Lecture 18

Whether we define them as "living" or "nonliving" or as an "infectious enzyme," we do know three major things about the nature of prions. These are..

(1) they have arisen in organisms during evolution (2) they are able to propagate themselves and the diseases they cause (3) they appear to be able to evolve and to adapt themselves to different hosts.

Give some examples where prions have been transmitted from infected tissues. (4)

-to patients taking injections of growth hormone harvested from human pituitary glands -from instruments used for brain surgery (prions can survive the autoclave sterilization process) -in corneal grafts -in electrode implants

What are the two means of prion transmission?

1. Direct contact with infected tissue. 2. Consumption of affected tissues.

What is practical consequence of our realizing the we don't need genetic material to evolve, as we have seen now in prions?

A practical consequence is the realization that therapeutic approaches aimed at stabilizing PrP or reducing PrP expression are less likely to be thwarted by emergence of drug resistance than those based on targeting PrPSc. BECAUSE THEY EVOLVE TO DRUGS AND HAVE SPECIFIC ADAPTATION TO THEIR ENVIRONMENTS.

There is a paper that looks at the "darwinian evolution" of prions in cell culture. What do they conclude? (3)

A prion strain, transferred from one species to another and subsequently returned to the original host, may in some instances have changed or "mutated". PrPSc are β sheet-rich conformers of PrPC which can occur as different strains. Each strain is associated with a different conformer of PrPSc.

What is the overall outcome of the prion disease? (2)

Accumulation of plaques in brain and loss of normal brain function.

What are amyloid plaques?

Affected areas contain microscopic insoluble amyloid fibrils and macrocrystalline arrays known as amyloid plaques.

How many mutations are known to be associated with PRPSc prion disease?

At least 20.

Why is mad cow disease such bad thing in herds of cows?

Because once one has it you have to kill the entire herd. If we eat the cow infected beef or brain, then we have potential to develop mad cow disease. This is also called scrappie. * We can't ship our beef to Europe - and the US.

What is the definitive diagnostic test for transmissible spongioform encephalopathy (TSE) or prion disease?

Biopsy of brain tissue. * We can't diagnose this until the person is dead. We don't have some diagnostic where we can say, you have mad cow disease. So the brain biopsy is too late. There is no cure and no treatment so it is potentially a really scary thing esepcially if it is in our food system.

What is the brain pathology of prion infection?

Brain pathology is spongiform encephalopathy—large vacuoles in cortex and cerebellum give brain a sponge-like appearance. You get these deposits of amyloid plaques.

How is PrPSc encoded?

By the host cell gene.

In this research, the researchers concluded that biologically cloned prion populations gradually become heterogeneous by accumulating "mutants": cell-adapted" outcompete "brain-adapted". How did they make this conclusion?

CPA shows that cell derived and brain-derived prions differed (the prions from the cells didn't infected the same way as the prions from the brain). This was carried over time to show it's an accumulation of mutations, it's not immediate. * We show that biologically cloned prion populations gradually become heterogeneous by accumulating "mutants": cell-adapted" outcompete "brain-adapted".

Mad cow disease can happen in two ways. What are they? (2)

Can arise spontaneously or by ingestion of infected tissue.

What are some of the symptoms prion infection?

Chronic, progressive, invariably fatal central nervous system degeneration.

The infectious agent of prion disease has what? And doesn't have what? * These are two very distinctive characteristics.

Has proteins. No detectable nucleic acid.

What are some questions that people are still asking that support the prion hypothesis (not viral)?

If a virus, how could it be genetically inherited? Why is it so resistant? Extremely purified and can't detect yet still infectious but no immune response? Bacteria and yeast can produce prion-like proteins.

Where are PrPC found?

In tneuronal synapses.

What is the prion hypothesis?

Infection with exogenous PrPSc would "seed" aggregation. Once the process begins, it is autocatalytic, as the product catalyzes formation of more of itself from the originally folded protein. * It makes a transititon to the scrapy form and they start sticking to each other and form this fiber, the amyloid fiber, and it's gets longer and longer.

