The Nanomechanics of Neurotoxic Proteins Reveals Common Features at the Start of the Neurodegeneration Cascade

Abstract

Amyloidogenic neurodegenerative diseases are incurable conditions caused by specific largely disordered proteins. However, the underlying molecular mechanism remains elusive. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these “neurotoxic proteins” triggers the pathogenic cascade. Using force spectroscopy with unequivocal single-molecule identification we demonstrate a rich conformational polymorphism at their monomer level. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the monomeric β-conformational change and neurodegeneration. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt or reverse multiple neurodegenerative diseases.

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Titinisagiantproteinresponsibleforstriated-muscleelasticity.Itcontainsase-
ries of ordered domains and a large disordered segment called the PEVK do-
main. It acts as an entropic spring and is thought to be responsible for the
generation of passive contractile force in muscle. The ordered domains belong
to the immunoglobulin (Ig) type C2 and fibronectin (FN) type III superfamilies.
We expressed a 171-residue-long fragment of the PEVK domain (polyE) and
an Ig domain (I27) in BL21 derivative E.coli Rosetta competent strains.
FTIR spectroscopy combined with a diamond anvil cell was used as a non-
perturbing method for investigating the secondary structures of these recombi-
nant proteins. Fluorescence spectra of I27 were also recorded.
PolyE preserves its disordered characteristics across a wide range of pressure
(0-16 kbar), temperature (0-100?C), pD (3-10.5) and in presence of several
cosolvents. Upon pressure treatment, titin I27 unfolds at 10.7 kbar at 30?C.
As the function of temperature we observed two transitions. At 50?C the sec-
ondary structure is loosened, and the protein transforms into a molten-globule
state. At 70?C the protein completely unfolds. Unfolding is followed by ag-
gregation at ambient pressure. Moderate pressures (>2 kbar), however, can
prevent the protein from aggregation.
temperature-pressure phase diagram of titin I27, which contains metastable re-
gions as well.
We determinedthe detailed
3211-Pos
Kinetic Enhancement of NF-KB/DNA Dissociation by IkBalpha
Vera Alverdi, Byron Hetrick, Joseph Simpson, Elizabeth A. Komives.
UCSD, San Diego, CA, USA.
The nuclear factor kappa B (NF-kB) family of transcription factors is involved
in inter- and intracellular signaling, cellular stress response, growth, survival,
and apoptosis. Specific inhibitors of NF-kB transcription including IkBa,
IkBb, and IkBε, block the transcriptional activity of p65 and c-Rel-containing
NF-kB dimers. DNA binding by NF-kB is inhibited by the ankyrin repeat pro-
tein kappa B (IkBa), which sequesters NF-kB to the cytosol. The mechanism
and kinetics of DNA binding inhibition by IkBa are still unknown, but we re-
cently demonstrated that IkBa enhances the dissociation of NF-kB from DNA
transcription sites. We are investigating the effect of IkBa on the association
and dissociation rates of the NF-kB/DNA complex using titration measure-
ments, stopped- flow fluorescence and Isothermal Titration Calorimetry
(ITC). We are using pyrene labeled DNA, and IkBa Tryptophan fluorescence
to study the fluorescence changes occurring during the enhanced dissociation
process. Our results show that IkBa increases the dissociation rate of the
DNA from the NF-kB complex in a concentration-dependent manner and
with high efficiency. We repeated the experiments using a different mutant
of IkBa, C186P/A220P (CPAP). We studied also the formation and dissocia-
tion of a forward- and a backwards-ternary complex between, IkBa-NFkB-
DNA using pyrene labeled DNA, and IkBa Tryptophan fluorescence. The rates
of association and dissociation of DNA, IkBa and CPAP to form the ternary
complexes were also compared to interpret the kinetics of the enhanced disso-
ciation process.
Board B72
3212-Pos
Functional Regulation of the Anti-Apoptotic Protein BCL-xL through
Post-Translational Modification of its Intrinsically Disordered Loop
Ariele Viacava Follis, Richard W. Kriwacki.
St. Jude Children’s Research Hospital, Memphis, TN, USA.
Several studies reported functional down-regulation of the anti-apoptotic pro-
tein BCL-xL as a consequence of phosphorylation or deamidation of amino
acids within its large intrinsically disordered loop. We meant to elucidate these
poorly understood mechanisms of apoptotic regulation, and at the same time
develop a case study of functional interplay between folded and disordered seg-
ments within the same protein. We present here preliminary results towards
a structural and mechanistic understanding of these phenomena. Our studies
suggest that the post translational modification of its intrinsically disordered
loop may trigger conformational rearrangements in the folded core of BCL-
xL that decrease its affinity for BH3-only protein partners.
Board B73
3213-Pos
Ultrabithorax, an Intrinsically Disordered Protein, Selects Protein Inter-
actions by Topology
Sarah E. Bondos1, Hao-Ching Hsiao1, Daniel J. Catanese, Jr.2,
Kristopher Jordy3, Kathleen S. Matthews3.
1Texas A&M Health Science Center, College Station, TX, USA,
