Experiments Establish 'Protein-only' Nature Of Prion Infections

[Lithos]

Two independent research groups have established conclusively that prions are proteins, and that they do not depend on genes or other factors for transmission of their traits. According to the scientists, the studies answer a nagging question that had raised doubts among some researchers about the validity of the so-called "protein-only" hypothesis of prion infectivity.

--more--

Very interesting. And additional proof that the current testing is woefully inadequate.

[UpInArms]

1. some more of this article

that is revealing:

In test tube experiments, the researchers demonstrated that the protein conformations produced at different temperatures propagated themselves as distinct strains -- providing templates for the folding of other proteins into the same shapes. Further structural analyses of two of the strains confirmed that the proteins were, indeed, folded differently.

When the researchers introduced the differently folded proteins into yeast cells, they found that inside cells, these proteins did indeed produce different prion strains that passed their properties from generation to generation. Finally, they showed that extracting prion protein from subsequent generations of yeast cells yielded protein with the same properties as the strain with which the cells had originally been infected.

[ret5hd]

2. what i find interesting about this is...

aside from the "disease" aspect...

is what we have here is essentially the simplest form of "life" yet discovered.

Per my half-forgotten HS biology classes, viruses (virii?) were the simplest form of life, and in fact were considered to occupy a state between being inanimate and life.

Prions seem to be simpler (no RNA or DNA covered by a protein shell, just a protein) but seem to carry all the other traits of a virus: needing a living cell for self-propogation, communicable, disease causing.

Maybe this is how life actually started? I don't know, just thinking.

[jpak]

3. A relict of replicating biomolecules from Earth's early history?

maybe

But I'm not sure if these critters could be called "life forms" .

They replicate, but do they metabolize (process energy for growth) or evolve under the influence of environmental factors???

Dunno - but still cool stuff...

:)

[ret5hd]

4. yeah, thats why i quoted the term "life". (nt)

[belle]

11. Huh. interesting.

[papau]

5. chemical bond manifold folding is not "life" - but it is interesting.

I still do not see how one gets an agent that refolds the maniford correctly - or alternatively stops a checmical bond action that causes the "normal" protein from going "prion".

Indeed if prion causes normal to go prion, why are not all "normal" turned into "prion" shaped protein?

but it's interesting. Indeed it is right up there with the question "What evolutionary goal is met by a one cell form of life becoming multi-cell?".

I suspect I will die with many questions still unanswerred!

:-)

[treepig]

7. a major conformation change is required

to change the healthy cellular form of the prion protein (shown on the left, below) into the form associated with prion diseases (shown on the right, below):



the reason that all healthy prion proteins don't spontaneously assume the incorrect conformation is that there is a considerable energy barrier between the two forms. for example, if you have a convertable sofa, it doesn't spontaneously interconvert between its two forms - active intervention, i.e., an input of effort/energy is required.

[papau]

8. Sound like ice and water and the need for heat - I don't see metabolism?

:-)

So at the wrong body temp proteins go south and we are dead? And what prevents this?

OR what permits it?

Indded if outside energy is the only source, what form is that energy limited too?


Indeed I am asking if the unsolved 1999 question "must be either a posttranslational modification or environmental factors involved in this event" has been solved

from: Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12633-8.

It has been known that the structural transition from PrP(C) to PrP(Sc) leads to the prion formation. This putative conformational change challenges the central dogma of the protein folding theory-"one sequence, one structure." Generally, scientists believe that there must be either a posttranslational modification or environmental factors involved in this event. However, all of the efforts to solve the mystery of the PrP(C) to PrP(Sc) transition have ended in vain so far. Here we provide evidence linking O-linked glycosylation to the structural transition based on prion peptide studies. We find that the O-linked alpha-GalNAc at Ser-135 suppresses the formation of amyloid fibril formation of the prion peptide at physiological salt concentrations, whereas the peptide with the same sugar at Ser-132 shows the opposite effect. Moreover, this effect is sugar specific. Replacing alpha-GalNAc with beta-GlcNAc does not yield the same effect.

