Identification and characterization of IAPP derived inhibitors of cytotoxic self-assembly and amyloidogenesis of islet amyloid polypeptide (IAPP) and ß-amyloid [beta amyloid] peptide (Aß) [A beta] [Elektronische Ressource] / vorgelegt von Li-Mei Yan

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• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2010
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 4 Mo

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Identification and characterization of IAPP-derived inhibitors
of cytotoxic self-assembly and amyloidogenesis of
islet amyloid polypeptide (IAPP) and β-amyloid peptide (A β)


Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der RWTH Aachen University zur Erlangung des akademischen Grades
einer Doktorin der Naturwissenschaften genehmigte Dissertation


vorgelegt von

Master of Science
Li-Mei Yan
aus Jilin, VR China


Berichter: Universitätsprofessor Dr. Aphrodite Kapurniotu (TU München)
Universitätsprofessor Dr. Werner Baumgartner

Tag der mündlichen Prüfung: 26. Aug. 2008



Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

Acknowledgement
First of all I would like to thank Prof. Dr. Aphrodite Kapurniotu for giving me the opportunity
to perform my Ph.D work under her supervision in the Laboratory of Bioorganic and
Medicinal Chemistry at the Department of Biochemistry and Molecular Cell Biology in the
Institute of Biochemistry of the RWTH Aachen. I thank her for her scientific advice, her
numerous helpful discussions and her constant supervision of my scientific work. Her support
and encouragement were especially valuable for my work. I also thank Prof. Kapurniotu for
performing transmission electron microscopy.
Special thanks also to Prof. Dr. Jürgen Bernhagen, leader of the Department of Biochemistry
and Molecular Cell Biology at the Institute of Biochemistry of the RWTH Aachen for
valuable advice and helpful discussions.
I would like to thank all my colleagues in the working group for providing a wonderful
working atmosphere. I enjoyed very much the great scientific freedom, excellent working
conditions, and the inspiring atmosphere at the institute.
During my work, I encountered numerous people who provided essential help and supported
me along the work in various ways. I would like to specifically thank Marianna Tatarek-
Nossol for performing pull-down assays, and for introducing me to cell culture, NuPAGE,
and western blot techniques. I would like to thank Aleksandra Velkova for numerous helpful
discussions and support. I would like to thank Erika Andreetto for peptide synthesis and for
kind and valuable suggestions. I thank Dr. Athanasios Kazantzis for introducing me to the
adenylate cyclase activation assays and for peptide synthesis; Marko Müsken and Heidi
Vasan for technical assistance in peptide synthesis and purification; Prof. Dr. Stefan
Stevanovic for MALDI measurements.
I am grateful to my friends in Aachen and Halle: Hongqi Lue, Li Li, Mei Yu, Jiang Kan,
Xianping Yang, Klaus Peters, Gisela Peters and Gabliela Tamba for their support and
friendship.
I would like to thank my parents who give me endless love and encouragement. Many thanks
to my sisters: Lisong and Lixin for their help and support, especially during my stay in
Germany. Most importantly, special thanks to my husband, Mai Luo, for his love, continuous
support, and understanding throughout these last years. His love has been the best support for
me to continue and finish my doctoral study in Germany.

i

Parts of this thesis have been published in:


Yan LM, Velkova A, Tatarek-Nossol M, Andreetto E, Kapurniotu A.
IAPP mimic blocks Abeta cytotoxic self-assembly: cross-suppression of amyloid toxicity of
Abeta and IAPP suggests a molecular link between Alzheimer's disease and type II diabetes.
Angew Chem Int Ed Engl. 2007;46(8):1246-52.


Yan LM, Tatarek-Nossol M, Velkova A, Kazantzis A, Kapurniotu A.
Design of a mimic of nonamyloidogenic and bioactive human islet amyloid polypeptide
(IAPP) as nanomolar affinity inhibitor of IAPP cytotoxic fibrillogenesis.
Proc Natl Acad Sci U S A. 2006 Feb 14;103(7):2046-51. Epub 2006 Feb 7.


