Life, says Professor Nick Hoogenraad, can pivot on something as simple as the shape of a protein molecule. Proteins form the machinery of cells and since a protein's function is highly dependent on its 3-D structure, misfolded proteins can be disastrous to a cell.
In fact, he says, it has recently become clear that the accumulation of unfolded proteins can kill. Professor Hoogenraad, head of the School of Biochemistry, points to a group of brain disorders that cause dementia and death, including Gerstmann-Straussler Schinker syndrome, New Guinea's "laughing disease", kuru, Creutzfeld-Jakob disease and the closely related "mad cow" disease BSE (bovine spongiform encephalopthy).
All involve an unusual class of infectious agents called prions. Prions, says Professor Hoogenraad, are basically misfolded proteins that aggregate in brain tissue, killing nerve cells.
When a protein misfolds, scientists tend to look for some subtle, inherited mutation in the gene that encodes it. Yet heredity can only account for a minority of cases of prion diseases, which means abnormal proteins can arise in the absence of any inherited genetic defect, says Professor Hoogenraad.
There is now considerable evidence to suggest that the basic error is not genetic but a problem of protein-folding, and finger of suspicion has been pointed at a remarkable family of molecules called molecular chaperones, which are critically involved in protein folding.
"The ultimate function of all living cells depends on proteins, but molecular chaperones are the overlords of the system", says Professor Hoogenraad. "Understanding how they work has enormous implications for our understanding of normal cell function, and what happens when things go wrong".
A protein's three dimensional shape, says Professor Hoogenraad, is as vital to its function as its underlying amino-acid chemistry. The basic role of molecular chaperones is to assist proteins to fold into correct shapes, then keep them that way.
A protein starts out as a chain of amino acids, whose sequence is specified by the DNA code of a gene. It is a central dogma in biochemistry that the amino-acid sequence contains all the necessary information for a protein to fold spontaneously into its final form. However, within the crowded environment of the cell, it is now clear they need help from molecular chaperones.
In 1986, Swiss Biochemist Dr Geoff Schatz, from the Biocentre in Basel, took a gene for a protein called DHFR and grafted in a DNA sequence that re-addressed its protein to a new cellular location: instead of floating in the watery cytosol of the cell's interior, it ended up inside the [G] mitochondria.
DHFR is a bulky protein, and Schatz was challenged to explain how it had squeezed itself through a tiny [G] pore in the mitochondrial membrane. Initially he suspected some enzyme - an "unfoldase" - had unravelled the protein, making it slender enough to thread through the pore.
Indeed, when he treated his cell culture with a compound to lock DHFR molecules permanently into their 3-D shape, they were prevented from finishing up inside mitochondria. Yet his "unfoldase" proved to be a ghost, for the real answer to the problem of protein shape and translocation across membranes lies in the association of proteins with molecular chaperones from the instant that the protein is made.
DHFR did not travel alone - Professor Hoogenraad says it is now clear the protein molecules are escorted to their mitochondrial destination by molecular chaperones, specifically a chaperone called heat-shock protein 70 (HSP70).
"HSP70 wraps around the protein and prevents it folding. The whole complex binds to a mitochondrial import receptor and the protein then travels through a pore where it then undergoes folding," he said.
The discovery of molecular chaperones has forced biochemists to revise their views about how proteins form and behave. Professor Hoogenraad says that the folding of proteins to their final, functional 3-D shape is driven by thermodynamic forces.
Cells have a watery environment, yet many of the amino acids in a protein are "water-hating" (hydrophobic) [G] - the protein solves this problem by hiding its hydrophobic amino acids in its interior, so only the "water-loving" (hydrophilic) [G] amino acids exposed to interact with the watery cytosol. In this state, tensions within the protein molecule are minimised.
Because of this, there is a "right" way and any number of "wrong" ways to fold a given protein. The folding process begins with a spontaneous rearrangement of the amino acid chain that hides its hydrophobic residues from water. If this doesn't occur, molecular chaperones intercede, embracing the unruly protein and preventing it from aggregating into an insoluble mess.
