Please welcome this month’s Scicurious Guest Writer: Emily Burns!

Lots of people set themselves goals – like things to do by the time you’re 30. Maybe it’s to find your dream job, meet the love of your life, or travel the world! For sufferers of Cystic Fibrosis, it’s living to see your 30th birthday. Even with all of the advances in medicine and technology, the average life expectancy of someone with Cystic Fibrosis is 33 years. Cystic Fibrosis is an inherited disease that mostly affects the lungs, but also the pancreas, liver and intestines. The body fluids we need – like the mucus in our lungs and intestines – are much thicker than normal, making it extremely difficult to breathe and digest food. Constant physiotherapy, breathing exercises, diet supplements and antibiotics are needed just to get on with daily life. And all of this suffering is caused by one tiny change in our DNA, which then messes up how one single protein folds into the right shape. It’s otherwise known as a protein misfolding disease.

How are proteins made?

There are over 2 million proteins in the human body, carrying out their individual tasks to keep us breathing, thinking – enabling us to live. But their production isn’t easy. It’s an incredibly intricate and specialised process that is constantly going on inside us. If it goes wrong, there are serious consequences to our health, with Cystic Fibrosis being a prime example. To understand proteins, you need to get your head around DNA. Imagine you have a massive instruction manual and each paragraph in that manual explains how to make a different tool. We read the paragraph, we make the tool, and the tool does its job. Our DNA is that instruction manual, and each of our genes is a paragraph within it. The gene is read, the tool – a protein within our body – is made, and this protein then goes on to do its job.

DNA is found within the nucleus of each cell. Depending on the signals the nucleus receives from its own cell and those around it, it will read certain parts of the DNA and send those genes out into the cell. But it can’t send it out as DNA; it has to convert it into messenger RNA (mRNA) first. This process is called transcription and involves an enzyme called RNA polymerase, which sits on the DNA and copies out the code into mRNA. While DNA is made up of 4 molecules known as the bases A, T, G and C; mRNA is made up of the bases A, U, G and C. Once the mRNA has been made, the ribosome copies that code into amino acids. Amino acids are the building blocks of proteins. There are 20 main amino acids that can be strung together in different sequences to create proteins. Every 3 bases make up the code for a different amino acid – for example, CUA codes for leucine. There are combinations of bases for start (AUG) and stop (e.g. UAG), telling the ribosome when to finish with the mRNA and move on with its chain of amino acids. AUG codes for methionine – it will always be at the start of a new protein. That whole process is called translation. In a way, it all comes back to the instruction manual metaphor: transcription is the process of the RNA polymerase transcribing out the paragraph into a form that can be carried out of the cell; while translation is the process of the ribosome translating the genetic code into the actual tool – from genetic information to practical proteins. That’s TWO lots of copying: from DNA to mRNA and from mRNA to amino acids. That’s a lot of opportunities for things to go wrong! While the body has mechanisms in place to prevent any mistakes being made, nothing is perfect.

How and why do they fold?

We now have this chain of amino acids floating about called a polypeptide. As it is, it can’t do an awful lot. So the polypeptide is carried to the Endoplasmic Reticulum (ER): the factory of the cell where all the magic happens. There’s a whole host of proteins within this factory called chaperones, which help the polypeptide to fold into its correct structure. Those 2 million proteins are going to be pretty useless unless they can interact with each other in some way, allowing signals to be passed along. These signals tell cells when to replicate, die, or differentiate into another type of cell – amongst a million other things! The structure of the protein allows other proteins to bind to it, so that they can recognise each other and signals can be transmitted. So without that shape, they may as well be blobs floating around.

It can get pretty complicated. Part of the CFTR protein (the NBD1 domain), solved in 2004 by Lewis HA et al (Embo J. 23: 282-293) (Source)

Sometimes, if there are mutations within the DNA, the wrong chain of amino acids will be put together. This is a problem for a few reasons: it can change the protein function; the shape (so that other proteins can no longer bind to it); or it can cause the protein to start sticking together in clumps, clogging up the cells. For these reasons, the ER checks the protein very stringently before allowing it to leave, and does so by seeing if it can fold into its correct structure. Changes in the amino acid sequence will mess with this ability to fold. These changes can come about in several ways. Your DNA can be damaged from environmental agents like ultraviolet light (sunshine) and radiation, which is why it’s so important to stay off the sunbeds! This damage messes with the DNA sequence, so when it’s copied out, the amino acid sequence is also going to be wrong. Also, the TWO lots of copying (transcription and translation) going on introduce a lot of room for error. While most of these errors are corrected during the RNA polymerase’s proofreading, once in a while the wrong amino acid sequence will be translated. Errors also occur during DNA replication: the process that happens every time a cell divides into a new one. The whole DNA sequence is copied out for the new cell by an enzyme called DNA polymerase (not to be mistaken for its buddy RNA polymerase, the one we’ve been talking about so far); which is thought to make about 120,000 mistakes every time a cell divides! Again, while the majority of these are corrected by proofreading, a few can slip through the net.

What happens when it goes wrong?

Any proteins that can’t be folded into their correct structure in time are sent to a proteasome, the cellular trash can, where they are chomped into bits. This quality control process is incredibly important, as we don’t want proteins getting out that don’t do their job properly. For Cystic Fibrosis, the protein in question is the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), and the most common mutation by far is ?F508. This means that the 508th amino acid in the CFTR sequence is deleted. This absolutely tiny change results in the protein taking ages to fold within the ER, leading to its demise in the proteasome.

