A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor

A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor

Patricia A. Bresnahan*†‡, Wes Yonemoto*†, Sharon Ferrell*,

Debora Williams-Herman§, Romas Geleziunas* and Warner C. Greene*‡

Current Biology 1998, 8: 1235-1238

Summary

Human immunodeficiency virus 1 (HIV-1) Nef downregulates CD4 expression by increasing endocytosis of surface receptors and decreasing CD4 trafficking from the Golgi network to the plasma membrane. Although HIV-2 binds to AP-1 and AP-2 adaptors through terminal tyrosine-based motifs, these motifs are not found in HIV-1, and thus the mechanism by which Nef interacts with cellular signaling is unknown. In this paper, the authors investigate the role of a highly conserved dileucine motif in HIV-1 Nef that acts as an internalization signal and is required in CD4 downregulation. In addition, the authors provide evidence that supports dileucine motif-dependent binding of HIV-1 Nef to the AP-1 adaptor, as well as an evolutionary difference between HIV-1 and HIV-2 structural motifs for binding cellular adaptors.

Results

CD4 expression was characterized for human 293 cells after transfection with either HIV-1 NL4-3 Nef or mutant Nef variants. Flow cytometry was used to measure the percentage of cells that were CD4+, and immunoblotting was used to confirm Nef expression for each of the test groups. The groups transfected with Nef, namely + Nef, + Nef E160A, and + Nef P167A, showed low amounts of CD4 surface markers. On the other hand, the groups transfected with mutant Nef showed expression of CD4 surface markers comparable to the CD4 only group with no Nef transfection. Thus, this data confirms that Nef downregulates CD4 and that dysfunctional Nef will not downregulate CD4.

Expression of CD8 and CD8-Nef chimeras on human 293 cells was characterized using FACS. The top figure shows that the percentage of CD8-positive cells for the mutant Nef LL/AA was comparable to that of CD8 transfection only and much higher than that of wild-type Nef, which indicates that “CD8-Nef LL/AA mutant is preferentially expressed at the cell surface compared to CD8-Nef.” The middle figure shows that internalization of CD8-Nef occurred nearly four times faster than that of CD8-Nef LL/AA mutant, which shows that the dileucine motif does have an effect on CD8 internalization rate. The bottom figure shows that cell lysates had similar amounts of CD8-Nef and CD8-Nef LL/AA.

Shortfalls:

-Although the paper does extensive characterization using immunoprecipitation, FACS, flow cytometry, immunoblotting, and SDS-PAGE, additional characterization through other techniques such as qRT-PCR showing amount of expression for wild-type Nef and mutant Nef LL/AA, would have helped to support their findings on Nef’s HIV-1 effect of CD4 downregulation.

-The experiments had low sample sizes (N=2 or 3 in most cases), and more experiments could have been done in order to validate the mean and standard deviations for the percentage of CD4+ and CD8+ cells.

Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-renewal

Citation: Garcia-Gonzalo FR, Izpisu´ a Belmonte JC (2008) Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-Renewal. PLoS ONE 3(1): e1384. doi:10.1371/journal.pone.0001384

Summary: Human embryonic stem cells (hESCs) have the potential to provide a much needed source of differentiated cells, which can be used in treating various human diseases. Currently there exist multiple problems limiting their use in regenerative medicine. One such problem is expanding the hESCs in vitro in a chemically-defined culture medium. Although many of the signaling factors controlling hESC self renewal and differentiation have been studied, there still does not exist an optimally defined medium that can adequately streamline the expansion of human embryonic stem cells.

Belmonte et. al. tested various chemically defined media (CDM) that robustly sustain long term hESC self renewal. He determined that the media currently used in various papers is unsatisfactory so they decided to enhance a published medium. The medium selected for optimization was N2/B27-CDM because it was inexpensive, easily prepared and had shown promising results in preliminary testing. The selected CDM was supplemented with a variety of additional molecules chosen from literature. A strong positive effect on the expansion of undifferentiated hESCs (HUES7 and HUES9) was observed when the CDM was supplemented with 15% knockout serum replacement (KOSR).