[treepig]

10. energy barriers could be overcome by input of outside energy

such as heat, but in biological systems, such barriers are generally overcome instead by actually lowering the barrier.

for example, the whole point of an enzyme is to stabilize the transition state of a chemical reaction (i.e., lower the energy barrier), or in some cases tunnel through the barrier, in either case to allow reactions to occur rapidly at physiological temperatures.

similarly, it is likely that one misfolded prion protein acts as a template to "catalyze" the misfolding of the second, the two misfolded proteins then catalyze the misfolding of two more (to give four), the four then produce eight, and so on. the point being that geometrical amplification of misfolded proteins can occur once the process gets started.

now consider that hundreds of millions (perhaps billions or trillions) or misfolded prion proteins are needed to cause clinical symptoms of a disease. clearly it will take a long time to go from 1 to 2 to 4 to 8 to 16 to 32 . . . (.etc.) to get all the way to the hundreds of millions stage (hence the long incubation period for the disease). anything that speeds up this process even slightly (or gives a higher number of starting misfolded proteins, such as eaten diseased animal brains), is therefore much more likely to allow the disease to occur before a person dies of other causes, such as old age (after all, it is a scientific certainty that everyone alive today who doesn't die of something else first will die of a prion-related disease).

in some cases, congenital mutations in the protein sequence makes the prion protein easier to undergo the conformational change form the healthy to diseased forms. in other cases, glycosylation apparantly can play a similar role in speeding up the process.

it might be noted that for years researchers produced prion proteins used in their studies in bacteria. bacteria do not glycosylate proteins in the same way the mammalian cells do, therefore the results of many, many studies are not very relevant to the development of human/mammalian disease (and probably contributed to the long time it took for the protein-only mode of infection to be accepted).

[papau]

13. Thanks - As is obvious, I am not a bio person - and you gave a

nice explanation that even I could understand.

:-)

I realize "mutations happen", and that reaction speed at physiological temperatures to get one misfolded prion protein, acting as a template, to "catalyze" the misfolding of the second, could be longer in some folks than in others - but this does leave room for a few papers to be written and even a few degrees to be awarded in the area of why, how, how fast is the reaction, and how is it slowed or stopped.

If I was young (and better at bio this time round :-) )it would be a top contender for a career choice!

[treepig]

17. hmm, "a top contender for a career choice"

yeah, if you like doing a hell of a lot of work for basically sub-mininum wage pay for 10 years of your life (grad school and post-doc where you're expected to work 80 hour weeks for $22,000/yr).

suppose that's why 60% of life science ph.d. students and post-docs are foreign.

it is an interesting field, tho!

[HereSince1628]

12. There is a LOT more written on the move to multicellularity...

And IMHO the questions about multicellularity are very different.

From a biologist's perspective several things are very interesting to me.

1) Prions do seem to have the capability to alter the shape of other biomolecules. Of course enzymes are proteins and they catalyze much of cellular metabolism. Altering a molecules "folding" seems not a far step for an enzyme.

A disease based on a "self-replicating" enzymatic capacity is very curious. It begs the questions--What makes these proteins vulnerable to "being turned" against their basic nature?

2) The capacity of an enzyme to alter a substrate so that it behaves like the enzyme is truly fascinating. This replicability of enzymatic capacity isn't the ability to cause a new protein to be made but rather the capacity to alter existing proteins. Hence it's not so much a form of vertical inheritance (parent to child, and unrelated to the central dogma of biology...creating mRNA templates that drive protein syntehesis) as it is a source of horizontal transfer of information/capability (a protein alters an existing protein). TO me an interesting question is...are biological systems "adapted" to protecting themselves from horizontal assaults of things such as prions?

[papau]

14. Great Questions - the horizontal transfer question is similiar to the

DNA sequence change that ties not back to parents but instead is a transfer from some other currently living thing. Again I know nothing real - only the fact the question was raised 50 years ago when I took a sip from the college firehose in this area.

