A possible new way to attack Parkinson’s disease

Alpha-synuclein is the main component of the Lewy body of Parkinson’s disease.  It contains 140 amino acids, and is ‘natively unfolded’ in that it has no apparent ordered secondary structure (alpha helices, beta pleated sheets) detectable by a variety of methods — far ultraviolet circular dichroism, Fourier transform infrared spectroscopy or NMR spectroscopy. When the protein binds to artificial membranes half of it forms alpha helices.   Amazingly, after a huge amount of work we don’t know what alpha-Synuclein actually does.  Knockouts have only minor CNS abnormalities.

However, alpha synuclein forms fibrils which bind to cell surface receptors with internalization and transmission to other cells, just like prions.   Two such receptors for alpha-synuclein fibrils are Lymphocyte Activation Gene E (LAG3) and Amyloid PrecursorLike Protein 1 (ALPL1).

LAG3 has 4 immunoglobulin like domains (D1 – D4).  It uses D1 to capture the carboxy terminus which is exposed and concentrated on the surface of the alpha-synuclein fibrils.

Interestingly the monomers are said to adopt a self-shielded conformation which impedes the exposure of the carboxy terminus.  Phosphorylation of serine #129 enhances the binding of alpha-synuclein preformed fibrils to LAG3 and APLP1.  So the carboxy terminus of alpha-synuclein is a promising traget to block Parkinson’s disease progression.

The prions within us

Head for the hills. All of us have prions within us sayeth [ Cell vol. 156 pp. 1127 – 1129, 1193 – 1206, 1206 – 1222 ’14 ]. They are part of the innate immune system and help us fight infection. But aren’t all sorts of horrible disease (Bovine Spongiform Encephalopathy aka BSE, Jakob Creutzfeldt disease aka JC disease, Familial Fatal Insomnia etc. etc.) due to prions? Yes they are.

If you’re a bit shaky on just what a prion is see the previous post which should get you up to speed — https://luysii.wordpress.com/2014/03/30/a-primer-on-prions/.

Initially there was an enormous amount of contention when Stanley Prusiner proposed that Jakob Creutzfeldt disease was due to a protein forming an unusual conformation, which made other copies of the same protein adopt it. It was heredity without DNA or RNA (although this was hotly contended at the time), but the evidence accumulating over the years has convinced pretty much everyone except Laura Manuelidis (about whom more later). It convinced the Nobel Prize committee at any rate.

JC disease is a rapidly progressive dementia which kills people within a year. Fortunately rare (attack rate 1 per million per year) it is due to misfolded protein called PrP (unfortunately initially called ‘the’ prion protein although we now know of many more). Trust me, the few cases I saw over the years were horrible. Despite decades of study, we have no idea what PrP does, and mice totally lacking a functional Prp gene are normal. It is found on the surface of neurons. Bovine Spongiform Encephalopathy was a real scare for a time, because it was feared that you could get it from eating meat from a cow which had it. Fortunately there have been under 200 cases, and none recently.

If you cut your teeth on the immune system being made of antibodies and white cells and little else, you’re seriously out of date. The innate immune system is really the front line against infection by viruses and bacteria, long before antibodies against them can be made. There are all sorts of receptors inside and outside the cell for chemicals found in bacteria and viruses but not in us. Once the receptors have found something suspicious inside the cell, a large protein aggregate forms which activates an enzyme called caspase1 which cleaves the precursor of a protein called interleukin 1Beta, which is then released from some immune cells (no one ever thought the immune system would be simple given all that it has to do). Interleukin1beta acts on all sorts of cells to cause inflammation.

There are different types of inflammasomes and the nomenclature of their components is maddening. Two of the sensors for bacterial products (AIM, NLRP2) induce a polymerization of an inflammasome adaptor protein called ASC producing a platform for the rest of the inflammasome, which contains other proteins bound to it, along with caspase1 whose binding to the other proteins activates it. (Terrible sentence, but things really are that complicated).

ASC, like most platform proteins (scaffold proteins), is made of many different modules. One module in particular is called pyrin (because one of the cardinal signs of inflammation is fever). Here’s where it gets really interesting — the human pyrin domain in ASC can replace the prion domain of the first yeast prion to be discovered (Sup35 aka [ PSI+ ] — see the above link if you don’t know what these are) and still have it function as a prion in yeast. Even more amazing, is the fact that the yeast prion domain can functionally replace ASC modules in our inflammasomes and have them work (read the references above if you don’t believe this — I agree that it’s paradigm destroying). Evidence for human prions just doesn’t get any better than this. Fortunately, our inflammasome prions are totally unrelated to PrP which can cause such havoc with the nervous system.

