送交者: Godfather 于 2006-1-24, 14:04:12:
OK, here we go again.
1. Given the fact that I cannot afford to waste more time (mid-terms are coming!), this is my final post on this incident despite what responses I will get and what the outcome of the incident will be. I have too much stuff to deal with… Brokenback Mountain is playing; Blue Devils lost their first game over the weekend; My i-Pod was broken; Martina Hingis comes back; Samuel Alito is facing vote; The housing market is cooling off; I have to wait for the school bus every morning since my Dad does not want to buy a $3000 used car for me… The worst thing is that my parents are always fighting on whether or not to go back to the motherland. After recent promotion from an Associate to Senior Associate at the community college, my Dad is quite confident that he could find a position at a middle school in the motherland where he believes that he should be able to get the benefits as a deputy head of a province pretty soon by doing real science. Wow, it sounds fantastic! After so many years at several community colleges from West to Midwest to East, I think he has got enough training and I also believe he is a great scientist because he can drink (often got drunk and blamed by Mom though). Another exciting thing is that my Dad has promised me that he will bring me to the private rooms at KTV and massage places. A small secret (don’t tell anybody else): so may beautiful young ladies over there to serve you. Fabulous! Unfortunately, my Mom does not want to go back. She has learned that we can get green cards someday if we join the organization. But, I think it’s just a woman’s opinion. After all, I think it would be wonderful if my Dad can do great science while enjoying Karaoke and massages everyday in the motherland. Believe or not, I will drive a BMW with the money he will be sending to me. I always believe he can make great contributions to the motherland!
2. I sincerely apologize to the readers for my mistake that I used AgrD3 to interpret AgrD1. Hoping such simple mistake would not be made during my mid-terms.
3. Thanks to my teachers for my AP bio and AP chem.
courses. Actually, I am hoping they would give me better grades.
4. Just forget all the craps such as vector, expression, streptomycin, purification procedure, LC/MS data, hydrophobicity, crystal structure, interaction, cell wall, so on… All of these are just speculative! Also, let’s forget about the immunogenecity of Colicins. Moreover, most of them are just dealt with technical issues, which is only about consistency and reproducibility. What we need is hard science to prove it! Please ignore all my previous posts on this incident. You all will forgive me for my previous misjudgments, right?
5. Here is the scientific proof for Dr. Qiu’s Fabrication on his Nature/Biotech. paper and related work without a need of experimental confirmation. If somebody wants to verify the information I provide here, please go check the references at the expense of your own time.
A. Background
a. Colicins and Colicin Ia: After so much discussions, most people have already been familiar with this class of bacterial toxins for their classification, structures and functions. Probably no need for me to repeat again. What I’d like to emphasize is that the bactericidal activity of Colicins including Ia is caused by the hydrophobic interaction between C domain and the phospholipids on bacterial membrane to form ion channels, thereby increasing intracellular ion concentrations. But, they are only produced in certain mutants of E. coli, so that they arenot active to E. coli unrelated bacteria such as gram-positive bugs like SA. Please see Qiu’s introduction and cited references.
b. Peromone is a class of peptide hormone first discovered in plants in 1950s. Later, it was found to exist in gram-positive Streptococcus faecalis in 1970s. The first bacterial pheromone was identified as an octapeptide in 1984. By the growth of interest and extensive studies, their involvement in the bacterial communications was suggested by two-component hypothesis and quorum sensing system in genera of Bacillus, Streptococcus, Enterococcus and Staphylococcus. They function as regulators and signaling molecules for sporulation and biofilm formation. Please see Qiu’s citations.
c. In the P2 region, Agr locus consists of the genes of agrB, agrD, agrC and agrA to produce respective proteins. AgrA may act on the promoter to regulate the expression. AgrB is a transmembrane protein involved in the processing of AgrD propeptide. AgrC is a membrane protein that serves as a receptor for AgrD and also is a histidine kinase to involve in the signal transduction. AgrD constitutes the pheromone precursor protein (propeptide). By the way, AgrD pheromone is also called autoinducing peptide (AIP) since it can trigger the autoregulation process in the quorum sensing system.
d. There are 4 groups of genes in the Agr locus from 4 different SA variants, and they share high homolog each other. As for the AgrD, of course, there are 4 corresponding sequences. In Qiu’s paper, he cited reference 10 as the source of the sequences. In fact, it only described the discovery of the forth group and they compared the newly discovered gene with previously reported other 3 groups. For AIP sequence, group I: YSTCDFIM (AgrD1); group II: GVNACSSLF (AgrD2), group III: YINCDFLL (AgrD3) and group IV: YSTCYFIM (did not mention by Qiu). The common and unique structure for the 4 pheromones is the existence of a cysteine residue in the middle. Replacement of cysteine with alanine by mutagenesis demonstrated the loss of activity, indicating the presence of cysteine is essential from sequence point view.
B. Qiu’s Idea
Colicin Ia can form ion channel to kill E. coli and related bacteria, but no effect on gram-positive bacteria that are more important for clinical infection. AIPs are secreted by gram-positive bacteria and involved in the quorum sensing system in which AgrD can bind to its receptor AgrC on cell membrane. Then, if one combines the protein and peptide together, the peptide can specifically bind to the receptor of Staphylococci while the attached Colicin Ia can cause cell death by channel-forming effect. That’s why it’s a targeted drug. Logical enough, right? Sounds good. Wonderful, let’s do it and make a breakthrough!
C. Proof of Qiu’s Fabrication
Certainly, the pheromones derived from AgrDs can bind to their respective receptors on a particular SA strain. However, Dr. Qiu could not understand why he needed (or was asked by reviewers) to confirm the existence of thiolactone. The reason is very simple! All pheromones found in SA so far have the lactone structure, which is absolutely required for its binding and subsequent signaling process. The thiolactone of AgrD1 is a linkage between the thio group of cysteine and the carboxyl group of the C-terminal residue, and AgrB has been suggested to catalyze the formation of such thiolactone. As a result, there are 4 AIPs from respective 4 groups of SA with thiolactone structure (AIP-I, AIP-II, AIP-III, AIP-IV, see review by Lyon G. J. & Novick, R. P. 2004, Peptides, 25, 1389-1403). Dr. Guangyong Ji at the Uniformed Services University of the Heath Sciences is an expert in this area. People can check his quality publications for references. Such cyclization under physiological conditions would not happen spontaneously as well.
Now, the question is what about in E. coli. One would wonder if there is an enzyme to catalyze the cyclization in E. coli. Unfortunately, E. coli, especially the strain Dr. Qiu used for expression, TG1, does not have posttranslational modifications according to common knowledge. In addition, there have been NO such reports so far. I did find a group of enzyme called methionyl-tRNA synthetase in E. coli, however, the reaction catalyzed by this enzyme is cyclization of homocysteine (linkage of thio group to its own carboxyl group), which is a mechanism to prevent non-natural amino acid to participate protein synthesis in nature.
Thus, having all these said, what do we have now?
a. An engineered protein fused with AgrD1 (AIP-I) and Colicin Ia
b. The C-terminal AgrD1 can cyclize the cysteine with final methionine after expression in E. coli.
c. This engineered protein can specifically target SA to exhibit its specific bactericidal activity.
d. The bactericidal specificity was due to AgrD1 and its thiolactone structure.
Please make your own judgment!!
Well, that’s the end of my presentation, and it’s time for me to buy/sell some stocks for the AP Eco class.