The biological function of the normally folded prion protein has yet to be identified. What do we know and what do we think it COULD be involved with?

It binds copper and could be involved in homeostasis.

When PrPSc comes in contact with PrPC, what happens?

It converts the PrPC into more of itself. These molecules bind to each other forming aggregates so it leads the cellular prions to become scrappie. So if you get scrappie, it'll induce all the other proteins to become scrappie.

The conformational change from PrPC to PrPSc involves formation of beta sheets. This change yields what difference to the new PrPSc protein?

It is insoluble and forms oligomers and aggregates. * Then once these oligomers all aggregate, they form the amyloid fibres and those are what accumulate in the brain and cause problems.

Proteins normally are digested down to amino acids in the gut. How can prions make their way through the gut and into the brain?

It it hypothesized that prion proteins circumvent the normal process of intestinal absorption by passing into the the Gut-Associated Lymphoid Tissue (GALT). * So there is a weakness in our intestine and that is how we get sick - they are really thin cells and so it is a weakness in our gut tissue that the prion proteins may be able to get through.

What happens when you have soluble and insoluble prion proteins all mixed up together?

It turns all the solubles into insolubles and you get this amyloid fibril.

Give two examples where we have seen people become infected through the consumption of prion affected tissues.

Kuru was transmitted through cannibalism in Papua New Guinea. Humans can contract the disease by consuming material from animals infected with the BSE (vCJD).

What are some clinical signs of transmissible spongioform encephalopathy (TSE) or prion disease? (9)

Loss of motoric functions (lack of coordination, ataxia, involuntary jerking movements), personality changes, depression, insomnia, confusion, memory problems, dementia, progressive tonic paralysis, death. * The plaque formation and neuronal loss makes your behaviour change and so that is why it is called mad cow disease.

What disease is associated with prions?

Mad Cow Disease.

What is the inherited prion disease?

Mutations in the PrP gene that favour the transition from the cellular form to the pathological form of PrP. * So this would be associated with the more spontaneous change in prions as opposed to consumption.

How do genetics of prion proteins play into the prion diseases?

Mutations in the prion gene can increase occurrence of disease. * For example, humans who are homozygous for valine at position 129 are predisposed for CJD disease and kuru.

Is there a cure for the transmissible spongioform encephalopathy (TSE) or prion disease?

No.

The point - prion proteins when exposed to drug or various cells have the opportunity to become heterogeneous, to mutate or to change. Are they mutating their amino acids here?

No. They are not really "mutating" the amino acids, it is just changing its structure This is an evolving population.

Is the prion hypothesis correct?

Not all are convinced and still believe that a undetected virus is responsible for conformational changed in PrPC. But for now it is the best explanation that we have.

While PrPC and PrPSc have the same amino acid sequence, they differ in their structural characteristics. Explain.

PrPC is rich in α-helical structure while PrPSc has substantially more β-sheet structure.

How is the PrPSc made?

PrPSc (prion protein) can catalyze its own formation from PrPC in animals.

PrPC is monomeric and protease sensitive. How does PrPSc compare?

PrPSc is resistant to degradation by protease and is multimeric.

How do prion diseases arise?

Prion diseases arise from the harmful function of the abnormal proteins; misfolded forms of proteins (rich in β-sheet structures) have a strong propensity to aggregate into insoluble material and form fibrils.

What are the four MAJOR points regarding the nature of prions. (4)

Prions are transmissible, replicable, and variable disease-causing agents that are distinct from viruses They have arisen in organisms during evolution They are able to propagate themselves and the diseases they cause They appear to be able to evolve and adapt themselves

What is the nature of the infectious prion agent? (3)

Prions are transmissible, replicable, and variable disease-causing agents that are distinct from viruses.

Give an example in real life where we have seen the transmission or propagation of prion disease.

Propagation of kuru disease in New Guinea natives (ritualistic cannibalism). It has been shown that this disease is in the cannibalistic culture (kuru - a form of mad cow disease).

What is the distinct characteristic of prions?