Tatarek-Nossol M, Yan LM, Schmauder A, Tenidis K, Westermark G, Kapurniotu A.
Inhibition of hIAPP amyloid-fibril formation and apoptotic cell death by a designed hIAPP
amyloid- core-containing hexapeptide.
Chem Biol. 2005 Jul;12(7):797-809.
ii Abstract
Abstract
Protein aggregation into cytotoxic oligomers and fibrils in vivo is linked to cell degeneration
and the pathogenesis of more than 25 uncurable diseases, which are often called amyloid
diseases, while the high aggregation propensity and insolubility of several bioactive
polypeptides and proteins in vitro prevents their therapeutic use. Aggregation of human islet
amyloid polypeptide (IAPP) into pancreatic amyloid is strongly associated with pancreatic
cell-degeneration and the pathogenesis of type II diabetes (T2D). IAPP is a 37-residue
polypeptide which acts physiologically as a neuroendocrine regulator of glucose homeostasis.
However, IAPP misfolds and self-associates into cytotoxic aggregates and fibrils even at
nanomolar concentrations. Brain amyloid plaque formation, cell degeneration and the
pathogenesis of Alzheimer’s disease (AD) are associated with cytotoxic misfolding and self-
assembly of β-amyloid peptide (A β). A β is a ubiquitary expressed, 40 (A β(1-40)) to 42
(A β(1-42)) residue polypeptide of yet unknown physiological function. A β is found in serum
and cerebrospinalfluid in subnanomolar concentration. Both A β and IAPP are short, natively
unfolded polypeptides which share a sequence similarity of 50% but have been so far thought
to be functionally unrelated. Clinical and pathophysiological observations indicate that the
two major cell degenerative diseases AD and T2D might be linked to each other. However,
direct evidence for a molecular link has been missing.
Compounds that block cytotoxic protein/polypeptide self-assembly and amyloidogenesis are
important targets of therapeutic intervention in protein aggregation diseases. The concept
underlying the here presented studies was based on the hypothesis that bifunctional soluble
IAPP mimics which combine bioactivity with the ability to block and reverse IAPP cytotoxic
self-assembly could be promising candidates for treatment of diabetes.
Using a recently developed minimalistic conformational restriction strategy, four double N-
methylated IAPP analogues, [(N-Me)G24, (N-Me)I26]-IAPP (IAPP-GI), [(N-Me)A25, (N-
Me)L27]-IAPP (IAPP-LA), [(N-Me)F23, (N-Me)A25]-IAPP (IAPP-FA) and [(N-Me)I26, (N-
Me)L27]-IAPP (IAPP-LI), have been designed and synthesized. Their design approach was
based on the hypothesis that NFGAIL is a crucial “amyloid core” and self-recognition
sequence of IAPP and consisted of the introduction of two N-methyl residues into selected
amide bonds within the NFGAIL region of full length IAPP.
iii Abstract
In fact, the studies presented in this thesis show that these four IAPP analogues are highly
soluble, non-amyloidogenic, and non-cytotoxic molecular analogues of a non-amyloidogenic
IAPP conformation and full IAPP receptor agonists.
In addition, the studies show that all four designed IAPP analogues are able to interact with
IAPP and to suppress IAPP cytotoxic self-assembly and fibrillogenesis. The potencies of the
effects of the different IAPP analogues differ significantly, consistent with a conformational
specificity of the IAPP-analogue interaction causing their inhibitory effects. Most importantly,
it is also shown that all four analogues are able to interfere with A β(1-40) and to suppress
with varying potencies A β(1-40) self-assembly and fibrillogenesis. Thereby, IAPP-GI is
proved to be the most potent analogue with regard to inhibition of self-assembly of both IAPP
and A β(1-40). These findings suggest that IAPP-GI and/or the other IAPP analogues might
become lead compounds for the development of therapeutics in T2D and/or AD.
In addition, the studies presented in this thesis also suggest that hetero-association of early
prefibrillar and likely non-toxic IAPP and A β(1-40) species into soluble hetero-complexes
attenuates cytotoxic self-association of both polypeptides. Hetero-association of early
prefibrillar and nontoxic IAPP and A β(1-40) species may “protect” both polypeptides from
pathogenic misfolding and self-association in vivo, offering thus a molecular link between the
pathogenesis of AD and T2D and suggesting that effective therapeutic strategies targeting
both diseases might be possible.
Taken together, the results in this thesis offer a proof-of-principle of a novel concept for
designing potent amyloid disease therapeutics and of a chemical engineering approach to
redesign a natively amyloidogenic and bioactive polypeptide sequence into a soluble, non-
cytotoxic, and bioactive analogue which is also a highly potent inhibitor of cytotoxic self-
assembly of the native amyloidogenic sequence it has been derived from. Therefore, the
inhibitor design concept tested in this thesis might applicable to other disease-related self-
associating polypeptides too.

iv Index
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