif “SSYRRTFGG” of desmin - DesS13F and DesR16C
- interfere with assembly by forming filamentous aggregates. Consistent with in vitro data, both
mutants fail to generate a bona fide filament system in cells lacking a type III IF cytoskeleton. In
cells expressing vimentin or desmin, both mutants fail to integrate into the endogenous filament
network and severely affect its cellular localization. The novel desminopathy-causing mutant
DesL377 22fs apparently interacts with wild-type desmin at dimer, tetramer and higher level of
filament organization in vitro, but leads to a disruption of the IF cytoskeleton in cells. This mutant
is not detectable in the myotubes of a heterozygous carrier even upon proteasome inhibition.
Two – DesR454W and DesK449T - out of the six tail mutants form abnormal filaments during in
vitro assembly and correspondingly generate aberrant filaments in cells devoid of type III IF
protein cytoskeleton. The desmin fragment Des(ESA) C244, resembling almost “first-half” of a
desmin molecule, has deleterious effects on filament assembly in vitro as well as in transfected
cells. It exhibits nucleoplasmic aggregates in two of the four investigated cell lines.
With respect to characterizing the association of desmin disease mutants with B-crystallin, data
from yeast two-hybrid analyses, electron microscopy (EM), cosedimentation assay and
viscometry distinctly suggest that the tail domain of desmin is pivotal in modulating its binding to
B-crystallin. We show that B-crystallin binds to wild-type desmin filament under optimized
buffer condition, but its binding to C-terminal deletion variants is either diminished or abolished.
We speculate that this occurs due to differences in hydrophobic surface properties and exposed
residues of wild-type desmin and its deletion variants. Des RDG is devoid of the conserved tripeptide motif “RDG”, yet it binds to B-crystallin with similar strength as desmin wild-type.
Thus, we propose that the prerequisite for binding of B-crystallin to desmin is the 3-dimensional
desmin protein conformation, which can be altered due to a mutation, and not the linear amino
acid sequence involving conserved motifs per se. The two tail mutants – DesI451M and
DesR454W - reveal weaker and stronger binding, respectively, to B-crystallin as compared to
wild-type protein. With respect to kinetics of binding, unlike desmin wild-type, DesR454W binds
to B-crystallin at all stages of assembly, and this probably results from its “open” filament
structure both alone and in an equimolar mixture with wild-type desmin. Data from R454W and
wild-type desmin transfection in 3T3 cells corroborate the in vitro data, showing that DesR454W
binds 50% more cytosolic B-crystallin than desmin wild-type. Hence, our data suggest that
mutations in desmin cause toxic gain-of-function, whereby the desmin mutants show enhanced
binding to B-crystallin. A plausible explanation for aggregate formation in desminopathy could
be such modified protein-protein interactions.
In summary, our data demonstrate the impact of desmin mutations not only on its structural
property, but also on its molecular interaction with B-crystallin. This adds to our understanding
of the molecular basis of desminopathy as we show for the first time that subtle alterations in the
nanoarchitecture of desmin filament are sufficient to induce aberrant interaction with an
associated protein B-crystallin. Such a modification might eventually contribute to the
pathogenesis of desminopathy.

Keywords: cytoskeleton, intermediate filament, desmin, mutations, myofibrillar myopathy,
desminopathy, small heat shock protein, B-crystallin, assembly, electron microscopy,
transfection, cosedimentation, viscometry.