The primordial structure of proteins

Is the tendency to misfold and form aggregates of fibrils and gels the true nature of proteins? Graeme O'Neill reports.

The progressive failure of cellular quality-control mechanisms that keep proteins in shape make the long-lived human species particularly susceptible to amyloidoses -- diseases caused by the accumulation of misfolded proteins, a British researcher told the Combio2005 conference in Adelaide last month.

Prof Chris Dobson said that amyloid diseases like Alzheimer's and Parkinson's ultimately occur because proteins require constant help from accessory molecules, including chaperones, to fold properly and retain their active form.

Dobson, whose research group at Cambridge University's Department of Chemistry investigates the structure and folding dynamics of proteins, said virtually all proteins have the potential to misfold and aggregate into amyloid deposits because without active stabilisation, their alpha helices tend to collapse into energetically minimal beta sheets and form fibrils, which polymerise into amyloid deposits.

The Cambridge team has been studying protein folding dynamics with nuclear magnetic resonance (NMR) spectroscopy. "We're interested in exokirubg, the links between the DNA-coded information and biological function," he said.

"When proteins come off the ribosome, they're unstructured chains of amino acids that must fold to become functional. We're very interested in the question of how they fold. Somewhat by chance we got into the intriguing things that can happen when proteins misfold.

"Ultimately, folding generates a protein's biological activity, but many proteins are translocated from the cytosol to their sites of activity in an unfolded or semi-folded state.

"Protein folding and unfolding is now known to be associated with a whole range of biological phenomena including trafficking to organelles, cell growth and division, and ubiquitination -- the targeting of misfolded or damaged proteins for degradation."

Aggregates, fibrils and gels

Dobson said proteins that misfold or fail to remain correctly folded, have been implicated in about 20 human diseases, including Alzheimer's disease, Parkinson's disease, variant Creutzfeldt-Jakob disease ('mad cow disease'), and even cystic fibrosis, which involves misfolding of a complex membrane protein.

In amyloid disease the misfolded proteins aggregate; in Alzheimer's, Parkinson's and Huntington's disease, they form neurotoxic amyloid plaques in the brain.

"One day a couple of physicians came from London to see us about several patients who had systemic amyloidosis -- they had kilograms of protein deposited in their tissues, and it turned out to be mutant lysozyme [a protective enzyme that lyses bacterial cell walls]."

A point mutation causes the lysozyme molecules to misfold, prolonging the persistence of an intermediate form that spontaneously aggregates into fibrils.

"The story would have stopped there, but for a chance observation by a postdoctoral researcher who put a lysozyme sample into an NMR machine and left it for several days. It turned into a gel -- and to my amazement, when we checked the NMR chute, it was stuffed full of fibrils -- it showed that a protein could misfold and end up as fibrils, which had previously been known only in amyloid diseases.

"We wondered if it might be a common property of normal proteins, so we took some ordinary proteins like myoglobin under denaturing conditions, and they also formed fibrils.

"So it's a generic phenomenon. Lots of proteins do it. A graduate student took homopolymers, like polythreonine and polylysine, where the same amino acid is repeated, and found he could convert them into amyloid fibrils -- so there's no particular sequence requirement for forming fibrils.

"The fibrils form beta sheet structures -- the main-chain hydrogen bonds to form beta sheets, which seem to be the dominant contributors to fibril formation, but the side-chain amino acids don't seem to contribute.

"What we think has happened is that proteins have been selected over evolutionary timescales to be very unusual, and packing together the side chains may force the main chain to adopt the structures that underpin a protein's function. But what they really want to do is form fibrils or gels.

"So if a protein escapes from these strictures under non-physiological conditions, it will tend to revert to a fibrillar structure. So we came to suspect that maybe amyloid diseases are not caused by aberrant side-sequence motifs, they're really just reverting to their primordial structures."

Just a few grams

Dobson said the proteins of certain species, like humans, appear to have a higher propensity to 'lapse' into fibrils. Interestingly, lysozyme aggregates form kilograms of amyloid, with relatively little impact on a patient's health, yet a few grams of alpha synuclein can kill dopaminergic neurons in the brain's substantia nigra region, causing the neurodegenerative disorder Parkinson's disease.

Dobson said previous experiments have shown that alpha synuclein is lethal to neurons in culture. But his team showed that adding amyloid fibrils from proteins unrelated to alpha synuclein are also lethal to nerve cells.

His team has shown that oligomers several tens of amino acids long, that have not yet formed fibrils, are even more toxic than fibrils. The minimal size for toxicity is around 20-mer.

"Maybe all proteins have a tendency to misfold and aggregate. As they go through these toxic intermediate stages, they are detected by chaperones and ubiquitins that help them fold correctly into non-toxic forms, or mark them for degradation. But as these regulatory mechanisms begin to fail with age, people's proteins become less stable, particularly if people have mutations that make particular proteins unstable."

"As we get older, we tend to accumulate proteins that are damaged in some way, and our regulatory and control systems are running down, so the aberrant proteins are escaping, precipitating from solution and aggregating into amyloid.

"We're now doing things that didn't happen in early human evolutionary history. Everybody is living longer, and half the people over 85 have Alzheimer's disease. Everybody has some fibrils after age 50 or 60, and prion diseases like scrapie in sheep are passed on through urine, blood or the placenta, through the practice of feeding young cows protein from the brain tissues of old cows."

Dobson said type 2 diabetes is due to humans eating very different diets to their ancestors, resulting in changes in hormonal and protein levels. Most patients on dialysis awaiting kidney transplants develop protein-associated disease, including amyloid deposits of beta-2 microglobulin in the joints and skeletal tissues.

"It's just the wrong size to slip through filters, and is concentrated 10-fold in the serum of patients on dialysis.

"If you think about it, evolution has sussed out the problem: it has evolved systems in which amyloid aggregation is bound to happen, but is kept in check during the reproductive years.

"By the time you've paid your college fees, these safety systems become redundant -- natural selection works in a way that determines your proteins can never be more stable than they need to be.

"A typical protein is about 300 amino acids long. Twenty different amino acids can be assembled in 10 different combinations, so the proteins we see are an infinitesimally small fraction of the potential repertoire, and they are very atypical in their behaviour.

"With their core structures and side chains, natural selection has exquisitely designed them to pack together in cells. While the temperature and pH of the cells remains within the normal range, stabilising mechanisms carefully maintain their shape and function, so they can persist for long periods of time before being turned over, minimising the aggregation of damaged proteins.

"But as we get older, these stabilisng mechanisms begin to fail, there is increasing reversion to unstable, toxic forms, and we develop amyloid diseases."