As to evolution- going from single cell to multi-cell - I have never heard of anyone with a "why"

Evolution happens because - all the reasons we apply to multi-cell - do not seem to apply to that monster of a change from single cell to multi-cell.

Yhis is yet one more area that I know next to nothing!

:-)

[treepig]

16. a big advantage to being multicellular

is that that allows cells to become specialized.

there is a limited number of things any one cell type can do. consider that the human genome contains about 30,000 genes - however in any particular cell type only about 5-6,000 of the genes are expressed (i.e, made into proteins) - the rest remain silent. somewhat interesingly, yeast - a unicellular eukaryote, only has about 6,000 genes in total. therefore, there seems to be a limit to what any individual cell can do or be.

however, in a multicellular organism, different cells can express different sets of genes, and become quite different from each other. on top of a basic set of several hundred "housekeeping" genes that are required by each and every cell to survive, each cell in your body is able to "pick and choose" (unconsciously, of course) which of the other 15-20% of the total 30,000 genes that it will use. by "picking and choosing" different set of genes, the human body consists of over 200 distinct cell types - the final result is quite remarkable (compared to a yeast cell, for example). many of these cell types are highly specialized and could in no way survive on their own - hence the benefits of multicellularity.

perhaps an analogy would be to an agarian society where everyone is a farmer (think, unicellular yeast cell). not really much of a society. now consider the current modern society (think, the human body) - chock full of a diverse array of highly interdependent specialized members, very few of whom could survive on their own, but together (for better or worse) the result is quite remarkable.

[HereSince1628]

21. For trad. zoologists this is important as all animals are multicellular

by definition...unicellular animal-like things aren't animals. So the step up to multicellularity is rather fascinating for zoologists.

Why would a perfectly good single celled organism become multicellular?

Sure multicellularity makes possible compartmentalization of tasks to specific cell lines...but if a single cell can do this perfectly well, why risk sharing that responsibility with a partner?

Can cellular partners always be counted on to be trustworthy?

Sexual reproduction (meiosis producing gametes) occurs in single celled organism. But sexual reproduction is much more common in multicellular organisms.

A single cell undergoing meiosis is committing a sort of suicide without death...it will divide into a number of "daughter" cells (4 is a common teaching model) each with only half the genetic complement to survive. Timing is obviously everything. If these haploid individuals don't find a partner they are genetically terminal.

A multicellular organism might gain an advantage somewhat over the critical nature of timing...a multicellular organism can have multiple reproductive options (potentially and actually a number of simple animals reproduce by both asexual and sexual means). In the classic sense (and the classic sense is somewhat undone by studies demonstrating the pluripotentiallity of cells) ann organism would have somatic cells to tend to the business of growing and surviving and germ cells to deal with sexual reproduction. Some cells undergoing meiosis don't "undo" the organism, allowing for the possibility to survive and engage in another bout of gamete making.
If the first gametes failed to find partners the chances improve with each iteration of gamete making.

Sexual reproduction has its own cost/benefit ratio. For animals the ratio seems to favor sex. For single celled organisms sexual reproduction is too frequent to be said to be uncommon, but it isn't the dominant form of reproduction. In animals which are relatively long-lived organisms whose lives may include/span several habitats, diversity in the gene pool seems to be a big deal. Sexual reproduction enhances diversity through mixing. Is that the benefit of multicellularity, or is that merely a useful by-product???

As to prions...there seems to be no DNA involved in the transformation of a perfectly good protein into a prion. That is both interesting and...well, wierd. A cow getting a prion isn't going to modify it's DNA, but its proteins could alter another cow's proteins if the prion is fed to another cow in the form of say brain protein added to feed supplements. Such a change in a protein is in itself outside the inheritance structure that makes evolution possible to discuss. But, resistance to protein folding into prion- like patterns might be a consequence of protein synthesis and protien primary, secondary and tertiary structure, hence DNA and its stored information might be important to prion susceptibility and resistance.

