Mechanisms of adipocytic differentiation by Factor of adipocyte differentiation 158

Abstract:

The pandemic of obesity, which affects one third of all Americans, predisposes individuals to high blood pressure, gallbladder disease, colorectal diseases, diabetes, cancer, and possibly Alzheimer’s disease. The recently discovered fad158 (factor of adipocyte differentiation158), is a novel gene, which positively affects early adipocyte differentiation. However, knowledge about the mechanisms by which fad158 modulates adipogenesis remains elusive. The objective of the proposed investigation is to determine either the extracellular or intracellular location of the leucine-rich repeats of fad158 (Specific aim 1) and to identify the proteins fad158 interacts with (Specific Aim 2). While a couple of studies have examined the expression patterns of wild type fad158, hardly any study has probed possible mechanisms by which fad 158 affects adipogenesis. Our hypothesis is that fad 158 has extracellular leucine-rich repeats that bind secreted factors and proteins in the membrane or the cytosol, thus modulating early adipogenesis. This hypothesis will be scrutinized by determining the location and orientation of the extracellular repeats of fad158 by immunogold labeling/western blot and by binding studies to discover binding partners of fad158. Culmination of our proposed studies will likely yield information about the orientation of C-terminal fad158 and identify potential binding interactions that orchestrate the early processes in adipogenesis. The long-term goals of this project are to provide therapeutic options for peptide inhibition of adipocyte differentiation, on the one hand, and for induction of adipogenesis in clinical scenarios such as mastectomy or reconstructive surgery, on the other.

Specific Aims

This proposal aims to unravel the mechanisms by which the novel gene fad158 (factor of adipocyte differentiation) promotes the pre-adipocyte differentiation that can lead to obesity, a global and national health problem. Leucine-rich repeats, found in many proteins, participate in protein-protein interactions and impinge on many physiological processes such as proliferation, migration, angiogenesis, and differentiation (Bell et al. 2003). Fad158 is induced during the first 6 hours of adipocyte differentiation in growth-arrested 3T3L1 cells. Its critical role in adipogenesis can be gauged by the fact that its over-expression induces oil droplet formation in a fibroblastic cell line while its knockdown inhibits adipogenesis. Increased cell size or cell number of adipocytes can result in obesity (Dang et al. 2009). Knowledge about the early stages of adipogenesis is scarce. Also scant is information about role of the leucine-rich repeats of fad158. Even though fad158 has been shown to be an early inducer of adipogenesis in mammalian cells, the molecular mechanisms of action of fad158 remain to be characterized.

Our long- term goal is to provide therapeutic options for peptide inhibition of adipocyte differentiation and for induction of adipogenesis in clinical scenarios such as mastectomy or reconstructive surgery. The objective of the proposed investigation is to determine either the extracellular or intracellular location of the leucine-rich repeats of fad158 (Specific aim 1) and to identify the proteins fad158 interacts with (Specific Aim 2). The central hypothesis is that fad 158 has extracellular leucine-rich repeats that bind secreted factors and proteins in the membrane or the cytosol (Specific aim 2), thus modulating early adipogenesis. This hypothesis is based on data (Tominaga et al.2004, Kubota et al. 2004), which indicate that fad158 is a membrane protein with leucine-rich repeats that can likely be cleaved by proteases to generate products that might interact with cellular proteins. Additionally, studies by Berditchevski et al (1996) support this hypothesis. Our rationale is that, confirmation of the cellular location of fad158 and identification of its binding partners will lead to new target proteins for the prevention of obesity and related complications.

We will test our hypothesis through the pursuit of the following specific aims:

1. (a)Determine the cellular location of fad158: Murine tissue will be used here. The working hypothesis is that this approach can determine the extracellular orientation of the leucine-rich repeats of fad158- the determination of which will direct specific aim 2. Immunolabeling the C termini of fad158 with Colloidal Gold-Antibody for Electron Microscopy:

(b)Western Blot to determine the presence of fad 158 in cellular fractions of preadipocytes. Membrane, cytosolic, and nuclear fractions of 3T3L1 or human preadipocytes will be separated by centrifugation and run on SDS-PAGE before being probed with antibodies raised against the C-terminal domain of fad158.

1. Affinity chromatography to Identify binding partners of fad158.

We will use GST-fusion protein of fad158 expressed in E coli in binding experiments followed by MS/MS to determine the sequence of proteins interacting with C-terminal fad158. Once we determine the orientation of the extracellular leucine-rich repeats of fad158, we will use whole cell lysates or any of the appropriate cellular fractions of differentiating and non-differentiating 3T3L1 adipocytes in binding studies with GST-tagged LRRs of fad158.