Proteinaceous infectious agent that contains no nucleic acid and consists only of a single species of protein called PrP. * A new kind of infectious agent that can transmit a disease without the intervention of informational nucleic acids.

Explain the solubility differences between PrPC and PrPSc.

Proteinase K (would typically degrade proteins) treatment of PrPC results in complete digestion of the protein whereas PrPSc is partially resistant to digestion with proteinase K and retains infectivity.

What is the principle behind protein misfiling disease?

Proteins can adopt an aberrant conformation that cause disease.

Amyloidosis

Refers to a variety of conditions in which amyloid proteins are abnormally deposited in organs/tissues. A protein is described as being amyloid if, due to an alteration in its secondary structure, it takes on a particular aggregated insoluble form similar to the beta pleated sheets.

How has research through experimentation proven that the prions contain NO nucleic acid but contains proteins.

Resist inactivation by procedures that modify nucleic acids (UV irradiation, nucleases, chemical inactivation). Somewhat sensitive to alkali, phenol, SDS (so things that break down proteins also disrupt the prions).

How is the prion hypothesis similar to natural occurrences we have seen before?

Similar to bacterial flagellum or fimbriae growth; tubulin polymerization, viral capsid assembly and crystal growth. * Bacterial fimbrae are the same. This is really similar in this insoluble property. So even though itforms this big tail, fimbrae cannot be solubilized - it really resembles these prion proteins. The process of polymerization is really similar. You get this tube or aggregate that stick all together.

What are prions?

Small, filterable infectious particles that contain protein but no detectable nucleic acid.

Swa selected for a resistant substrain, whereas, in its absence, the susceptible substrain outgrew its resistant counterpart. How did the researchers find this?

So they took the brain homogenates of prion infected animals, stuck into a cell line and the took the lysate from that. Then they did three different trials. They looked at the % of PrPSc-+ cells propagated with swa, without swa, or with swa and then removed from the swa treatment. In the presence of swa, swa-resistant prion variants selectively grew.So over time when swa is present over generations, they saw group increases so we get resistant scrappies. So over time, at the very end, we can see that we have almost equal amounts of resistance protein at the end. The percent of scrappie postiive cells increase when swa was present. * This sounds a lot like bacterial resistance. When you take the drug away, the susceptible bacteria come back because they don't want to produce resistance markers if they don't have to.

So what is the overall "life cycle" of a prion.

So what happens is that we all probably have prions being expressed. We have the gene for these prion proteins and its only when an insoluble protein comes in and a spontaneously protein becomes insoluble and attacks the native prion protein (the normal protein that is not diseased) and then that precipitates this aggregation - it precipitates all the normal proteins and that forms the plaques under your brain.

What does the PrPSc look like and what does it do in the human body?

So you get these insoluble fibrils that deposit in the brain and that is what causes all the symptoms. PrPSc aggregates and accumulates in diseased brain.

Prions show the hallmarks of Darwinian evolution. Explain.

Subject to mutation, as evidenced by heritable changes of their phenotypic properties, and to selective amplification, as documented by the emergence of distinct populations in different environments. So this showed that we don't need genetic material to evolve.

The PrPSc attacks the native prion PrPC. What is the outcome of those?

The PrPSc attacks the native prion PrPC, changes its conformation into an abnormal form and causes an exponential production of insoluble proteins; they aggregate and form the fibrillar structure.

What are the overall general characteristics regarding PrPSc? (5)

The abnormal, disease-producing protein is called PrPSc (for scrapie) has the same amino acid sequence as PrPC. has dominant secondary structure β-sheets (THIS IS THE BIG DIFFERENCE - it has the same amino acids) is insoluble because of the beta sheets secondary structure is multimeric (it forms together into these multimeric fibrils) and resistant to digestion by proteases.

What did the study of prions really show us that we have never thought to be true through all of our scientific studies of biology and evolution?

The existence of prions suggest that information coded in the three-dimensional structure of a protein can be transferred from organism to organism and propagate itself without the intervention of a nucleic acid.

