Our observations consistently show that E34K, which is much less conservative than F71Y substitution, is also more disruptive. Notably, the most disruptive mutation, LR, is non-amyloidogenic. These results support the idea that structural destabilization alone is not sufficient to make the protein amyloidogenic, and suggest that local protein structure and dynamics in sensitive regions are important.
MD simulations also revealed significant mutational effects on the conformation of Trp72 and suggested that alternative conformations of Trp72 and perhaps Trp8 at the bottom of the helix bundle were mainly responsible for the differences in the Trp packing among the variant proteins observed by near-UV CD Fig. Such perturbations are expected to increase solvent exposure of the nearby Tyr18 and other residues from the major amyloidogenic segment LeuLeu22, thus favoring protein misfolding.
In all cases high yields of protein were obtained. Establishing the expression and purification methods was essential to perform the proposed studies and also set the basis for new relevant projects that will continue after the reporting period. Gel filtration reported on the apparent stability of the tetramers showing that, in common with hTTR, sbTTR and lampTTRs form homogenous tetramers and retain a tetrameric conformation in solution Fig.
Because the TTR tetramer is generally very stable and does not loose structure with heating up to C, we used Urea 3M to destabilize the tetramer before performing a melting scan. These results suggest that during evolution from agnathan to humans, TTR not only gained affinity for THs but also apparently became more amyloid-prone and, paradoxically, more thermostable.
Increased stability may have evolved as a protective mechanism against amyloidosis. These results were confirmed by electron microscopy Fig. These results suggest that, surprisingly, fibrillation propensity of TTR correlates directly with protein thermostability. The structure of lampTTR has not been previously described and its markedly longer N-terminus may suggest an alternative or more stable configuration. To obtain information on lampTTR structure and possibly on the localization of the N-terminal within the structure we tried to crystalize the protein. Crystal screens were based on previously published conditions for h and sb TTR.
Crystals shaped as needles were obtained in 2. The obtained crystals will be tested by x-ray and data will be collected to produce a lampTTR 3D structure. The corresponding sequences were cloned into a pET11a vector, expressed in E. Since CRTAC1 forms high molecular weight aggregates spontaneously in solution, the observed thermostability may also be a consequence of a high thermoresistance of the formed aggregates. The presence of high molecular aggregates was confirmed by size-exclusion chromatography SEC Fig. However, for dlCrtac1b, SDS-PAGE revealed that the protein in the higher retention volume fraction contained a mixture of both aggregates and monomers suggesting that monomeric dlCrtac1b was highly unstable and aggregated immediately after isolation.
No significant change in the aggregation patterns of the CRTAC1 proteins analysed was identified during 3 weeks incubation not shown. Because CRTACs are aggregation prone and hard to crystallize, we focused specifically on the most conserved and potentially more soluble beta-propeller region this region has been crystallized in other beta-propeller proteins. Knowing the expression and purification of the proposed proteins might require thorough optimization we set the goal to produce soluble and sufficient amounts of proteins within this project and continue with the crystallography trials after the project.
For this task we established a new collaboration that will continue after this project with the group of Prof. Liebermann at the Georgia Institute of Technology. Liebermann has extensive experience on the crystallization of difficult beta-propeller proteins. After extensive optimization, we could obtain a significant amount of protein in the soluble phase Fig.
WP1- Apolipoprotein AI misfolding Amyloid diseases, including neurodegenerative and systemic amyloidosis, affect millions of patients worldwide and are a major public health challenge. Apolipoproteins, a family of lipid surface-binding proteins are overrepresented in amyloidosis. Hereditary apolipoprotein A-I apoA-I amyloidosis is a life-threatening type of systemic amyloidosis due to specific amino-acid mutations in the apoA-I protein.
Mutant apoA-I proteins aggregate and deposit, often as N-terminal fragments in various organs, causing organ damage. So far, the molecular events triggering protein aggregation in this and other amyloid diseases are not completely understood. We contributed to elucidate the molecular basis for the amyloidogenic properties of the apoA-IE34K variant and compared its conformation and dynamics with those of other disease-causing variants, Phe71Tyr amyloidogenic and LeuArg non-amyloidogenic.
Clinical Mobile Resources
We found that the mutations reduced protein stability and altered aromatic side chain packing and caused local helical unfolding. Our findings exemplify how diverse mutations alter local conformational dynamics in similar sensitive regions of a protein molecule. This phenomenon is probably not limited to apoA-I, but may extend to other globular proteins and their disease-causing variants This contribution to elucidation of early steps on protein unfolding, along with improved diagnostics, can advance molecular understanding and therapeutic targeting of amyloid disorders.
The study of amyloidosis as a basis to understand the process so effective treatments can be identified is of considerable socio-economic interest. This is particularly so in Europe where the average age of the population is shifting upwards and makes diseases of the aging population relevant from a series of socio-economic perspectives. The costs of amyloidogenic diseases for society are considerable in terms of health care needs, loss of work, injury but also because of their impact on the structure and functioning of societies.
