Apolipoprotein AI Pro-peptide Cellular Localization Cliff Bolinger and Leslie Frost Department of Chemistry, Marshall University, Huntington, WV 25755. Introduction
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Cliff Bolinger and Leslie Frost
Department of Chemistry, Marshall University, Huntington, WV 25755
Increased levels of HDL cholesterol, commonly referred to as “good cholesterol”, has been shown clinically (and in non-human models) to decrease levels of LDL cholesterol, or “bad cholesterol.” The main constituent of HDL, Apolipoprotein AI (Apo AI), makes up 65% of the HDL complex. It has been documented that high levels of Apo AI and HDL in serum have an inhibitory effect on the development of atherosclerosis. Apo AI is the only lipoprotein that has a pro-peptide containing six amino acids on its N-terminus which is cleaved by an unknown enzyme in the serum resulting in mature Apo AI (without the pro-peptide) and pro Apo AI (with the peptide still attached) circulating in the serum. The pro-peptide appears to have no bearing on Apo AI functionality, but we have speculated that it does in fact play a role in cholesterol maintenance. The goal of this project was to identify the cellular localization of the peptide using a confocal microscope to gain a better understanding of its possible functions.
Images and Data
Fig. 1 5uL of dilute peptide was added to 1000uL of media on the epithelial cells. Localization occurred within minutes. The nucleus of the imaged cell was magnified and then imaged along the z-axis to provide further information on the localization within the nucleus. It is apparent that there is an inhomogeneous distribution within the nucleus.
Materials and Methods
Our peptide was custom synthesized and tagged with FITC at the N-terminus by Cambridge Research Biochemicals. The peptide along with the fluorescein control both have an excitation wavelength of 494 nm and an emission wavelength of 525 nm. The LDS-751 control has an excitation wavelength of 558 nm and a peak emission wavelength of 710 nm. Both the controls and the peptide were diluted to the desired concentrations (.1uM for time course studies and .125uM for others) and stored in the freezer until needed. We obtained cells by simply swabbing cheek epithelial cells after cleansing and then layering them onto a cover slip. The cells were then covered with in media at 37C. Our experiments were conducted on a BioRad mrc 1024 confocal microscope. The first was a live cell imaging technique on the confocal microscope where the desired fluorophore was introduced to the cells in media and observed over time to determine cellular localization. This was done in a small imaging chamber to allow continuous exposure to media and fluorescent molecules. The next experiment consisted of cells soaked in media overnight along with the desired fluorophores at .125uM concentrations. These cells were also used for live cell imaging to determine uptake and localization of multiple fluorophores within the same cell. The final experiment was done by “freezing” the cells’ uptake of a specific fluorophore with methanol in different time increments to determine differences between the uptake of the peptide versus fluorescein alone. These cells were attached to a slide with Gel Mount™ and then stored in the fridge until time of imaging.
Fig. 3 These images are the same cells viewed on the confocal microscope with different optical filters. The left (green) image of our peptide was taken with a 522 DF32 filter and the right (red) image of LDS was with a HQ598/40 filter. The images show co-localization within the nucleus and suggests that our peptide localizes with more efficiency because the concentrations of the peptide and LDS were roughly equivalent.
Fig. 3 The graph shows the intracellular accumulation as measured by fluorescent intensity of epithelial cells over the time course. The cells were “frozen” (fixed) on cover slips at each time point with methanol and mounted onto a slide.
Results and Discussion
From the three different experiments conducted, it is evident that thefluoresceinlabeled peptide both enters into cells at a faster rate than the fluorescein alone and localizes within the cell’s nucleus once it is within the cell membrane. Fig. 1 shows the labeled peptide’s compartmentalization within only the nucleus. Once the peptide is within the nuclear membrane, it appears to have further specific distribution. This suggests that the peptide is interacting with the cell’s DNA, perhaps initiating a LDL concentration lowering pathway in the body. Fig. 2 confirms the nuclear compartmentalization of the peptide through its co-localization with LDS-751, a known DNA fluorescent stain. Fig. 3 shows that with the peptide bound to FITC, the cellular uptake occurs on an semi-linear scale whereas the fluorescein alone seems to spike immediately in intensity and then remains steady or drops off to very little present in the cell. We are not sure for the reason of the fluorescein’s odd pattern of uptake, but the notable trend is the steady and possibly regulated uptake of the peptide. Together these experiments show that the peptide is indeed being localized within the nucleus of cells, giving reason for further study into its biological function.
We would like to try different arrangements of the same six amino acids fluorescently tagged to ensure that the specific function of the peptide is unique to its sequence. We would also like to experiment with DNA microarrays to determine which genes are upregulated by the peptide. Within the near future our lab would like to initiate some rat trials to have a better understanding of the peptide’s impact on cholesterol levels in living organisms.
Dr. Michael Norton, Chris Barry, and especially David Neff…Confocal Genius