Inhibition of Bacterial Mutagenesis through Polyubiquitination


  Abstract- Bacterial cells can have DNA damage due to transcriptional error, or through the effect of an antibiotic. The SOS response is a bacterial cell program for coping with DNA damage, in which the is arrested, and DNA repair is induced. The repairs have high probability in leading to mutagenesis in the bacteria, which can lead to antibiotic resistance. The RecA protein in bacteria is responsible for the activation of the SOS response; therefore, making it a target for inhibition. I elected to use the ubiquitination system, natively used for apoptosis, as a means of targeted degradation of the RecA protein in bacteria prone to mutations. Polyubiquitination of misfolded proteins leads to the breaking down of the protein with the aid of proteasomes, which break down unnecessary proteins through a chemical reaction known as proteolysis. Using random forest-predictors, I determined a statistically high likelihood of ubiquitination of the RecA protein in MRSA, Tuberculosis, and other high risk bacterial infections. I hypothesized that I could foster ubiquitin-tagging on RecA by forcing the protein to misfold. Chaperones are proteins which interact with each other to prevent specific sets of proteins from misfolding. CHIP (C terminus of HSC70-Interacting Protein) is a biomolecule that inhibits interactions between the chaperones of RecA. Adding CHIP, ubiquitin, and 26s proteasomes into the bacterial system, should theoretically lead to the degradation of the RecA protein inside the bacteria. I tested my hypothesis by conducting an assay for monitoring CHIP-mediated ubiquitination, and conducted analysis on the assay using SDS-Page gel electrophoresis, and Western-blotting. The resulting data showed signs of polyubiquitination on the RecA protein, with chains of five or more ubiquitin, showing high drug potential. Adding an antibody drug conjugate, containing all the necessary components of a CHIP-mediated ubiquitination reaction, to common antibiotics can lead to the inhibition of bacterial mutagenesis, and higher antibiotic drug potency.


I.    INTRODUCTION


During the cell cycle, if a cell's DNA ever undergoes any damage, it activates the SOS response, a cell program designed to repair DNA. Unfortunately, the SOS response is extremely error prone, and most of the time ends up leading to mutagenesis. In fact the SOS response is one of the major causes of bacterial mutagenesis. Due to this, bacterial cells can sometimes mutate in a manner that gives them resistance to many antibiotics. There are two main proteins involved in the SOS response, LexA, and RecA. LexA makes sure that SOS response remains off when the cell is healthy, and RecA makes sure that SOS response is activated when the cell's DNA is damaged. Degradation of the RecA protein can prevent the activation of the SOS response, greatly reducing the risk of mutation. One such way that proteins such as RecA can be antagonized, is through the human ubiquitination system. The ubiquitination system consists of the proteins ubiquitin and proteasomes. Ubiquitin tags any protein which is misfolded, signaling that something is wrong with the protein. If any protein is polyubiquitinated, then proteasomes will come in and destroy that protein though a process called proteolysis. In order to destroy the RecA protein in bacteria using the ubiquitination system, RecA must be forced to misfold. Proteins have specialized molecules with them, called chaperones, which prevent them from misfolding. Without the presence of chaperones, proteins will not be able to maintain their structure, and misfold. CHIP (C terminus of HSC70-Interacting Protein) is a special biomolecule, that inhibits the interactions between the chaperones specific to the RecA protein. So the presence of CHIP in the bacterial system, would cause the misfolding of RecA, leading to the forced ubiquitination of the protein.


Image I

Diagram explaining SOS response mechanics [1] 


II.   Background

A.    Using

In order to see whether ubiquitination was the proper means to be implemented for the degradation of RecA, I used UbPred’s Random Forrest Predictor to predict the potential of ubiquitination of the RecA proteins of various bacteria. I did this on the RecA of bacteria which are known for having high risk of mutation, such as MRSA, Tuberculosis, and others. I also repeated the process on the RecA of E. Coli, which through a BLAST protocol, I decided was similar enough to the RecA of MRSA and Tuberculosis that I would be using it as my substrate in my experimentation. The RecA of all of the bacteria showed statistically high probability of ubiquitination on four or more lysine sites. Based on the largely positive results on all of the RecA Proteins, I chose to move forward with using ubiquitination as my main tool for targeted degradation.


The ubiquitination system is based on the fundamental of antagonizing cells and proteins which are no longer necessary, or are causing more damage than they are worth. So in order to force ubiquitin tagging on the RecA protein of the bacteria, I had to inflict some sort of problem on the RecA protein, that would make it appealing for the ubiquitin to bind to. The most common reason ubiquitin will bind to a protein is because the protein is misfolded, affecting it’s ability to efficiently complete it’s task. Based on this information, I made it my goal to implement the targeted misfolding of RecA, to promote its ubiquitination. Proteins have specialized molecules with them, called chaperones, which prevent them from misfolding. Without the presence of chaperones, proteins will not be able to maintain their structure, and misfold. To force the misfolding of RecA, I first targeted the chaperones of RecA. RecA has a special class of chaperones, called heat-shock proteins. Specifically, it has molecules of HSP 70, or Heat-Shock Protein 70. There are many Ubiquitin E3-Ligases that inhibit interactions between specific classes of chaperones, and promote the ubiquitination of the protein being supervised by the respective chaperones. By searching the catalog of UBPbio, a company which specializes in producing ubiquitin-proteasome biotechnologies, I was able to find one ligase, CHIP or (C terminus of HSC70-Interacting Protein), that had the ability to inhibit interactions between HSP 70 chaperones. I proceeded to use CHIP as the method for inducing RecA ubiquitination.