Figure 1 shows that the human embryonic stem cell lines (HUES9 or HUES7) cultured in N2/B27-CDM with 15% KOSR were positive for undifferentiated hESC markers such as alkaline phosphatase (ALP) and TRA-1-60. The same result was shown for the positive control group using mouse embryonic fibroblast-conditioned medium (MEFCM). Whereas the negative control of only using N2/B27-CDM lacked the pluripotency markers ALP and TRA-1-60.

Figure 1: Knockout serum replacement stimulates hESC self renewal. (A) HUES9 cells cultured for five passages in the indicated media were fixed and stained for alkaline phosphatase (ALP) activity (top row) or stained with propidium iodide to visualize DNA (bottom panel). (B) HUES7 cells cultured for five passages in the same media as above were stained with an anti-TRA-1-60 antibody (green) and with DAPI to visualize DNA (blue).

The next step involved looking at KOSR’s patent to ascertain the chemical composition of KOSR to better identify the molecules associated with the strong positive expansion of hESC self-renewal. KOSR consisted of small organic molecules, trace elements and three proteins, namely insulin, transferrin and lipid-rich albumin. Lipids associated with albumin were the only non-chemically defined component of KOSR. Thus the active ingredients of KOSR were passed through filters with molecular weight cutoffs creating two fractions. The higher molecular weight fractions demonstrated the same ability as KOSR in their abilty to sustain hESC self-renewal. It was shown that the active ingredient of KOSR is a molecule greater than 50 kilo Daltons. The molecule that best fit that billing was lipid-rich albumin.

Then lipid rich bovine serum albumin known as AlbuMAX was tested to determine its effect on hESCs. The 15% KOSR corresponds to 1% AlbuMAX, thus this amount was added to N2/B27-CDM. This demonstrated the same effect that N2/B27 + 15% KOSR proving that AlbuMax was the active ingredient in KOSR that provides a positive expansion of undifferentiated human embryonic stem cells (figure not shown) Belmonte et. al. then used flow cytometry to show the expression of TLA-1-60 was dependent on lipid components of AlbuMAX and not the protein components. This was done by using bovine pancreatic trypsin on KOSR (or 10% AlbuMAX). HUES7 cells were cultured on the following media:

1. mouse embryonic fibroblast conditioned medium (MEFCM)

2. N2/B27-CDM

3. N2/B27-CDM + KOSR (C+K)

4. N2/B27-CDM + BSA(lipid-poor)

5. N2/B27-CDM + AlbuMAX (C+A)

6. N2/B27-CDM + TrypsinKOSR (C+TA)

TRA-1-60 expression has been lost in most of the cells cultured with N2/B27-CDM alone and N2/B27-CDM+BSA (lipid-poor), whereas TRA-1-60 expression was shown in the media (1,3,5,6). This was also confirmed by Figure 3D where OCT4 and NANOG (undifferentiated hESC markers) were present in media (1,3,5,6) but not in the the N2/B27-CDM by itself. Figure 3E is from an SDS-PAGE and coomassie blue staining to prove that protein from KOSR and AlbuMAX were properly degraded from the respective media and thus not the dependent source of hESC self-renewal.

Figure 3. Lipid-rich BSA (AlbuMAX) is responsible for the effect of KOSR on hESC self-renewal. (D) HUES9 cells cultured in the indicated media for five passages were used to analyze Oct4 and Nanog mRNA expression by quantitative RT-PCR. (E) Trypsinization of KOSR and AlbuMAX leads to the disappearance of BSA and other protein bands from these fractions, as observed by SDS-PAGE and Coomassie blue staining.

To further ensure the integrity of the human embryonic stem cell lines (HUES7 and HUES9) cultured in AlbuMAX, were tested for pluripotency by seeing if they retained the pluripotency markers (ALP, TRA-1-60, Oct4 and Nanog). This was done by the creation of embryoid bodies and determining if they can differentiate into the three germ layers (ecto-, endo-, mesoderm). Thus hESC were cultured for seven passages in N2/B27-CDM + AlbuMAX and controls. The embryoid bodies after careful preparation were transferred to gelatain-coated plates (0.1% gelatin for 2 hours at 37 deg C) in DMEM +10 % FBS (fetal bovine serum) and allowed to differentiate for 20 additional days, changing medium as necessary. After 10-15 days in adhesive culture, there were visible neurons and cardiomyocytes. At the end, RNA was isolated and quantitative RT-PCR was used to illustrate the expression of differentiated cell markers in each respective germ layer. Therefore illustrating that the pluripotency was maintained after the prolonged use of N2/B27-CDM + AlbuMAX.