In regards to expected outcomes, the work purported in the aims above is likely to pinpoint the orientation of the LRRS of fad158 (Specific aim 1) - information that can then be used to assist in the identification of its binding partners (Specific aim 2), which will be the proteins that play crucial roles in the early stages of adipogenesis. Especially, a better understanding of the actions of fad158 can help clinical applications requiring induction of adipogenesis in reconstructive backbone surgery and mastectomy. The results we expect to obtain will have a significant positive impact, because the identified proteins will yield new targets for preventive interventions besides enhancing our understanding of basic biological process of adipocyte differentiation.

Significance and Innovation

Fad158, which positively affects adipocyte differentiation, has transmembrane regions and 8 leucine-rich repeats (LRRs) (Kubota et al. 2004). Leucine-rich repeats have been known to indulge in protein-protein interactions and to modulate physiological processes such as proliferation, cell migration, angiogenesis, hormone receptor interactions, cell adhesion, and differentiation (Tominaga et al. 2004, Kubota et al. 2004). Leucine-rich repeats have two important functions. First, LRRs are involved in beta sheet formation in proteins; second, in many proteins, LRRs bind other proteins, including membrane proteins (Funderburgh et al. 1993). In other words, LRRs help ensure proper folding of proteins, thus facilitating interactions with other proteins. Experiments involving transfection of EGFP-fad158 chimeric plasmids have shown that GFP-labeled LRR fragments of fad158 distribute throughout the cell, indicating the possibility of proteolytic cleavage (Tominaga et al. 2004). Moreover, fad 158 is indispensable for adipocyte differentiation since over-expression of fad 158 induces oil droplet formation in the murine fibroblastic cell line, NIH-3T3, and antisense knockdown of fad158 inhibits adipogenesis in murine 3T3-L1 preadipocytes (Tominaga et al. 2004). However, the molecular mechanisms of action of fad158 are not known. This proposed research will provide in-depth knowledge about the orientation of its leucine-rich repeats- and yield information about its binding partners. The significance of our contribution resides in the fact that our approach can provide the information needed to develop strategies aimed at pharmacological inhibition of proteins involved during adipogenesis. Antibody-based inhibition or chemical inhibition of proteins is a goal that has been achieved by the pharmaceutical industry in the treatment of diseases. On the other hand, there are some clinical scenarios where induction of adipogenesis is a desirable goal. For instance, patients who have undergone mastectomy can benefit from the growth of fatty tissue. Similarly, it is helpful to have subcutaneous fat production at the sites of reconstructive surgery. It is evident that the success of our proposal will have a critical impact on the development of preventive strategies against obesity and type II diabetes- a closely related problem. We also expect that our research can advance biological research by paving the way for the development of siRNA against fad158 and for the development of another murine model of adipogenesis. Moreover, the research proposed herein will significantly enrich our understanding of the basic biological process of adipocyte differentiation.

While it is well known that increased body fat mass in obesity-a global and national health problem- can result either from hypertrophy (cell size increase) or hyperplasia (cell number increase) of adipocytes, there remains a paucity of preventive strategies against obesity. Our research strategy is highly innovative because it relies on Immunocytochemistry/biochemistry, and on the proven capabilities of proteomics. This proposal will utilize a novel approach to first identify the location of the leucine-rich repeats of fad158 (Specific aim 1), intracellular or extracellular, and then to unearth the binding partners of fad158 (Specific aim 2) before embarking on additional efforts to examine the role of the binding partners in signaling pathways and networks (Specific aim 3). The results obtained from our proposed experiments will lead to the development of antibodies against fad158, will help identify novel proteins involved during adipogenesis, and will highlight new signaling moieties. Antibody targeting and peptide inhibition are currently being pursued as therapeutic strategies in the treatment and prevention of inflammatory and autoimmune diseases.

Approach Section:

General Methods:

Murine 3T3-L1 preadipocytes, available from ATCC, will be kept in DMEM with calf serum. The differentiation media consisting of 10% FBS, 10ug/ml insulin. 0.5mM 3-isobutyl -1-methylxanthine, and 1 uM deexamethasone will be added to post-confluent cells (2 days). Human preadipocytes will be separated from fat pads to be obtained from Plastic Surgery Units at Lawrence Memorial Hospital or KUMC. Murine NIH-3T3 cells will also be maintained in DMEM supplemented with calf serum. Murine fat pads will be obtained from Animal Care Unit at the University of Kansas, Lawrence.