Professor Hoogenraad says protein folding occurs by trial and error - if the protein folds the wrong way, it is captured by a chaperone, which then briefly relaxes its embrace to allow the protein to try again. Transient errors may occur simply because proteins are very large, unstable molecules - "They have to be, to be sufficiently flexible to interact with other molecules," he said.
Even when a protein is correctly folded, its inherent instability means that external stresses can threaten to unravel the amino acid chain, with potentially serious consequences for cell function.
For example, a body temperature rise of just a few degrees Celsius during a fever or vigorous physical exercise can destabilise proteins. When a cell overheats, molecular chaperones called heat-shock proteins (HSPs) intervene, catching and stabilising the unravelling proteins until the crisis passes.
Cells slowly accumulate mutations as they age, which led Professor Hoogenraad to wonder what would happen if such mutations resulted in permanent instabilities in certain proteins, or compromised the stabilising role of the chaperones themselves?
He and other researchers in the field suspect that diseases which are characterized by the accumulation of protein aggregates, such as Alzheimer's disease or [G] prion diseases like Creutzfeld Jakob disease, mad cow disease and kuru could all be caused by incorrect protein folding. Since molecular chaperones play an indispensable role in this process, their involvement in the development of these diseases appears likely.
"We know that the energy differences between a folded and unfolded protein is very small," he said. "Proteins are very unstable because they must be able to change shape when they interact with other molecules".
"If a cell senses that its proteins are destabilising, it switches on genes that increase production of molecular chaperones".
"With prion diseases, the prion particle is actually an altered conformation of a normal protein - structural domains called alpha helixes collapse into structures called beta sheets. This abnormally shaped protein then interacts with other, normal sister molecules, forcing them to change their shape as well".
"The forms with beta sheets aggregate spontaneously and form deposits that can kill neurons. In theory, a chance mutation in a single, aging cell line could catalyse a runaway reaction of protein misfolding in healthy cells - a prion disease".
"That's how a prion disease like mad cow disease can be infectious even though the prion has no genetic material to help it replicate".
"The prion form of the protein is actually highly stable - so stable that it can survive cooking and the action of digestive enzymes to reach the brain".
Professor Hoogenraad says research with yeast cells has provided the first firm evidence implicating molecular chaperones in the conversion of normal proteins to prion particles.
So, how important are molecular chaperones to living cells? "Every one of the many thousand of proteins in a cell probably requires help from a chaperone to fold normally and to maintain its stability. The concentration of proteins in an individual cell is very high - and in the mitochondria, so high that proteins form a gel with almost no free water".
"Quite aside from the crucial role played by molecular chaperones in keeping the protein machinery of the cell in good order, they also play a key role in ensuring that proteins get to exactly the right destination in a cell. They also control the process of protein targeting".
"Proteins must move around to do their work. Thus, some proteins are needed in the nucleus, some in the mitochondria and others in each of the other compartments of the cell. Not only do proteins need a group of organising proteins to ensure they fold correctly, so they don't react accidentally with the wrong molecules, they also need to get to the right destination".
Professor Hoogenradd and his colleagues have been focusing on a family of molecular chaperones found in cellular organelles called mitochondria - mitochondria provide cells with biochemical energy in the form of ATP.
A mitochondrion's machinery is constructed from about 1000 different proteins - all but about a dozen of them imported from outside the organelle. Not surprisingly, given that proteins arrive inside in an unfolded state, the mitochondria requires its own complement of molecular chaperones - these too are encoded by nuclear genes, assembled in the cytosol and then imported by the mitochondria.
Professor Hoogenraad in collaboration with Professor Peter Hoj and a team of colleagues, have isolated and cloned a group of mitochondrial chaperones including chaperonins 60 and 10.
"Chaperonin 60 is a very large protein with a barrel-like structure, and chaperonin 10 forms the lid of the barrel" said Professor Hoogenraad.
"An unfolded protein enters the chaperonin 60 "barrel" and the "lid" clamps shut; the captive proteins anchors itself to the barrel's interior with its hydrophobic domains and folds into its 3-D shape. This requires multiple rounds of clasp-and-release, driven by pulses of biochemical energy from ATP. When the protein is folded, the lid opens and the protein is ejected".