What is the prion hypothesis concerning?

The formation of infectious and pathogenic prions from normal PrPC.

What are the overall general characteristics regarding PrPC? (5)

The normal protein is called PrPC (for cellular) is a transmembrane glycoprotein has dominant secondary structure α-helix and that makes is soluble is easily soluble is monomeric because it is soluble and easily digested by proteases because it is soluble.

What kinds of cells are the prions expressed in?

The prions are expressed in cells of healthy humans and animals.

So the normal protein undergoes a conformation change to yield the new beta sheets. This is the disease associated protein with a misfolded structures. What are the outcomes from this point? (3)

The proteins can aggregate or not and then go to gain toxic activity or to lose biological function. We are unsure.

Explain what we mean when we are talking about soluble versus insoluble proteins.

The soluble and insoluble proteins means that the soluble proteins are dissolved. So you can see white particular matter when they are insoluble. You can visually see this. From a chemical point of view- you cannot take enzymes to something that is insoluble. It's really hard - because enzymes need to be working with something that is soluble and often these big precipitates are so insoluble that even chemicals won't degrade it.

What is significant about the abnormal prions, the mutated prions (PrPSc) in healthy infected cells? (3)

Their abnormal conformations (PrPSc) are insoluble, resistent to digestion and aggregate.

What is the host immune response to prion infection?

There is no host immune response - no inflammation.

When researchers knocked out the prion gene to see what role it was playing, what did they find?

There was no change in the mice function, so they have no idea what the role or function of that gene is. * Do we need the gene? We don't know. But it is expressed so it must be good for something but we don't know yet what for.

How are prion proteins (PrPC) encoded?

They are encoded by the host genome.

How are human prion diseases acquired?

They can be transmitted or inherited.

There is a paper that looks at the "darwinian evolution" of prions in cell culture. How did they carry out their study? (Explain the second half).

They examined prion characteristics using their cell assay. Then the generated a chronically infected cell population by exposing a line of cells to a prion protein and let them double. They were going to study their infected cell lines with swainsonine (swa) which inhibits chronic infection of cells.

What were the overall conclusions from this research of prions.

They found that the prions that grow through a cell and then later infect the a cell are different from the prions that hadn't gone through a cell, showing that there is some kind of change that happens. They then found that this change or mutations happens over a period of time, through the small accumulation of mutations or change. They found that there is a selective pressure for prions to mutate in the presence of drugs to continue growing through evolution and adaptations.

Development of heterogeneity in cloned prion populations: Selective pressure results in the emergence of different mutants as major constituents of the evolving population. How did the researchers find this?

They have a cell and we've got this PK1[22L] homogenate (the prion that has been grown through a cell line). We have the 22L prions in there. So if we have the one in there that has that mutation such that it was swa resistant. They found that one that had the mutations. They followed it and they exposed it to swa or no swa. With swa exposure, all the prions mutated or had that structural change. If swa wasn't present, that didn't happen. The selection pressure of that drug causes it to change. The next one they took, they found the presence of the mutations. And when they added swa or didn't, they found the exact same thing. The selection pressure of that drug forced it to become that "mutated" structure. In the absence of the sea drug, the same cells would not produce that saw resistant prion because they are lazy - they won't do work that they don't have to.

There is a paper that looks at the "darwinian evolution" of prions in cell culture. How did they carry out their study? (Explain the first half).

They used an assay that can identify a scrappie protein. It's just a way to measure how much scrapie is there to make comparisons. They used different cell lines to represent different hosts and they used a bunch of different scrapie "strains" of proteins. They take the cell lines and expose them to different dilutions of different prion proteins from different hosts. They are just brain homogenates from different host (an animal that was infected with the prion and they have the brain homogenate so they are different prion proteins). They measure the amount of scrapie positive cells to make a comparison of whether it is there or no it is not there. Then they are using an ELISA and are measuring the scrapie protein.

The misfolded protein aggregates and causes disease by either of two ways - discuss.

This can happen by the gain of toxic activity or Loss of biological function.

