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A "general hypothesis" of amyloid formation. Image Credit: Alessandro Laio and Fahimeh Baftizadeh Atomistic simulation of aggregation of small polipeptides

Wrong molecular turn leads to type 2 diabetes

Computers at the United States Department of Energy’s (DOE) Argonne National Laboratory help explain how proteins misfold creating tissue-damaging plaques leading to type 2 diabetes.

Structures called amyloid fibrils are also implicated in neurodegenerative conditions such as Alzheimer’s and Parkinson’s, and in prion diseases like Creutzfeldt-Jacob and mad cow disease.

The research pinpoints a critical intermediate step in the chemical pathway leading to amyloid fibril formation. With a new target in view, future work could lead to a possible treatment, such as designing an inhibitor to interfere with a harmful pathway. The research results helped reconcile earlier research data that until now appeared contradictory.

An amyloid fibril is a large structure consisting of misfolded proteins.

Such fibrils form plaques, or areas of tissue damage, that researchers can see with microscopes. Fibrils are believed to arise when proteins deviate from their normal 3D structure, misfold and clump together with other misfolded proteins.

Like puzzle pieces, proteins are only useful when they have the correct shape.

As fibrils form very strong, misfolded clumps, scientists believe hope lies in heading off folding errors — not in attempting to dismantle misfolded proteins.

The researchers used two main approaches to identify and understand the misfolding pathway. A sophisticated technique dependent on 2-D infrared spectroscopy, followed the chemical sequence of events leading to fibril formation. It was developed by University of Wisconsin-Madison professor Martin Zanni. His technique can measure extremely fast processes using very small samples.

Then Zanni’s measurements from molecular simulations were used to arrive at a complete picture of the early events leading to amyloid formation. The interpretation was conducted by Juan de Pablo and Chi-Cheng Chiu from the University of Chicago’s Institute for Molecular Engineering.

De Pablo and Chiu composed, ran and interpreted large-scale computer simulations of protein pathways in real time. They essentially created a model of the molecular steps involved in fibril formation using Intrepid, an IBM Blue Gene/P computer system at the Argonne Leadership Computing Facility (ALCF), as well as resources at the University of Chicago Research Computing Center.

“Using only one of these two methods would have been like running a race with only one leg. By combining both computation and experiment, we can get answers faster and more dependably.”

Juan de Pablo, PhD, University of Chicago, Institute for Molecular Engineering.

Together, researchers located an entire step that had been missing in protein misfolding, and the absence of which had been fueling confusion. An earlier study had indicated a missing intermediate step might be a floppy protein loop – incompatible with the end result being a tough fibril. Researchers felt fibrils should come from a rigid structure — a β-sheet.

The new data showed that both structures occur in reaction to changes over time. Transient rigid β-sheets form, then morph into floppy protein loops, which then form more β-sheets. The final β-sheets bind together and stack up to form rigid, damaging fibrils.

The focus now will be to target this new series of steps. With more data, researchers might be able to design an inhibitor drug which binds to the offending protein, blocking the molecule and halting fibril formation.

Next, Juan de Pablo intends to learn more about the particular protein stages implicated in type 2 diabetes. He has examined the basic units and small aggregates consisting of two, at most three, molecules. “Now we need to understand how these small aggregates disrupt cell membranes,” he adds. “We also want to decipher how the fibril grows from such a small nucleus.”

He is pushing forward with plans to investigate bigger systems by using more supercomputing. He was recently awarded computing time on Argonne’s IBM Blue Gene/Q, called Mira, the newest resource available to users at ALCF. Mira is a 10-petaflops [a petaflop is the ability of a computer to do one quadrillion floating point operations per second] computer performing 10 quadrillion calculations in each second of computing time.

De Pablo, Zanni and collaborators will apply their computer methods to determine the intermediate steps in diseases other than type 2 diabetes, including neurodegenerative diseases such as Alzheimer’s. Scientists attribute more than 20 human diseases to the formation of amyloid fibrils. The misfolding of a specific protein—a different one for each disease—is what triggers the problematic intermediate β-sheet.

“We want to understand the broader origins of the misfolding and aggregation problem, which we can do by looking at a wide range of molecules associated with different diseases.

"The eventual goal is to answer a few vital questions: What are the early stage misfolding events and small aggregates that form? How do they form? And how can we design inhibitors to stop them from forming?”

Juan de Pablo, PhD

The findings are described in a paper published in the Proceedings of the National Academy of Sciences, titled “Mechanism of IAPP amyloid fibril formation involves an intermediate with a transient β-sheet.” Also contributing to the research were scientists from the University of California-Irvine and the State University of New York at Stony Brook.

Significance
There is an enormous interest in the mechanism by which proteins misfold and aggregate into amyloid fibrils. Amyloid has been implicated in many human diseases, but the mechanism of aggregation is not understood. Intermediates have been postulated to play an important role in the process, but there have been very few direct measurements that provide specific structural details. The use of isotope labeling and 2D IR methods has allowed the characterization of a critical intermediate generated during amyloid formation by islet amyloid polypeptide, the peptide responsible for amyloid formation in type 2 diabetes. Identification of this intermediate provides a structural explanation for the lag phase and may explain why some species develop amyloid deposits of hIAPP while others do not.

Abstract
Amyloid formation is implicated in more than 20 human diseases, yet the mechanism by which fibrils form is not well understood. We use 2D infrared spectroscopy and isotope labeling to monitor the kinetics of fibril formation by human islet amyloid polypeptide (hIAPP or amylin) that is associated with type 2 diabetes. We find that an oligomeric intermediate forms during the lag phase with parallel β-sheet structure in a region that is ultimately a partially disordered loop in the fibril. We confirm the presence of this intermediate, using a set of homologous macrocyclic peptides designed to recognize β-sheets. Mutations and molecular dynamics simulations indicate that the intermediate is on pathway. Disrupting the oligomeric β-sheet to form the partially disordered loop of the fibrils creates a free energy barrier that is the origin of the lag phase during aggregation. These results help rationalize a wide range of previous fragment and mutation studies including mutations in other species that prevent the formation of amyloid plaques.

Support for this research was provided by the National Science Foundation and the National Institutes of Health.

The Argonne Leadership Computing Facility is supported by DOE’s Office of Science.

Argonne National Laboratory seeks solutions to pressing national problems in science and technology. The nation's first national laboratory, Argonne conducts leading-edge basic and applied scientific research in virtually every scientific discipline. Argonne researchers work closely with researchers from hundreds of companies, universities, and federal, state and municipal agencies to help them solve their specific problems, advance America's scientific leadership and prepare the nation for a better future. With employees from more than 60 nations, Argonne is managed by UChicago Argonne, LLC for the U.S. Department of Energy's Office of Science.

- See more at: http://www.anl.gov/articles/wrong-molecular-turn-leads-down-path-type-2-diabetes#sthash.qJjnCDQS.dpuf