Professor Hoogenraad and his colleagues decided to pursue two fundamental questions relating to chaperone activity: how does the cell "know" its mitochondrial compartment is stressed, and what internal signal prompts the chaperone genes in the nucleus to switch on and rescue rickety proteins?
It is a fair bet, he says, that stress is sensed by a mechanism that detects the accumulation of unfolded or malfolded proteins. So he and his colleagues set out to create an experimental model in which unfolded proteins would selectively accumulate within just one part of the cell, the mitochondria. They would then try to determine how the chaperone genes, located in the nucleus, receive this stress message and respond by making the appropriate chaperones.
Dr Ryan Martinus, a post-doctoral fellow in the lab thought the best way to stress mitochondria was to remove the DNA that codes for a protein in the mitochondria that forms part of the ATP-synthesis machinery, cutting off ATP production.
Professor Hoogenraad says cells with this deletion, called Rho zero cells, continue growing as long as they have an ATP source independent of the mitochondria. "They look normal, but divide a little more slowly than normal. Most importantly, they are able to import proteins normally, but many of these fold incorrectly".
"When you ask what happens to the molecular chaperones in these cells, you find that chaperonin 60 and 10 are present in higher levels - just these, and no others".
"Somehow the stress of accumulating unfolded proteins is communicated to the nucleus by a set of messenger molecules that we have yet to identify. The nucleus responds by making large amounts of chaperonins 60 and 10 to help the proteins to fold - but they can't in this experimental system, because we have simply overwhelmed them".
"If the mitochondria are able to communicate their stress to the nucleus, it means there must be a signalling pathway - probably, a receptor in the mitochondria senses the accumulation of unfolded proteins, and triggers a cascade of events that transmits a signal to the nucleus. So the chaperone 60 and 10 genes must contain a DNA element that responds to this signal by switching on the genes".
To test this idea, the La Trobe researchers needed to analyse the chaperonin 60 and 10 genes - which at the time, had not been isolated from mammals.
One of Professor Hoogenraad and Professor Hoj's PhD students, Michael Ryan, managed to isolate the gene for chaperonin 10 and decided to sequence the entire gene, to see if he could identify the element that allowed the gene to respond to heat stress.
"Mike began sequencing the DNA segment from both ends at once", Professor Hoogenraad said. "To our surprise, he found the chaperonin 10 at one end, and the chaperonin 60 gene at the other".
"The two genes sit head-to-head on one chromosome, joined by a common, bi-directional promoter that regulates both genes at once. This type of gene arrangement is almost unheard of in mammalian cells".
"When we looked at the shared regulatory sequence, we found a heat shock element. Mike isolated the regulatory sequence, attached [G] reporter genes to either end and inserted the whole thing back into mammalian cells".
"When he put the cells into a hot water bath, both reporter genes switched on. So he was able to confirm identity of the switch that turns on the chaperonin 60 and 10 genes, but we still don't know what switches it on - nor do we know which switch triggers the mitochondrial-specific stress response".
Graham Garth, a PhD student is now trying to identify this switch. Together with Ryan Martinus and Dr Per Hansen, they are using Rho zero cells to define the signalling pathway between the mitochondria and the cytoplasm.
There are other facets to the molecular chaperone story. Another postgraduate student, Margaret Clarke is investigating why individuals with autoimmune diseases like diabetes and multiple sclerosis have high levels of antibodies in the bloodstream to molecular chaperones - notably chaperonins 10 and 60.
"If there are antibodies, it means these chaperonins, which are normally found only inside mitochondria, are somehow getting into the bloodstream and being targeted by the immune system. Why should mitochondrial proteins be ending up in the wrong place?"
"Are the antibodies a symptom of some underlying problem, or are chaperones somehow involved in the onset of auto-immune diseases?"
"In certain bacterial diseases, including tuberculosis and leprosy, the immune system's antibody attack appears to be directed not at the invading microbes' surface proteins but against their molecular chaperones, particularly chaperonin 60".
"Why should the chaperones in these microbes provoke an immune response, when all bacteria and indeed all cells possess them?" asks Professor Hoogenraad. "And could we protect people against autoimmune disease by immunising them with their molecular chaperones?"
"An understanding of how chaperones work has enormous implications for our understanding of how the cell works, responds to stress and how a number of important diseases occur".