Then to determine which major lipid species were present in AlbuMAX (thus responsible for hESC self-renewal), high performance liquid chromatography (HPLC) was used. Based on previous reports and on the HPLC performed the following candidate lipids were selected for testing:

1. lysophosphatidylcholine (LPC)

2. lysophosphatidic acid (LPA)

3. sphingosine-1-phosphate (S1P)

4. prostaglandin E2 (PGE2)

5. chemically defined mix of unsaturated and saturated free fatty acids (FFA).

*Keep in mind that the candidtate lipids were selected to account for lipids that may be present in minute qualities and weren’t present quantifiable during the lipid analysis. “Given that the active molecules in AlbuMAX need not be those that are more abundant and therefore appear more prominently in the lipid analysis, we also decided to test several lipids which we thought were good candidates to mediate the observed effect (Belmonte).” The candidate lipids were added to N2/B27-CDM media. To illustrate the effect of each lipid, they were first stained with alkaline phosphotase (ALP) activity and DNA stained with propidium iodide Figure (5A). Then RNA was extracted from the cells in each respective medium and analyzed using quantitative RT-PCR to test for the expression of NANOG and OCT4 (undifferentiated hESC markers). As evidenced by figure 5B, lysophophatic acid (LPA) and sphingosine-1-phosphate (S1P) show OCT4 and NANOG expression. LPA and S1P show comparable expression of the hESC markers to that of the control group N2/B27- CDM + AlbuMAX.

Figure 5. Testing of candidate lipids. HUES9 cells were grown in N2/B27-CDM alone, with 1% AlbuMAX or with the indicated lipids for six passages. At the end of this period cells were (A) stained for ALP activity and their DNA visualized with propidium iodide or (B) RNA was extracted from cells and the expression of Oct4 and Nanog mRNAs was analyzed by quantitative RT-PCR. Lipids used are: lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P), prostaglandin E2 (PGE2) and a chemically-defined mixture of saturated and unsaturated free fatty acids (FFA) known commercially as chemically defined lipid concentrate (Invitrogen).

Significance: Chemically defined media (CDM) is an important avenue in stem cell technologies. The N2/B27-CDM supplemented by KOSR was instrumental in the expansion of hESC self-renewal. The KOSR components were analyzed and tested to determine which active ingredients caused the strong positive effect on the expansion of undifferentiated hESCs. After a series of experiments it was determined that lipid-rich albumin (AlbuMAX), was the dependent factor in the media. Although HPLC was performed and various lipid candidates were selected to determine the lipids responsible for the hESC self renewal, it was difficult to determine specifically the lipid(s) responsible. Although the lipid analysis was inconclusive, it is of paramount importance to know that lipid-rich albumin is capable of having a strong positive effect on the expansion of hESC.

It is also important to note that additional stem cell technologies will be benefit from this piece of information, specifically those pertaining to biomaterials (3D niches of adhesion molecules and scaffolds) as well as bioreactors (that can provide a microenvironment for the expansion of hESC in robust and reproducible chemically defined media).

Critique: For the most part, I was really impressed with the step by step determination of the active ingredient in knockout serum replacement (KOSR). The protocols were properly thought out and anytime a possible question was raised, it was answered by a positive or negative control. With that said, I would like to bring attention to the correlation between figure 5A and 5B. Specifically the results in the N2/B27-DCM + LPA experiment. It demonstrates expression of hESC markers (NANOG and OCT4) yet lacked alkaline phoshatase activity which is another known hESC marker. How is this possible?

Also, I’m aware that different combinations of signaling factors can strongly influence the cell fate of hESC, but what is puzzling is why they would isolate specific ligands and attempt to discern which lipid is most responsible for expansion of hESC. I’m sure they understand that OCT4, NANOG, and SOX2 are connected and are responsible for the cell-fate determination of hESCs. In their defense they did mention that they might try SP1 and LPA concurrently to determine its effect on expansion of hESC. I’m sure that currently there is research aimed at testing various combination and concentration of promising signaling factors.

It is this research coupled with the knowledge of cell adhesion to biomaterials, cell viability in chemically defined media, as well as cell adaptations to environmental cues that will continue the promise of hESC in regenerative medicine.