Into the Fold

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Biomedical engineer Rohit Pappu directs the new Center for Biological Systems Engineering, where he and a team of key investigators use network approaches to solve problems underlying complex diseases such as cancer and neurodegeneration.

Huntington’s disease is cruel and devastating. The inherited disorder’s signature is the wasting away of brain nerve cells, leading to a host of nightmarish symptoms and outcomes. In Huntington’s, a portion of DNA known as a CAG repeat occurs 30 to 120 times rather than the 10 to 28 times that it does in normal cells. As the gene passes through families, the CAG repeats often get longer, hastening the development of disease at increasingly younger ages. Symptoms include uncontrolled movements, fidgeting, hallucinations, paranoia and dementia. One in 10,000 people of European stock is affected by Huntington’s. There is no cure, and while some drugs show promise, no known way exists to prevent the disease from getting worse.

Rohit Pappu, PhD, professor of biomedical engineering and director of the new Center for Biological Systems Engineering, studies proteins involved in the development of Huntington’s disease and related neurodegenerative motor control disorders. All involve an ensemble of recently recognized eccentric proteins, known as intrinsically disordered proteins (IDPs), and share the common theme of protein aggregation, or clumping, leading to neuronal death and disease. Perhaps the best-known example of protein aggregation is the beta amyloid plaques seen in the brains of Alzheimer’s disease patients.

IDPs comprise approximately one-third of proteins the human body draws upon to perform its myriad functions and carry out the dictates of its genes. Within the past 10 years, a consensus formed among a group of scientists that these proteins did not have a single, predictable structure. Researchers found instead that proteins shift their shapes, organize networks of other proteins, are relevant to biological complexity — giving rise to higher function in us humans (and distinguishing us from other species) — and also promote disease, lots of disease. IDPs have been implicated in cancers, cardiovascular diseases and a whole range of neurodegenerative disorders, including Parkinson’s disease, Alzheimer’s (AD), Huntington’s, groups of brain ataxias and a form of muscular dystrophy.

Organizing a network himself, Pappu in the past year has helped assemble a group of eight researchers, located on the second floor of Brauer Hall on the Danforth Campus. Each is devoted to an area of biomedical science with the common goal of understanding the essence of biomolecular and cellular networks.

This team was recruited from institutions nationwide. Members include Mark Anastasio, PhD, professor of biomedical engineering; Maxim Artyomov, PhD, assistant professor of pathology and immunology; Jan Bieschke, PhD, assistant professor of biomedical engineering; John Cunningham, PhD, assistant professor of biomedical engineering; Kristen Naegle, PhD, assistant professor of biomedical engineering; Barani Raman, PhD, assistant professor of biomedical engineering; Joshua Swamidass, MD, PhD, assistant professor of pathology and immunology; and Pappu.

CONNECTING AGGREGATION AND NEURONAL DEATH

At the crux of IDP activity is a complex phenomenon known as protein folding. This phenomenon transforms proteins from a do-nothing, one-dimensional chain of vastly different combinations of 20 protein building blocks, called amino acids, into a three-dimensional, interactive entity that goes to work in building and maintaining all of the things necessary to give traits and sustain life.

Our genome arguably has about 35,000 genes, generals that plan the actions of more than 100,000 proteins, foot soldiers that accomplish the missions.

Picture one such foot soldier as a gingerbread man spread flat on a cookie sheet. Let’s say he suddenly, magically springs to his feet and takes 3D shape — be it as menacing as a Freddy Kruger figure or as graceful as a Baryshnikov, with endless different shapes in between. That’s an extremely simplified explanation of how proteins transform to interact, articulate, communicate and mate, if you will, with other proteins and components of proteins, such as many different kinds of drugs. Biologists have known about protein folding for about 70 years, yet have only been able to determine the folded structure of about 10 percent of all known proteins. More needs to be known because protein mis-folding is implicated in 20 different diseases, ranging from Huntington’s and AD to cancer, to name just a few.

A 100-amino acid protein folds in about a minute. Laboratory tools such as molecular dynamics simulations, nuclear resonance spectroscopy (NMR) and fluorescence microscopes have revealed some baseline information, and researchers have built on known folding data for decades. But the overwhelming amount of research on protein folding and IDPs is theoretical, involving fast mathematics, fast computers. Pappu, who has worked on protein dynamics, especially folding problems, since coming to Washington University in 2001 after postdoctoral work at Johns Hopkins University, estimates that 90 percent of his work involves computationally derived theories and simulations.

“The whole specter of IDPs has turned the protein world topsy-turvy,” he explains. “The prevailing dogma had been that a particular protein folds into a particular singular shape, but in IDPs that’s out the window. Moreover, IDPs don’t fold on their own, as presumably most other proteins do. They rely on networks of other IDPs to signal that they should fold, and then they take on highly unpredictable, changing structures, and in some cases operate in partially unfolded states. This ‘disorderliness,’ the partially unfolded state, is thought to contribute to protein aggregation, building things like plaques in AD patients.”

Pappu’s most profound impact in IDP research over the past five years has come in advances in the understanding of Huntington’s disease and the gatekeeping role of flanking sequences around the polyglutamine stretch in the protein huntingtin. Here’s how the disorderliness is thought to work in Huntington’s disease. Nearly three-fourths of a biological cell is made up of water. Pappu and coworkers showed that polyglutamines are hydrophobic. In a cell, then, they aggregate, collapsing upon themselves, forming globules that want no part of the watery cell, only the company of like-minded molecules. This aggregation goes down with the ship, falling out of the cell solution, leading to insoluble deposits and eventual neuronal death signature of Huntington’s disease. The connection between aggregation and neuronal death remains unresolved.

In one advance, Pappu and his collaborators found a way to reverse the globule formation by, simply put, adding lots of electrostatic charge to the ends of polyglutamine stretches.

In another, they discovered that on either side of the CAG polyglutamate stretch there are flanking protein sequences that in healthy cells actually prevent the polyglutamines from associating with themselves, and thus forming the deleterious globules. In Huntington’s disease, these “gatekeeping” sequences, or chaperones, are somehow overwhelmed. Pappu is building on this finding to work out the mechanisms of aggregation and looking at the potential impact of different gatekeeping sequences.

The National Institutes of Health has funded his work, which has the potential for eventually preventing aggregation in nine different motor control diseases.

“We’ve shown that there are gatekeepers, and we’re now working out the gatekeeping potential of different sequences,” he says. “We’re looking closely at the quality control machinery and how it tries to protect the cell from aggregations. One big challenge is connecting the mutation to aggregation to disease.”

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Source: Washington University in St. Louis



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