We are interested in protein misfolding, amyloid formation and disease, both on the molecular level and in the cellular perspective.
The toxic mechanism of misfolded protein aggregates inducing neuronal degeneration is largely unknown, and this understanding it is clearly a future goal for our research.
Our objective is to inhibit the formation of the toxic species utilizing different approaches including small-molecule binding and interections with molecular chaperones. A special laboratory for prion research was opened in October of 2005 at LiU for the Hammarstrom research group.
We work with a number of proteins prone to misfolding:
and the molecular chaperones:
• BiP (Hsp70)
• GroEL (Hsp60)
• GroES (Hsp10) and other HSP10 chaperones
and fluorescent proteins:
• e-GFP
• e-CFP
• e-YFP
• mNeonGreen
We also work with the fly Drosophila melanogaster as research model for neurodegenerative disease, typically animals expressing Aβ and Tau.
Microscope images from Drosophila melanogaster models of Alzheimers disease. The Alzheimer associated protein Aβ has been expressed in different cell types in the central nervous system of the fly and formed amyloid. The amyloid is stained with green pFTAA. Cell nuclei are coloured red. Image from Jonson et al Cell Chemical Biology 2018
The basis for selective vulnerability of certain cell types for misfolded proteins in neurodegenerative diseases is largely unknown. This knowledge is crucial for understanding disease progression in the CNS. Cell specific expression of human Aβ1-42 associated with Alzheimer’s disease in Drosophila neurons resulted in concentration dependent severe neurodegenerative phenotypes, and intraneuronal ring-tangle like aggregates with immature fibril properties. Unexpectedly, expression of Aβ1-42 from a pan-glial driver produced a mild phenotype despite massive brain load of Aβ1-42 aggregates, even higher than in the strongest neuronal driver. Glial cells formed more mature fibrous aggregates, morphologically distinct from aggregates found in neurons, and was mainly extracellular. Our findings implicate that Aβ1-42 cytotoxicity is both cell and aggregate morphotype dependent.
In recent years it has become evident that impaired protein folding plays a key role in a wide variety of diseases.
There are considerable overlaps between misfolding causing loss-of-function and gain of toxic function. In many cases, it is not clear which of the mechanisms that are the dominating cause of pathology. It is therefore important to have a holistic view of the folding and misfolding areas to understand the molecular basis underlying these diseases.
Common denominators for these proteins are: Aberrant folding, partially folded intermediates, conformational plasticity and protein aggregation.
The amyloid diseases is a part of a larger group of conformational diseases, many of which are termed misfolding diseases. This group includes for example devastating diseases such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease.
Neurodegeneration is associated with amyloid diseases of the central nervous system, and most often the direct cause of symptoms. It is not known how neurodegeneration commences following misfolding of these proteins and many are the suggestions as of which of the multitude of misfolded self-assembled protein aggregates are the most toxic species.
It is likely that neurons are particularly sensitive to this type of proteinaceous toxins. Part of our research is focused on addressing this question. This is done by combining conformation sensitive, fluorescent, amyloid targeting probes that can be used both in the test tube and on tissue sections for microscopy. We address the test tube samples with biophysical analysis and compare disease properties of different amyloid conformations in animal models or in real patient samples displaying the same amyloid conformations, determined by fluorescence microscopy.
Conformation sensitive fluorescent probes can be used to detect amyloid and discriminate between different types of amyloid within one amyloid deposit. Small changes in the chemical structure of the probes modulate their binding. Image adapted from Zhang et al Journal of Medicinal Chemistry 2019
Using conformation sensitive fluorescence probe and a range of fluorescence imaging techniques we can delineate differences in amyloid plaques with high resolution and specificity. Image adapted from Nyström et al Journal of visualized experiments 2017
Amyloids can be found in virtually any organ of the body and over 35 different proteins are known to form amyloid in humans.
Familial Amyloidotic Polyneuropathy (FAP), also known as Skellefteåsjukan, is caused by amyloid formation of Transthyretin (TTR). FAP is hereditary and carriers of the disease associated mutant TTR will invariably come down with the disease. Successful basic research has paved the way for the treatment of this disease by stabilizing the culprit protein, the mutated TTR, and thereby preventing it from misfolding into amyloid.