Historical note: Stanley Prusiner was a year behind me at Penn Med graduating in ’67. Even worse, he was a member of my med school fraternity (which was more a place to get a decent meal than a social organization). Although I doubtless ate lunch and dinner with him before marrying in my Junior year, I have absolutely no recollection of him. I do remember our class’s medical Nobel — Mike Brown. Had I gone to Yale med instead of Penn, Laura Manuelidis would have been my classmate. Small world

A primer on prions

Actually Kurt Vonnegut came up with the basic idea behind prions in his 1963 Novel “Cat’s Cradle”. Instead of proteins, it involved a form of water (Ice-9) which had never been seen before, but one which was solid at room temperature. Unfortunately, it also solidified all liquid water it came in contact with effectively ending life on earth.

Now for some history.

The first Xray crystallographic structures of proteins were incredibly seductive intellectually, much as false color functional magnetic resonance (fMRI) images are today. It was hard not to think of them as the structure of the protein.

Nowaday we know that lots of proteins have at least one intrinsically disordered (trans. unstructured) segment of 30 amino acids ore more. [ Nature vol. 411 pp. 151 – 153 ’11 ] says 40%, and also that 25% of all human proteins are likely to be disordered (translation; unstructured) from end to end — basic on a bioinformatics program.

I’ve always been amazed that any protein has only a few shapes, purely on the basis of the chemistry — read this if you have the time — https://luysii.wordpress.com/2010/08/04/why-should-a-protein-have-just-one-shape-or-any-shape-for-that-matter/. Clearly the proteins making us up do have a relatively limited number of shapes (or we’d all be dead).

The possible universe of proteins from which our proteins are selected is enormously large. In fact the whole earth doesn’t have enough mass (even if it were made entirely of hydrogen, carbon, nitrogen, oxygen and sulfur) to make just one copy of the 20^100 possible proteins of length 100. For the calculation please see — https://luysii.wordpress.com/2009/12/20/how-many-proteins-can-be-made-using-the-entire-earth-mass-to-do-so/ — if you have the time.

So, even though it is meaningful question philosophically, just how common proteins with a few shapes are in this universe, we’ll never be able to carry out the experiment. Popper would say it’s a scientifically meaningless question, because it can’t be experimentally decided. Bertrand Russell would not.

Again, if you have time, take a look at https://luysii.wordpress.com/2010/08/08/a-chemical-gedanken-experiment/

Which, at long last, brings us to prions.

They were first discovered in yeast, and were extremely hard to figure out as they represented something in the cytoplasm which contained no DNA and yet which was heritable. The first prion was discovered nearly 50 years ago. It was called [PSI+] and it produced a lot of new proteins in yeast containing it (which is how its effects were measured) Mating [ PSI+ ] with [ psi-] (e.g. yeast cells without [ PSI+ ] converted the [ psi-] to [ PSI+ ]. It couldn’t be mapped to any known genetic element. Also [ PSI+ ] was lost at a higher rate than would be expected for a DNA mutation. The first clue that [ PSI+ ] was a protein was that it was lost faster when yeast were grown in the presence of protein denaturants (such as guanidine).

It turned out that [ PSI + ] was an aggregated form of the Sup35 protein, which basically functioned to suppress the ribosome from reading through the stop codon. If you need background on what was just said please see — https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/ and the subsequent 4 posts. This is why [ PSI+ ] yeast produced longer proteins.Things began to get exciting when Sup35 was dissected so domains could be found which induced [ PSI+ ] formation. Amazingly these domains spontaneously formed visible fibers in vitro resembling amyloid in some respects (binding the dye Congo Red for one). Then they found that preformed fibers, greatly accelerated fiber formation by unpolymerized Sup35 — beginning to sound a bit lice Ice 9 doesn’t it. Yeasts have many other prions, but the best studied and most informative is the one formed from Sup35.

So that’s how prions were found (in yeast) and what they are — an aggregated form of a given protein in a slightly different shape, which can cause another molecule of the same protein to adopt the prion proteins new shape. Amazingly, we have prions within us. But that’s the subject of the next post.