Vol.12(2)

PART I

I would like to talk about the emerging field of biomolecular imaging, which comprises different biophysical techniques applied in observing either the time-average or dynamical picture of the nano-world within the cell. Please do not be confused with the already common but still improving medical devices in tissue scanning, like the ultrasonar, MRI, etc. Proteomics comes in as a post-genomics era, which is then followed closely by the interactomics. Genomics is still important, e.g. in the study of SNP and LOH in cancer patients. However, as gene therapy has never gained the expected success, people have to target the culprit molecular components, i.e. proteins. By understanding which protein interacting with which protein, we are able to elucidate the pathway, and plan our targets. It's customary to design either small-molecule compound or humanized antibody to block these faulty proteins. And this is the current big-money business.

Is it the end of story? Obviously not. As some of the "golden-bullets" produced by pharmaceutical giants had failed the clinical trial due to occurence of death, we are held back and start to consider the importance of looking deeply into the action at the molecular scale. Of course, there are several reasons that lead to the same goal, like answers to certain biological puzzles have been elusive, understanding of the dynamic factor in protein functions, etc. One of these puzzles is the conversion process of PrPC into PrPSc as well as where does it happen within the neuron (for your note, PrPSc is the causative agent of the incurable BSE, commonly known as mad cow disease, as well as other brain-wasting diseases). And this problem has been in my mind for over a year.

About a year ago, I handed in an assignment for the module "Protein Engineering". In that writing, I proposed to mutate four residues in the alpha-helical core of PrPC so as to engineer a non-native CCXXCC motif into this protein. A bi-arsenical fluorophore designed by Roger Tsien's group in UCSD is able to fluoresce by excitation only upon forming covalent bonds with the thiol groups of the four cysteine in helical topology [Griffin, B.A., Adams, S.R., and Tsien, R.Y. (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 281:269-272]. I argue that the PrPSc conversion process may somehow has high enough free energy to disrupt the helical topology (please be reminded there are four covalent bonds there) and results in loss of signal. We may even change the scheme to a FRET system to observe gain of signal instead. By this way, we should be able to trace in real-time by confocal microscopy where the conversion happens. Of course, I am always proud of my idea. But in the end, I got only average grade for that module.

Just a few days ago, I came across this publication [Roberti, M.J., Bertoncini, C.W., Klement, R., Jares-Erijman, E.A., Jovin, T.M. (2007) Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged alpha-synuclein. Nat Methods. 4(4):345-51]. Well, I am happy about it. It shows that I got the right guess what other people have in their minds and who are able to successfully produce the results, though there is a bit difference between our approach. Amyloid formation of alpha-synuclein is an Alzheimer's (AD) pathology, which might be closely related to PrPSc conversion. Besides, I have also listed other related publications:

(i) Martin BR, Giepmans BNG, Adams SR, Tsien RY. (2005) Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nature Biotechnol. 10:1308-1314.
(ii) Sigurdson CJ, Nilsson KP, Hornemann S, Manco G, Polymenidou M, Schwarz P, Leclerc M, Hammarström P, Wüthrich K, Aguzzi A. (2007) Prion strain discrimination using luminescent conjugated polymers. Nat Methods. 4(12):1023-30.
(iii) Liu B, Archer CT, Burdine L, Gillette TG, Kodadek T. (2007) Label transfer chemistry for the characterization of protein-protein interactions. J Am Chem Soc. 129(41):12348-9.