III.   Materials and Methods




IV.   Results

A.    Results Breakdown

I conducted several CHIP-mediated ubiquitination reactions, and tested their success through gel electrophoresis, and Western Blotting. When running gel electrophoresis, I loaded a sample of unreacted RecA, as a control, and a sample of RecA that had gone through the entire ubiquitination reaction via assay. The gel showed that most of the sample in the lane containing unreacted protein had landed around 50 kDa, while most of the sample in the lane containing the reacted RecA had landed around 90 kDa.This showed that the reacted protein had been successfully tagged by several ubiquitin, increasing it’s size to almost 90 kDa. However, the reacted lane was also showing several other places where very small amounts of the sample had landed. Since it was a gel, it could be showing other components of the ubiquitination reaction, so to verify that the smaller components were not actually RecA, I conducted a Western Blot. The anti-RecA antibody I used in my Western Blot confirmed that all the RecA in the sample was deposited either at the 90 kDa site or the 50 kDa site, and that the other lines showing up in the gel were not RecA. To further prove that the line occurring at the 90 kDa site was a polyubiquitinated version of RecA and not a dimer, and that the line occurring around 50 kDa in both lanes was pure RecA, I conducted another Western Blot using an anti-Ubiquitin antibody. The results from this Western Blot, showed that the sample which went through the entire ubiquitination reaction was in fact ubiquitinated, and there was no presence of ubiquitin anywhere on the membrane, except at the 90 kDa site in the reacted sample lane.


B.    

A.    Figures and Tables

   Position figures and tables at the tops and bottoms of columns.  Avoid placing them in the middle of columns. Large figures and tables may span across both columns. Figure captions should be centered below the figures; table captions should be centered above. Avoid placing figures and tables before their first mention in the text. Use the abbreviation “Fig. 1,” even at the beginning of a sentence.

  Figure axis labels are often a source of confusion. Use words rather than symbols. For example, write “Magnetization,” or “Magnetization (M)” not just “M.”  Put units in parentheses. Do not label axes only with units. In the example, write “Magnetization (A/m)” or “Magnetization (A·m1).” Do not label axes with a ratio of quantities and units. For example, write “Temperature (K),” not “Temperature/K.”

  Multipliers can be especially confusing. Write “Magnetization (kA/m)” or “Magnetization (103 A/m).” Figures labels should legible, about 10-point type.


B.    References

   Number citations consecutively in square brackets [1]. Punctuation follows the bracket [2]. Refer simply to the 


III.   UNITS


  Use either SI (MKS) or CGS as primary units. (SI units are encouraged.) English units may be used as secondary units (in parentheses).  An exception would be the use of English units as identifiers in trade, such as “3.5-inch disk drive.”

  Avoid combining SI and CGS units, such as current in amperes and magnetic field in oersteds. This often leads to confusion because equations do not balance dimensionally. If you must use mixed units, clearly state the units for each quantity that you use in an equation.


IV.   SOME COMMON MISTAKES


  The word “data” is plural, not singular.  The subscript for the permeability of vacuum0 is zero, not a lower case “o.”  In American English, periods and commas are within the quotation marks, like “this period.”  A parenthetical statement at the end of a sentence is punctuated outfside of the closing parenthesis (like this).  (A parenthetical sentence is punctuated within the parentheses.)  A graph with a graph is an “inset,” not an “insert.”  The word alternatively is preferred to the word “alternately”  (unless you mean something that alternates).  Do not use the word “essentially” to mean “approximately” or “effectively.”  Be aware of the different meanings of the homophones “affect” and “effect,” “complement” and “compliment,” “discreet” and “discrete,” “principal” and “principle.”  Do not confuse “imply” and “infer.” The prefix “non” is not a word; it should be joined to the word it modifies, usually without a hyphen.  There is no period after the “et” in the Latin abbreviation “et al.”  The abbreviation “i.e.” means “that is,” and the abbreviation “e.g.” means “for example.”  An excellent style manual for science writers is [7].  


ACKNOWLEDGMENT


  The preferred spelling of the word “acknowledgment” in America is without an “e” after the “g.”  Try to avoid the stilted expression, “One of us (R. B. G.) thanks …”  Instead, try “R.B.G. thanks …”  Put sponsor acknowledgments in the unnumbered footnotes on the first page.


REFERENCES


[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1174825/

[2]http://www.ubpbio.com/index.php/product/e3-ubiquitin-ligases/misfolded-protein-ubiquitination-kit.html

[3]http://www.abcam.com/reca-antibody-ab63797.html?productWallTab=Questions

[4]http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2004/ popular.html

[5]  R. Nicole, “Title of paper with only first word capitalized,” J. Name Stand. Abbrev., in press.

[6]  Y. Yorozu, M. Hirano, K. Oka, and Y. Tagawa, “Electron spectroscopy studies on magneto-optical media and plastic substrate interface.” IEEE Transl. J. Magn. Japan, vol. 2, pp.740-741, August 1987 [Digests 9th Annual Conf. Magnetics Japan, p.301, 1982].

[7]  M. Young, The Technical Writer’s Handbook.  Mill Valley, CA: University Science, 1989.