This and other successful treatment strategies has proven that targeting protein misfolding can prevent or cure amyloid diseases.
Many different protein cause amyloid formation and disease in the human body. However, amyloid originating from the same protein does not always adopt the same amyloid structure. This conformational polymorphism is one of our research topics. Image from Fändrich et al, Journal of Internal Medicine 2018
For most sufferers, COVID-19 is a respiratory infection with symptoms like the common cold. However, some people develop a serious medical condition that affects many parts of the body, including the brain. Some people also suffer from persistent or delayed symptoms, called post COVID or long COVID.
When we follow the development around acute and long COVID with the eyes of an amyloid scientist, we see many similarities between the COVID phenotypes and the molecular processes that occur in amyloid diseases. Amyloidosis is caused by a specific and very stable structural protein change that is associated with a variety of proteins and diseases as described above. Through test tube experiments, we have strengthened our hypothesis that the spike protein on the surface of the SARS-CoV-2 virus forms amyloid structures when it is cleaved by neutrophil elastase, an inflammatory enzyme that is important for defending us against infection. Neutrophil elastase is abundant in infected airways in COVID patients.
There are many reports of coagulation disorders in COVID-19. Patients do not appear to respond as expected to anticoagulant therapy. Thus, vascular symptoms are frequently reported, but few studies present molecular mechanisms that explain these enigmatic and devastating conditions. In test tube experiments with pure proteins, we have shown that the amyloid fibrils formed when the spike protein is cleaved by neutrophil elastase can impair the ability of the body's regulatory system to break down blood clots at a normal rate. Something that could explain the enrichment of microscopic blood clots that are found in many people who suffer from long COVID.
In our further research, we aim to investigate the relationship between the amyloid formation we discovered and the coagulation problems, the neurological impact and multi-organ damage described in severe and long COVID / post COVID. It is of particular interest to understand how the protein sequence of the spike protein in the numerous SARS-CoV-2 variants influences the amyloidogenicity of the protein in relation to this putative amyloid pathogenesis.
The SARS-CoV-2 virus is covered by Spike proteins (in red) which enable infection of the host cells. Neutrophil elastase (yellow) is an important part of the first line of defence of the immune system. When the Spike protein is cleaved by Neutrophil elastase, the resulting protein fragments form amyloid that potentially have negative effect on blood coagulation, neurodegeneration and systemic diseases.
Amyloidogenesis of SARS-CoV-2 Spike Protein
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The prion diseases Creutzfeldt-Jakob’s disease and Bovine Spongiform Encephalopathy (Mad cow disease) are often associated with amyloid deposition. The term Prion is an abbreviation for PRotein Infectious ONly. Prions, in the classic sense, are protein infectious agents composed of the prion protein PrP. However, the debate is ongoing regarding prion properties also exhibited by amyloid assemblies comprised of other proteins.
The prion protein particle was the first example of an infectious agent that lacks nucleic acid, as opposed to conventional infectious agents such as parasites, bacteria or viruses. Prions cause a variety of diseases in humans and animals including bovine spongiform encephalopathy (BSE) or mad cow disease, Creutzfeldt-Jakob disease (in humans), Chronic wasting disease (in deer and elk) and scrapie (in sheep). Collectively prion diseases are in medical terms named transmissible spongiform encephalopathies or TSE´s due to the transmissibility of the diseases and the pathological feature of microvacuole formation in affected areas of the brain.
A dramatic feature of the transmissible spongiform encephalopathies, TSEs, is the rapid and efficient neuronal degeneration process. Misfolding of the prion protein (PrP) is a crucial step in these diseases. Here, the PrP misfolding process entails the conversion of a helical protein to a largely insoluble β–sheet rich state.
But, how can a misfolding process be infectious? The novel and controversial concept of the prion hypothesis is the propagation of protein conformations through structure based templated folding presented by Stanley Prusiner and is replicated in vitro (see Figure below).