The prions are proteins that carry information for self-reproduction. What does this contradict?

This contradicts everything we've ever known - the central dogma of modern biology.

What is PrPSc?

This is a glycoprotein, encoded by the host cell genome that is a mutated prion protein (leads to prion disease).

Transmissible spongioform encephalopathy (TSE)=prion diseases. What are these diseases?

This is a group of progressive conditions that affect the brain and nervous system of humans and animals and are transmitted by prions.

Recently, it has been discovered that BSE (one of the prion diseases) had been transmitted to humans in Europe after consumption of infected beef, producing a variant of the CJD called vCJD (another prion disease). What is the significance of this?

This is showing us that the transmission of the prion disease is not just cow to cow but it is cow to human as well.

Transmitted prion disease is otherwise known as...

Transmissible spongioform encephalopathy (TSE).

What prion proteins do uninfected animals express and where do they express these proteins? (4)

Uninfected animals express an isoform, PrPC, on the surface of neurons, lymphocytes, follicular dendritic cells, and other tissues.

What is the pathology of transmissible spongioform encephalopathy (TSE) or prion disease? (3)

Vacuolar degeneration, neuronal loss, and amyloid plaque formation.

What do we think causes the cell damage and prion disease?

We don't really know - it is not yet clear if these aggregates are themselves the cause of the cell damage or are simply a side effect of the underlying disease process.

Why are prions especially scary?

We have a new infectious agent that is scarier than anything else because we don't know how to control that - we don't' know how that works. Prions proteins by themselves can adapt and evolve and so that challenges what we've been taught because we though that we adapt through our genetic information.

What is the function of PrPC?

We know that it binds copper but we don't know what this means and what it's function is.

When we are talking about mutations in the prions, what are we really talking about?

When we talk about the mutations, we are actually talking about changes in the structure because there is no genetic information!

What are the hosts of prions?

Wild and domesticated ruminants (sheep and goats: scrapie; cattle: mad cow disease); mink, cats. Humans (there are a bunch of different diseases, including mad cow disease, kuru and neurodegenerative diseases (amyloid diseases).

Knowing that the prions are transmissible, how are the prions spread between mice in a lab?

You can actually transmit the disease through mice by giving them insoluble plaques. They just eat it and this produces the plaque formation. OR disease can be readily transmitted to mice by intracranial injection of brain homogenate taken from prion-infected animals.

The conformational change from PrPC to PrPSc involves formation of...

beta sheets.

The prions have been experiemtnally transferred from infected brain products into mice, hamsters, chimpanzees from ___________ _______________.

direct inoculation

Prions are proteins that can cause...

fatal brain diseases

PrPSc contains _________ α helices and _________ β strands than PrPC.

fewer more

The point - prion proteins when exposed to drug or various cells have the opportunity to become _________________.

heterogeneous (i.e. to mutate or to change)

Proteins can adopt an aberrant conformation that cause disease; what two mechanisms must be considered? (2)

loss of function of the native protein or gain of toxic activity of the aberrant conformation

The prion hypothesis proposes that...

misfolded forms of a cellular protein can catalyze the refolding of properly folded native protein molecules into similarly misfolded conformations.

Prions, albeit devoid of a nucleic acid genome, are subject to what three things? (3) * As concluded from this study of prions.

mutation and selective amplification and environmental adaptation.

Prion proteins become infectious and pathogenic (PrPSc) as a result of...

protein conformational changes.

The infectious agent of prion diseases contains what? But no detectable what?

proteins Nucleic acid.

The prions are proteins that carry information for...

self reproduction.

PrPC is monomeric and protease ______________ (sensitive/insensitive).

sensitive

While PrPC and PrPSc have the same amino acid sequence, they differ in their _______________ characteristics.

structural

Human prions are encoded by...

the host genome.

PrPC= Predominantly what secondary structure?

α-helix.

PrPSc= What is this secondary structure make up?

β-sheets (40%), α-helix (30%). * So they have all the exact same amino acid sequence. This is JUST a change in the secondary structure.