While mutations can cause a number of problems, I’m going to concentrate on those that cause proteins to either not reach their correct final structure (as seen in Cystic Fibrosis), or clump together and form aggregates (which is the case for Alzheimer’s Disease).

Like ?F508 in CFTR, there are some mutations that cause the protein to fold at a slower rate. If given the time, the protein would finally reach the correct structure and be allowed to go on its way. But, with the ER being so strict, the protein gets thrown out and degraded. Poor folding doesn’t just affect how much of the protein gets to the cell surface; it can also get the ER in a pretty bad state. Within our body, every single one of those 2 million proteins needs to be made in abundance, making the ER a pretty busy place to be. If a protein is taking its time, the ER can become dangerously clogged up and stressed, causing the Unfolded Protein Response (UPR). The UPR is policed by 3 main proteins: Ire1, PERK and ATF6. It is the responsibility of these 3 proteins to tell the nucleus to stop sending out anymore DNA for translation, except for that which codes for ER chaperones, who can help clean up the mess inside the ER. This gives the ER a bit more room to breathe. If it doesn’t work and the ER stress can’t be alleviated, the UPR tells the cell to initiate apoptosis: cell suicide. This idea of ER stress causing disease is the foundation of Type II Diabetes and certain neurodegenerative disorders, such as Pelizaeus-Merzbacher disease.

While the primary causes Alzheimer’s and Parkinson’s is still not known, one of the theories suggests that cellular and ER stress results in the cell death that we see. They are known as amyloid diseases, as they’re caused by the accumulation of amyloids in cells. We usually think of amyloids as being associated with Alzheimer’s, so you might think that they were a particular type of protein, but that’s not quite it. Instead, amyloids are protein delinquents: any protein that can form a beta sheet can become an amyloid. When a mutated protein misfolds, the side chains of amino acids (that dictate the specific fold) are no longer so important: the main chain of the polypeptide now causes these amyloid fibres to stick together. These amyloid fibres are formed regardless of the original folded protein structure (meaning that they form the same fibrous shape for every protein) and can penetrate the cells, causing cell stress and death.

Amyloid fibres stained in green. Source: Zerd/Flickr

Current Research

With so many diseases being caused by protein misfolding, scientists around the world are trying to build up a clearer picture about what’s going on. The more we know, the more chance we have of preventing protein misfolding and coming up with better treatments for diseases like Cystic Fibrosis. Recently, scientists from Universities and the pharmaceutical industry got together at the Oxford Symposium on Rare Diseases to discuss protein misfolding and aggregation in particular.

One of the main messages that seemed to be flying about at the symposium was that neurodegenerative diseases caused by misfolding proteins need to be targeted before major symptoms arise. While it might be possible to rescue neurons that have been minimally affected, we need to treat them as early as possible. There is no point in giving a drug that prevents protein aggregation to a patient where the damage is already done; the same way we don’t give statins to a patient with late stage heart disease.

Another interesting point came from Professor David Ron, who questioned our need to know all of the details, versus our need to treat a disease. Type II Diabetes, caused by beta cells dying from too much ER stress, comes about from an excessive level of insulin production. While we know this much, there are a lot of questions scientists don’t know the answer to: why does ER stress affect beta cells and not cells producing glucagon? Is insulin harder to make than other proteins? Are alpha cells more robust against ER stress? Are beta cells harder to regenerate? Or does it matter? Should we just focus on what we do know: reduce protein levels, reduce ER stress and prevent cell death? Or does it matter more to focus on mechanism?

Of course it matters. In my eyes at least, ‘science’ is a label we put on our thirst for understanding the world around us and ourselves. But with so many questions needing to be answered, it’s important to move forward with treatment development at the same time. Luckily, this seems to be the general consensus, which is why we’re just into the start of a phase III drug combination trial for Cystic Fibrosis.

With academia and pharma moving forward together on research and treatments, I hope we can push past that 30th birthday by a long way.


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Salminen A, Kauppinen A, Suuronen T, Kaarniranta K, Ojala J (2009). ER stress in Alzheimer’s disease: a novel neuronal trigger for inflammation and Alzheimer’s pathology. Journal of Neuroinflammation, 6(41). doi:10.1186/1742-2094-6-41

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Zhang Y, Nijbroek G, Sullivan ML, McCracken AA, Watkins SC, Michaelis S, Brodsky JL (2001). Hsp70 molecular chaperone facilitates endoplasmic reticulum-associated protein degradation of cystic fibrosis transmembrane conductance regulator in yeast. Molecular Biology of the Cell, 12(5), 1303-1314.

Harding HP, Ron D (2002). Endoplasmic reticulum stress and the development of diabetes: a review. Diabetes, 51(S3), S455-461.

Nishikawa S, Brodsky JL, Nakatsukasa K (2005). Roles of molecular chaperones in Endoplasmic Reticulum (ER) Quality Control and ER-Associated Degradation (ERAD). The Journal of Biochemistry, 137(5), 551-555. Doi:10.1093/jb/mvi068

Emily is a PhD student in the laboratory of Professor Neil McDonald of the London Research Institute, working on the structure and folding pathway of receptor tyrosine kinases. She did her undergrad at the University of Manchester and spent a year in Germany working in Boehringer Ingelheim’s Structural Biology department. She hopes to graduate next year and move onto the next science adventure.