PrP - The full-length soluble domain of mature human PrP protein is composed of 209 amino acids (res. 23-231) and is, in the natively folded PrPC form, folded into two domains. The N-terminal unstructured domain comprising the initial 90 residues has very high flexibility and affinity for Cu2+. The C-terminal globular domain folds into four helices and two short β–strands. The structure of the disease causing and infectious misfolded PrPSC particle is not known in detail due to the insolubility of the protein. PrPSC adopts a stable oligomeric structure with a large amount of β–sheet structure. Prion strains appear to be encoded in the PrPSC structure.
The prion protein can convert into its disease causing amyloid conformation both through a sporadic route and by templated seeding by adding a small fraction already formed amyloids. The spontaneous, unseeded reaction is significantly slower than the seeded reaction. Image adapted from Nyström et al, Journal of Biological Chemistry 2012
Prion diseases, also termed TSEs (transmissible spongiform encephalopathy) are known to afflict a large number of mammals. The disease is mediated either by sporadic events of misfolding of the endogenously expressed PrP, or by transmission of preformed, misfolded prion particles from a source outside of the host.
BSE in cattle, Scrapie in sheep, MSE in mink and Chronic wasting disease in wild cervids has been known for several decades. More recently, prion disease has been discovered in dromedary camels in North Africa, demonstrating again the wide spread of prion disease among mammalian species.
The three-dimensional fold of native PrP from a wide range of mammals is well conserved. However, the amino acid sequence can vary. There are a number of single nucleotide polymorphisms (SNPs) that are hypothetically protective against infection by prion disease. Dogs and pigs appear resistant to prion disease, while human, cat and cow all are susceptible to prion infection.
We have shown that, although being resistant to prion disease, both dog PrP and pig PrP readily form amyloid fibrils in the test tube and the resulting fibrils are capable of enhancing amyloid fibril formation of any of the PrP sequences we tested, regardless of species origin, prion susceptible or resistant.
We study prion protein sequences from several different mammals and how their similarities and differences dictate their ability to form amyloid and interfere with each other. Image adapted from Nyström and Hammarström Scientific Reports 2015
One intriguing feature about prion biology is the strain phenomenon and how it is coupled to a species barrier that moderates which species can infect and be infected by which.
Although very similar in native three-dimensional structure, PrP can misfold into numerous misfolded states and thereby cause disease with different clinical attributes as well as histologic lesion profiles. Often, the strain rather than the amino acid sequence dictates the course of disease, as well as intra- and inter species transmission. When a prion strain is transmitted from one species to another, it can reside in, and transmit between individuals of the new host species without causing clinical disease, for several generations before it eventually has adapted to its new host. The novel, adapted strain can then cause disease with different clinical symptoms than were seen in the original donor species.
Prions strains rather than amino acid sequence dictate their infectivity. Prions are known to be able to adapt to novel species, generating new infectious strains.
Since 2016 it has been known that Chronic Wasting disease is present in Europe, after the discovery of the disease in a reindeer in Norway. As of March 2019 the disease is also a fact in Sweden. Already at this early stage of investigation there are strong indications that the CWD prion strains found in Scandinavia are different from those found in North America.
We run a BSL3 lab dedicated to the studies of prions, specializing in experiments in the intersection between recombinant proteins and tissue samples.
It is now well accepted that misfolded proteins are severely detrimental to the cell. Evidence for this can be found within every cell from E. Coli to humans - in the absolutely vital machinery of the molecular chaperones.
Molecular chaperones constitute a natural defense against misfolded proteins. Chaperones can bind and sequester misfolded proteins, stretch them apart to provide a new chance for productive folding or present the misfolded protein to the degradation machinery.
We have shown that the molecular chaperones BiP (Hsp 70) and the bacterial chaperonin GroEL/ES (Hsp 60/10) recognize partially folded substrate proteins (a.k.a. clients) and remodel their structure. Partially folded proteins are structurally plastic and can in the absence of chaperones result in aberrant aggregation (misfolding). Chaperones can correct misfolded protein structure through binding induced unfolding and provide a new chance for correct folding. We have shown that Hsp10, albeit small, is potent as an unfoldase and a protein folding guide and also as inhibitor of protein amyloid formation in the test tube.
Intriguingly we also discovered that minute amounts of HSP10 can accelerate A尾 and PrP fibril formation suggesting a new pathway under chaperone depletion conditions.