Specific Aim 1:

(a)Determine the cellular location of fad158: Immunolabeling the C termini of fad158 with Colloidal Gold-Antibody for Electron Microscopy:

Introduction. This experiment will determine the localization of fad158 using gold immunolabeling and electron microscopy. The objective for this aim is to find the orientation of the leucine-rich repeats of fad158 and thus narrow down the search for the binding partners of fad158. The working hypothesis here is that fad 158 uses its leucine- rich repeats to bind secreted factors and proteins in the membrane or the cytosol (Specific aim 2), thus modulating early adipogenesis. We will test our hypothesis by using immunolabeling of the C-terminal leucine-rich repeats of fad158 to localize the C-terminus. The rationale behind our approach here is that knowledge about the orientation of fad158 will help in finding its binding partners and help further our understanding of any proteolytic cleavage that fad158 might undergo in vivo by proteases such as matrix metalloprotease. Completion of Specific aim 1 will narrow down the signaling networks that could be targeted for drug therapy against obesity.

Justification and feasibility. The orientation of fad158 is not known. A compelling reason behind our approach is that knowledge about the cellular localization or orientation of fad158 will further research efforts aimed at preventing the increased rates of adipocyte differentiation that predispose to obesity.

Research Design. A polyclonal antibody against murine fad158 will be raised in rabbits. ProteinA-colloidal gold particles will be purchased. Protein A-gold-antibody complex will be obtained by incubating the protein-gold suspension in antisera (Hisano et al. 1984). Tissues from murine fat pads will be collected and processed by conventional freezing. Blocks will be fixed in 2-4 % paraformaldehyde, subjected to serial dehydration in acetone, and embedded in Epon. Ultrathin sections will be cut on a microtome, mounted on grids, incubated in ovalbumin to block non-specific binding, immunostained with gold-anti-fad158, and examined under a Transmission electron microscope. Controls will include tissue sections not incubated with the antibody. Tissues treated with protease inhibitors will serve as negative controls.

Observation of gold staining in any cellular compartment or in the extracellular region will indicate the orientation and localization of fad158-LRRs. No staining will be expected in tissue sections not treated with antibody. In cells treated with protease inhibitors, we expect not to see staining for fad158-LRRs in cellular compartments since a protein with transmembrane domains has to be cleaved before its fragments can possibly enter the cytoplasm.

Expected Outcomes. As can be clearly seen from the description above, our goal in this section is very simple: we are only interested in finding the cellular localization and orientation of fad158. Immunolabeling fad158 in murine tissues followed by electron microscopy can certainly identify the cellular location of fad158. While preservation of membranes can be a challenge in fixing and cryosectioning procedures, organelles such as mitochondria, polysomes, ER, Golgi, and secretory granules can be easily distinguished in the cytoplasm. Another problem that may arise would be the detection of staining of the ER region in cells not treated with protease inhibitors. Were the ER to be stained positively, Thus, lack of staining in the cytoplasm will indirectly confirm an extracellular orientation for fad158 and the vice versa.

Potential Problems and Alternative Strategies. Preservation of membrane structure will be a major challenge. Since Tominaga et al. (2004) have already found this protein in mammalian cells using GFP-transfection, we are highly optimistic that our method of immunolabeling with gold particles will lay out the orientation of the C-terminal domain of fad158. Even if membrane ultrastructure is disrupted, we still expect to see gold staining for fad158-LRRs by electron microscopy. While it is quietly likely that there may be a portion of the C-terminus traversing the membrane, that very fact would still not preclude an intracellular or extracellular orientation of fad158. If conventional staining were to result in loss of antigenicity or loss of ultrastructure, a freeze-substitution method will be employed to prepare the tissue for electron microscopy.

Timeline. Immunostaining and electron microscopy experiments, depending on the time needed to raise antibodies against fad158, should be completed in less than six months.

(b)Western Blot to determine the expression of fad 158 in cellular fractions of preadipocytes.