[treepig]

22. to clarify your statement

"As to prions...there seems to be no DNA involved in the transformation of a perfectly good protein into a prion."

it should be noted that prions can be perfectly good proteins - more specifically, the normal cellular form of a prion (PrP(c)) is a perfectly good protein, it's only the misfolded form (PrP(sc)) that is "bad" - so perhaps we could give all the prion-bashing a break?

it still remains somewhat of a mystery what the "perfectly good" prion proteins does in a cell, one possibility is that they prevent brain damage:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14753456

other studies have suggested cellular signalling roles for prions:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14756798

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=14625887

[troublemaker]

18. re: As to evolution- going from single cell to multi-cell

Multi-cellular critters most likely started off as colonies of single cells. I imagine that circumstances favored clustered cells and then cells on the outside had a different environment from those on the inside -- next thing you know you've got favorable colonies with slightly differentiated cells... maybe cells that stick together form more cohesive colonies, and so on.

Just a guess. (Granted, from someone that genuinely believe life probably started as crystalline clays)

[papau]

19. logical -Doesn't quite work - the inner cell would not always be an inner

cell as the the environment re-arranged the cluster.

The specialized concept suggested by Treepig - given a limit of 6000 per cell - is also logical. and also has same problem.

but random single to multiple experiments would indeed follow this path.

I guess my question is why give up the strong cell wall and Independence in order to try a multi cellar existence? It is almost like God said - OK - now do multi-cell - 'cause there does not seem to be much if any survival push to do so for the original 6000.

[treepig]

20. here's what real scientists have to say

in real scientific papers:

J Biosci. 2003 Jun;28(4):523-8.

Title: On the origin of differentiation.

Bonner JT.

Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 08544, USA.

Following the origin of multicellularity in many groups of primitive organisms there evolved more than one cell type. It has been assumed that this early differentiation is related to size the larger the organism the more cell types. Here two very different kinds of organisms are considered: the volvocine algae that become multicellular by growth, and the cellular slime moulds that become multicellular by aggregation. In both cases there are species that have only one cell type and others that have two. It has been possible to show that there is a perfect correlation with size: the forms with two cell types are significantly larger than those with one. Also in both groups there are forms of intermediate size that will vary from one to two cell types depending on the size of the individuals, suggesting a form of quorum sensing. These observations reinforce the view that size plays a critical role in influencing the degree of differentiation.



Anat Rec. 2002 Nov 1;268(3):327-42.

Title: Origin of multicellular organisms as an inevitable consequence of dynamical systems.

Furusawa C, Kaneko K.

Center for Developmental Biology, The Institute of Physical and Chemical Research (RIKEN), Kobe, Japan.

The origin of multicellular organisms is studied by considering a cell system that satisfies minimal conditions, that is, a system of interacting cells with intracellular biochemical dynamics, and potentiality in reproduction. Three basic features in multicellular organisms-cellular diversification, robust developmental process, and emergence of germ-line cells-are found to be general properties of such a system. Irrespective of the details of the model, such features appear when there are complex oscillatory dynamics of intracellular chemical concentrations. Cells differentiate from totipotent stem cells into other cell types due to instability in the intracellular dynamics with cell-cell interactions, as explained by our isologous diversification theory (Furusawa and Kaneko, 1998a; Kaneko and Yomo, 1997). This developmental process is shown to be stable with respect to perturbations, such as molecular fluctuations and removal of some cells. By further imposing an adequate cell-type-dependent adhesion force, some cells are released, from which the next generation cell colony is formed, and a multicellular organism life-cycle emerges without any finely tuned mechanisms. This recursive production of multicellular units is stabilized if released cells are few in number, implying the separation of germ cell lines. Furthermore, such an organism with a variety of cellular states and robust development is found to maintain a larger growth speed as an ensemble by achieving a cooperative use of resources, compared to simple cells without differentiation. Our results suggest that the emergence of multicellular organisms is not a "difficult problem" in evolution, but rather is a natural consequence of a cell colony that can grow continuously.



Artif Life. 1999 Winter;5(1):1-15.

Title: On the evolution of multicelluarity and eusociality.

Bull L.

Faculty of Computer Studies and Mathematics, University of the West of England, Bristol, BS16 1 QY, UK.