2Baylor College of Medicine, Houston, TX, USA,3Rice University,
Houston, TX, USA.
Interaction between two structured proteins requires both complementary to-
pologies to generate a sufficient interface and surface groups capable of form-
Board B74
ing bonds within this interface to stabilize the complex. When one (or both)
partners is intrinsically disordered as a monomer, but folds upon interaction,
the same rules for partner selection - complementary topology and surface
chemistry - are expected to apply. However, many proteins do not fold
even when forming stable protein interactions, creating ‘‘fuzzy’’ protein com-
plexes. In these cases, the extreme instability of one partner may preclude
forming a well-defined interface. Despite the apparent lack of constraints,
these proteins specifically and reliably select the correct protein partners in
vivo. To understand the rules that determine partner selection when forming
fuzzy complexes, we have evaluated protein interactions formed by the Dro-
sophila melanogaster Hox transcription factor Ultrabithorax (Ubx). Ubx inter-
acts in vitro with 29 other proteins. All of these interactions require the
intrinsically disordered regions within Ubx. Surprisingly, despite the extreme
lack of structure within these regions, Ubx appears to select protein interac-
tions by topology: 22 of the 29 partners include one of five protein folds
out of the nearly 1200 folds listed in SCOP. These data suggest that topology
remains a constraint even in fuzzy complexes. Although some Ubx partners
bind equally well to both large intrinsically disordered regions within Ubx,
many partners clearly prefer binding to the disordered alternatively spliced
microexons. Partners preferring the microexon region bind various Ubx splic-
ing isoforms differentially. Consequently, surface chemistry is likely impor-
tant for these interactions. Together, our data suggests that both topology
and surface chemistry are key criteria for partner selection, even in fuzzy
complexes.
3214-Pos
The Nanomechanics of Neurotoxic Proteins Reveals Common Features at
the Start of the Neurodegeneration Cascade
Rube ´n Herva ´s1, Javier Oroz1, Albert Galera1, Oscar Gon ˜i1,
Alejandro Valbuena1, Andre ´s M. Vera1, Angel Go ´mez-Sicilia1,
Vladimir N. Uversky2, Margarita Mene ´ndez3, Douglas V. Laurents3,
Marta Bruix3, Mariano Carrio ´n-Va ´zquez1.
1Cajal Institute/CSIC, Madrid, Spain,2University of South Florida College of
Medicine, Tampa (Florida), FL, USA,3Instituto de Quı ´mica-Fı ´sica
Rocasolano/CSIC, Madrid, Spain.
Amyloidogenic neurodegenerative diseases are incurable conditions caused by
specific largely disordered proteins. However, the underlying molecular mech-
anism remains elusive. A favored hypothesis postulates that a critical confor-
mational change in the monomer (an ideal therapeutic target) in these
‘‘neurotoxic proteins’’ triggers the pathogenic cascade. Using force spectros-
copy with unequivocal single-molecule identification we demonstrate a rich
conformational polymorphism at their monomer level. This polymorphism
strongly correlates with amyloidogenesis and neurotoxicity: it is absent in
a fibrillization-incompetent mutant, favored by familial-disease mutations
and diminished by a surprisingly promiscuous inhibitor of the monomeric
b-conformational change and neurodegeneration. The demonstrated ability to
inhibit the conformational heterogeneity of these proteins by a single pharma-
cological agent reveals common features in the monomer and suggests a com-
mon pathway to diagnose, prevent, halt or reverse multiple neurodegenerative
diseases.
Board B75
3215-Pos
High Resolution Characterization of Tertiary Contacts in Intrinsically
Disordered Amyloidogenic States of a-Synuclein Provides New Scaffolds
for Structure-Aided Drug Discovery
Carlos W. Bertoncini, Santiago Esteban-Martı ´n, Xavier Salvatella.
Institute for Research in Biomedicine, Barcelona, Spain.
The realization that transient population of partially unfolded conformations
precedes the toxic aggregation of several amyloidogenic proteins has raised
major interest in the design of compounds that could prevent protein misfold-
ing. Long-range tertiary contacts offer a unique opportunity for the imple-
mentation of structure-based drug discovery strategies to find inhibitors of
pathological protein aggregation. Representation of such transient contacts
has, however, traditionally invoked the generation of low resolution and
highly heterogeneous ensembles of structures that are impractical for in silico
use. Here we show that it is possible to determine a single structural fold that
describes at high resolution all tertiary contacts transiently established by the
intrinsically disordered protein (IDP) a-synuclein under low and high amy-
loidogenic conditions. To generate the models we use paramagnetic relaxa-
tion enhancement (PRE) data as it directly probes transiently formed
tertiary contacts, while being insensitive to other ensemble descriptors,
such as size distribution, which are of little interest in docking studies. In
our calculation strategy we refuse to comprehensively describe the conforma-
tional ensemble of the IDP (i.e. fulfilling average size and size distributions)
Board B76
Wednesday, February 29, 2012
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