WP2- Transthyretin misfolding Transthyretin amyloidosis is another type of amyloid disease with high prevalence in countries like Portugal and Sweden. Human Transthyretin transports thyroid hormones in the blood in a tetrameric conformation. When the tetramer is destabilized the protein unfolds and aggregates into amyloid fibrils that deposit in organs or the nervous system causing severe and lethal polyneuropathies and cardiomyopathies.
We had previously characterized TTR evolution using fish TTR models and hypothesized that N-terminal region, which was remodeled during evolution, could be involved in amyloid formation. In the STAMYEV project we provided evidences that the N-terminus is involved in the protein stability and modulates amyloid formation during evolution.
Table of contents
We found that TTR from the ancient agnathan, lamprey does not form fibril despite the lower stability of the protein. With this work we set up the grounds to further investigate in detail the molecular interactions in lamprey TTR that prevent the protein to enter the amyloid-formation pathway. We hope in the future to translate this information into targeting human TTR amyloid formation in disease and provide insights into development of new therapies. The host in Boston works in direct collaboration with the Boston University Amyloidosis centre where basic science is combined with clinical interventions.
Boston is a large hub of pharmaceutical companies, several of them directly involved in finding therapies for TTR amyloidosis, and working directly with the Amyloidosis centre in clinical trials.
Recent Advances in Transthyretin Evolution, Structure and Biological Functions
Any future outcomes from this project have the potential to impact the patients. I will be working at the laboratory of Professor Liao, to help develop a project focused on Transthyretin amyloid cardiomyopathies. In this new project we will develop animal models of transthyretin amyloidosis and we will further test the N-terminal hypothesis in vivo, including different mutants. CRTAC is putatively involved with diseases of the human cardiovascular, haematological, neurological, respiratory and urinary systems and potentially in multiple sclerosis.
With our studies we found that CRTAC1 forms high molecular weight thermo-stable aggregates and revealed for the first time that such aggregates can consist of amyloid-like structures that might be related to its disease-association.
Overall, we provided new insights into the potential biological function of CRTAC1 in vertebrates and its possible role in disease. We have opened up the pathway for targeted studies in human to establish the link between the aggregating potential of this protein and amyloidogenic pathologies.
Recent Advances in Transthyretin Evolution, Structure & Biological Functions
Within my future projects at Stanford School of Medicine I will aim to further develop such studies. Overall, the project has had impact in diverse areas such as fundament biology, medicine, health, society and has carried out the groundwork for new targeted interventions in amyloid diseases. Amyloid related disorders represent a tremendous medical and socio-economic problem, specifically in ageing societies, such as in Europe or North America which are also two major hubs of amyloid research.
The understanding of amyloid formation is a clear priority in the context of degenerative diseases and our studies that are aimed towards understanding the evolution of the protein structures and dynamics of amyloidogenesis directly contribute to that goal. Specifically, we helped to: 1 establish a novel molecular mechanism of apoA-I misfolding in Apolipoprotein amyloidosis and atherosclerosis, 2 support the novel hypothesis that TTR N-terminus plays a key role in TTR misfolding in amyloid, and 3 establish a link between evolutionary adaptation and amyloid formation by fish and human TTR.
- How to Draw Sports World (Sports Related Beginners Drawing Tutorial) (how to draw comics and cartoon characters Book 36);
- Mr. Simples Problem.
- The Unspoken (Krewe of Hunters, Book 7).
- Recent Advances in Transthyretin Evolution, Structure and Biological Functions!
- Download Recent Advances In Transthyretin Evolution Structure And Biological Functions.
- Realmfall Act 1: The Scarab Princes?
Buy eBook. Buy Hardcover. Buy Softcover. FAQ Policy. About this book There is a strong interest in transthyretin TTR in connection with protein evolution, medical and clinical research. This monograph will bring the reader up to date on the latest developments and discoveries.
- Download Recent Advances In Transthyretin Evolution Structure And Biological Functions?
- Download Recent Advances In Transthyretin Evolution Structure And Biological Functions 2009.
- Among Murderers and Madness.
- Similar titles.
Show all. Resource Ecology. Restorative Therapies in Parkinson's Disease. Retinal and Choroidal Angiogenesis. Retrotransposition, Diversity and the Brain. Reviews and Protocols in DT40 Research. Reviews in Fluorescence Reviews of Environmental Contamination and Toxicol. George W. Ware, Lilia A. Albert, D. Crosby, Pim Voogt, O.
Hutzinger, James B. Knaak, Foster L. Mayer, D. Morgan, Douglas L. Park, Ronald S. Tjeerdema, David M. Whitacre, Raymond S. Yang, Francis A. Lilia A. Albert, Pim Voogt, Charles P. Gerba, O. Yang, George W. Ware, Herbert N. Nigg, Daniel R. Doerge, Francis A. Reviews of Environmental Contamination and Toxicology. Reviews of Physiology Vol Reviews of Physiology Biochemistry and Pharmacolog.