In this article versions of the abstract NKC model are used to examine the conditions under which two significant evolutionary phenomena - multicellularity and eusociality - are likely to occur and why. First, comparisons in evolutionary performance are made between simulations of unicellular organisms and very simple multicellular-like organisms, under varying conditions. The results show that such multicellularity without differentiation appears selectively neutral, but that differentiation to soma (nonreproductives) proves beneficial as the amount of epistasis in the fitness landscape increases. editorial note - that's a succint way of stating my rambling post above This is explained by considering mutations in the generation of daughter cells and their subsequent effect on the propagule's fitness. This is interpreted as a simple example of the Baldwin effect. Second, the correspondences between multicellularity and eusociality are highlighted, particularly that both contain individuals who do not reproduce. The same process is then used to explain the emergence of eusocial colonies.



Annu Rev Genet. 2001;35:103-23.

Title: Building a multicellular organism.

Kaiser D.

Department of Biochemistry and of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA.

Multicellular organisms appear to have arisen from unicells numerous times. Multicellular cyanobacteria arose early in the history of life on Earth. Multicellular forms have since arisen independently in each of the kingdoms and several times in some phyla. If the step from unicellular to multicellular life was taken early and frequently, the selective advantage of multicellularity may be large. By comparing the properties of a multicellular organism with those of its putative unicellular ancestor, it may be possible to identify the selective force(s). The independent instances of multicellularity reviewed indicate that advantages in feeding and in dispersion are common. The capacity for signaling between cells accompanies the evolution of multicellularity with cell differentiation.

Advantages ofMulticellularity
Certain advantages accrue simply from a larger size. The larger Gonium, Eudorina,
and Volvox colonies escape from predation by filter-feeding rotifers and small
crustaceans (23). Daughter colonies of Volvox, which would be small enough to be
eaten by these animals, are kept internally, protected inside their mother colony.
Another important advantage is that the larger colonies can absorb and store essential
nutrients more efficiently (23). Inorganic phosphate is often a limiting nutrient
for algae (3). Large multicellular algae have an advantage in phosphate uptake,
storing any excess as polyphosphate in the extracellular matrix that separates the
cells (25). Other nutrients may also be retained in the matrix, such as minerals, ions,
and water that would help protect against desiccation. Many algae are dispersed
by waterbirds (23); the larger colonies may have a better chance than unicells to
be carried.

[papau]

23. imposing an adequate cell-type-dependent adhesion force? Catch-22?

:-)

just funning!

:-)

It is clear that multi-cell happens when you start with uni-cell. And a gel - or adhesion force - will indeed keep a colony of uni-cell together. But what turns a "reproducing cell colony" with break-aways into a multi-cell with reproduction of multi-cell?

It is not clear that "evolution" with its "improve survivability" concept is the force that causes the event.

The concept that a post multi-cell "differentiation to soma (nonreproductives) proves beneficial as the amount of epistasis in the fitness landscape increases" would work if it applied to uni-cells.

Indeed, if this were true at the unicell level then there would be a reason we could call "evolution" for uni-cells to join up.

Again it is obvious the multi-cell arises from uni-cell. It is just the why that is missing - or perhaps better said - Our current concept of evolution seems to need some tweaking - the same way it was tweaked recently to reflect bursts of new species followed by millions of years of stability and no"evolution" - evolution is just not linear - ok -

So what tweak to the evolution theory explains the move from unicell to multicell?

[treepig]

6. hardly latest breaking news

considering that experiments had established the 'protein-only' nature of prion infections to the satisfaction of the nobel prize selection committee before 1997, the year the prize was awarded to the rather paranoid, but nevertheless brilliant, STANLEY B. PRUSINER

http://almaz.com/nobel/medicine/1997a.html

anyhow, the authors continue to make it sound like a big mystery what strain-specific forms of prion propagation occur. that's because they generally ignore fairly-long-established evidence that sugars attached to the proteins (note the lack of sugars on the structures shown in post #7) play a key role in this process. the role of glycosylation in prion diseases is described in a number of reputable papers, including:


from J Biol Chem. 2002 Sep 27;277(39):36775-81.

Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a protease-sensitive host glycoprotein, prion protein or PrP-sen, to a protease-resistant form (PrP-res). PrP-res molecules that accumulate in the brain and lymphoreticular system of the host consist of three differentially glycosylated forms. Analysis of the relative amounts of the PrP-res glycoforms has been used to discriminate TSE strains and has become increasingly important in the differential diagnosis of human TSEs. However, the molecular basis of PrP-res glycoform variation between different TSE agents is unknown. Here we report that PrP-res itself can dictate strain-specific PrP-res glycoforms. The final PrP-res glycoform pattern, however, can be influenced by the cell and significantly altered by subtle changes in the glycosylation state of PrP-sen. Thus, strain-specific PrP-res glycosylation profiles are likely the consequence of a complex interaction between PrP-res, PrP-sen, and the cell and may indicate the cellular compartment in which the strain-specific formation of PrP-res occurs.

from Proc Natl Acad Sci U S A. 1999 Nov 9;96(23):13044-9.

Prion protein consists of an ensemble of glycosylated variants or glycoforms. The enzymes that direct oligosaccharide processing, and hence control the glycan profile for any given glycoprotein, are often exquisitely sensitive to other events taking place within the cell in which the glycoprotein is expressed. Alterations in the populations of sugars attached to proteins can reflect changes caused, for example, by developmental processes or by disease. Here we report that normal (PrP(C)) and pathogenic (PrP(Sc)) prion proteins (PrP) from Syrian hamsters contain the same set of at least 52 bi-, tri-, and tetraantennary N-linked oligosaccharides, although the relative proportions of individual glycans differ. This conservation of structure suggests that the conversion of PrP(C) into PrP(Sc) is not confined to a subset of PrPs that contain specific sugars. Compared with PrP(C), PrP(Sc) contains decreased levels of glycans with bisecting GlcNAc residues and increased levels of tri- and tetraantennary sugars. This change is consistent with a decrease in the activity of N-acetylglucosaminyltransferase III (GnTIII) toward PrP(C) in cells where PrP(Sc) is formed and argues that, in at least some cells forming PrP(Sc), the glycosylation machinery has been perturbed. The reduction in GnTIII activity is intriguing both with respect to the pathogenesis of the prion disease and the replication pathway for prions


from: Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12633-8.

It has been known that the structural transition from PrP(C) to PrP(Sc) leads to the prion formation. This putative conformational change challenges the central dogma of the protein folding theory-"one sequence, one structure." Generally, scientists believe that there must be either a posttranslational modification or environmental factors involved in this event. However, all of the efforts to solve the mystery of the PrP(C) to PrP(Sc) transition have ended in vain so far. Here we provide evidence linking O-linked glycosylation to the structural transition based on prion peptide studies. We find that the O-linked alpha-GalNAc at Ser-135 suppresses the formation of amyloid fibril formation of the prion peptide at physiological salt concentrations, whereas the peptide with the same sugar at Ser-132 shows the opposite effect. Moreover, this effect is sugar specific. Replacing alpha-GalNAc with beta-GlcNAc does not yield the same effect.

[Lexingtonian]

9. no, this doesn't prove anything particularly important

This result hasn't actually been in much doubt at all- the demonstration is elegant, the gain in knowledge/certainty is pretty close to null. And I think this fellow Jonathan Weissman is a guy I knew when he was in grad school.

The real dispute is what kind of pathogenic action misfolded PrP contains- whether it e.g. enables a viral infection or disrupts a particular kind of wild type signalling pathway. Pathogenic plaques are not unique to prion diseases- there is a kind associated with the pancreas and correlated with diabetes. All this amusement about the particulars fo the folding is a specialists' game. (Forced to guess, I'd say both peptides are secreted calcium channel blockers by function.)

[papau]

15. Do all prion diseases express as pathogenic plaques?

Is a prion "infection" ever without plaque?


And before I forget - thanks for the info!

:-)