Introduction. In this experiment, the location of the C-terminal leucine-rich repeats of fad158 in mammalian tissues will be determined. Our objective here is to determine the cellular location of the LRRs of fad158 and thus expedite our search for proteins that might bind fad158 during adipogenesis. In order to meet our objective, we will test our hypothesis that fad158 uses its C-terminal leucine-rich repeats to bind proteins and positively regulate adipogenesis. In these experiments, western blot will be used to separate the various membrane components of 3T3L1 cells before subjecting these fractions to SDS-PAGE followed by immunoblotting with antibodies against C-terminal fad158. The rationale behind our approach here is that knowledge about the location of fad158-LRR will help in finding its binding partners and advance our understanding of any proteolytic cleavage that fad158 might undergo in vivo by proteases such as matrix metalloprotease. Realization of Specific aim 1 will pinpoint the signaling networks that could be candidates for drug therapy against obesity.

Justification and feasibility. While literature abounds with studies on protein and gene expression of adipocytes, hardly any information exists about the cellular location or the binding partners for fad158- a positive regulator of the early stages of adipogenesis (Kubota et al. 2004, Renes et al. 2005, and Tominaga et al. 2004).

Research Design. Cytosolic, nuclear, and membrane fractions from human preadipocytes will be separated by centrifugation. Samples will be run in triplicate. Cells transfected with anti-sense mRNA versus fad158 will serve as negative controls. The protein form GAPDH gene will be used as a loading control. Protein content will be determined by BCA assay. Microgram quantities of proteins will need to be obtained from these cells. Antibodies will be raised against LRR fad158; then, Western blot will be used to determine the expression of fad158 in the cytoplasm, in the nucleus, and in the membrane during the early stages of 3T3L1 differentiation. Detection of the bound protein will be accomplished by chemiluminescence.

One important clue that western blot can provide here is about any possible traversing of the cytoplasm by fad158. It is also possible that the C-terminal portion of the protein will be found in both the cytoplasmic and the membrane fractions. If that were to be the case, it would imply that proteolytic cleavage might be occurring and would necessitate closer examination of results obtained from cells incubated in protease inhibitors or treated with anti-sense mRNA. Identifying binding partners of fad158 will help in pinpointing a role for fad158 in modulation of signaling pathways during the early stages of adipocyte differentiation and assist in research efforts geared toward linking proteins into pathways and networks.

Expected Outcomes: We expect to find LRRs of fad158 in the membrane fractions of 3T3L1 cells. Furthermore, these experiments can also inform us if some portion of the C-terminal region of fad158 resides in the cytoplasm or if cleavage of the protein sends the C-terminus to the cytoplasm. This knowledge will enhance our efforts to better understand the signaling and transcriptional networks impinging on adipogenesis.

Potential Problems and Alternative Strategies.

Should this hypothesis be rendered invalid by unexpected outcomes, we can resort to immunofluorescence or immunohistochemistry to localize the C-terminus leucine-rich-repeats of fad158. During western blotting, BSA will need to be used in place of casein in order to prevent the possibility of the phosphoprotein casein detected by the antibody. Non-specific binding of the antibody will be a major concern here. Controls will be included. Moreover, protease inhibitors will be needed and samples will need to be kept on ice. An alternative approach to determine cytosolic localization of fad158 would be to transfect GFP-labeled expression vectors for the open reading frame of fad158 into 3T3L1 cells or into human adipocytes and then probe the localization with fluorescence microscope (GFP does not easily distribute to the membrane). Confocal microscopy with a fluorophore- labeled antibody against fad158 can be used as an alternative. An alternative approach to determine cytosolic localization of fad158 using transfection of GFP-labeled expression vectors for the open reading frame of fad158 into 3T3L1 cells or into human adipocytes has been used to probe the localization with fluorescence microscope (GFP does not easily distribute to the membrane)(Tominaga et al. 2004).

Timeline. Western blot experiments should be completed in 3 months.

Specific Aim 2: Identifying binding partners of fad158 by affinity chromatography:

Introduction. Protein-protein interactions play important roles in adipocyte differentiation. Our aim is to unearth binding partners of fad158 in cell lysates of murine 3T3L1 cells undergoing differentiation. The working hypothesis behind this approach is that t fad 158 uses its leucine- rich repeats to bind secreted factors and proteins in the membrane or the cytosol (Specific aim 2), thus modulating early adipogenesis. Protein expression in E coli can provide sufficient quantities of proteins for use in experiments. The GST-fusion system is one of most widely used systems utilized in the expression and purification of recombinant proteins in E coli. GST pull-down experiments can help identify interactions between a probe protein and unknown targets, thus providing confirmation of suspected interactions (Nature Methods, Vol 1, No.3, p 275-276, Dec 2004). Fad158 inserts representing fad-LRR (Leucine rich repeats, amino acids 406-803) will be amplified and cloned into the 3 prime-end of pGex vectors with the expression of the inserts under the control of IPTG-inducible tac promoter. All pGex vectors come with an internal Lac1q gene, coding for a repressor protein, which binds the TAC promoter region until induction by IPTG. The pGex vector gives high expression of the fusion protein upon IPTG induction. The rationale behind our binding experiment is that once the protein fad158 is immobilized with a GST tag, it will be feasible to uncover binding partners for fad158 by passing whole cell lysates or cellular fractions through the tagged protein. Prior to passing cell lysate, affininty purification will be performed on Cibacron Blue and Proteinase K columns to remove major contaminant proteins such as albumin and IgG. Results from these experiments ought to aid in furthering knowledge about adipocyte differentiation and help in the design of preventive strategies and therapeutic options against obesity.

Justification and feasibility. To date, there are no known experimentally determined binding partners for fad158. While gene and protein expression of 3T3L1 cells has been well characterized, these attempts have only scratched the surface by providing a list of proteins that undergo expression changes (Renes et al. 2005, Imigawa et al. 1999). Our approach will instead focus on binding interactions in which fad158 may participate.

Research Design. Mammalian source of prey protein and bacterial expression of fad158 will be used to assay

GST

Bait expressed as GST-fad158 fusion for binding partners of fad158 in E coli.

SDS/PAGE, tryptic digestion, MS/

Whole cell lysates from human preadipocytes or 3T3L1 cells exposed to an adipogeneic cocktail (insulin, dexamethasone, methyl xanthine, PPAR-gamma ligand) for 0, 1, 3, 6, 12, 24 hours and 1, 2, 4, 6, and 8 days will be used in this GST pull-down assay to identify interacting partners of fad158. The column will be washed and eluted.

We will first use one-dimensional SDS-PAGE, which is a good technique for separating membrane proteins. The protein will be treated with ammonium bicarbonate and acetonitrile, reduced with DTT at 56 degrees C for 4 minutes, alkylated with iodoacetamide for 30 minutes--, and then digested overnight at 37 degrees C with sequencing grade trypsin. At this stage, the sample is ready for LC/MS/MS and sequence alignment by BLAST. (Chan W et al. 2005. JBC. 25; 23741-47Oster M. et al. 2005. J Cell Sci. 118; 4667-78; Ingham, R et al. 2005. Mol Cell. Bio. 25, 7092-106).

Expected Outcomes. The foregoing research stratagem will inform us about potential binding partners for fad158. On the basis of previous studies on binding interactions, it is our expectation that binding partners for fad158 will be found in both the cytosolic and membrane fractions. An expected denouement of these findings will be the implications of some new proteins in signaling networks that are activated during adipocyte differentiation.

Interpretations of results:

Potential Problems and Alternative Strategies: It is possible that challenges and unlikely situations may be encountered in these experiments. If the GST-fusion system does not yield expected results, we will use yeast-two hybrid or other pull down experiments. Like other techniques, GST-fusion system also has some pitfalls associated with it. These include basal expression from a lac promoter situated between the TAC promoter and the 3 prime end of the lac1q promoter, co-purification of host proteins with the fusion protein from over-sonification, formation of inclusion bodies- a complex between foreign fusion protein and bacterial DNA breakdown of the fusion protein due to over-sonification. Basal expression can be minimized via catabolite repression achieved through the use of 2 % glucose. The formation of inclusion bodies can be prevented by reducing the incubation temperature to between 20 to 30 degrees Celsius, shortening the induction time, and enhancing aeration. Dnak is a common E coli protein which co-purifies with fusion proteins; fortunately, Dnak can be separated using ion exchange chromatography after purification with glutathione Sepharose. Another concern stems from the use of one-dimensional electrophoresis. 1 DE is a better technique for membrane protein separation but complex protein mixtures are difficult to separate by 1DE. In case of any difficulties encountered with 1DE, we will try 2DE using detergents during sample preparation so as to improve the solubility of membrane proteins and afford the separation of membrane proteins- an outcome difficult to achieve with 2DE. Another challenge will be the visualization of proteins from gels for mass spectrometry; staining agents are commercially available to aid in protein visualization. This method will most likely identify some binding partners of fad158 in cells undergoing early adipogenesis.

Timeline. Protein expression in E coli followed by gel electrophoresis and MS/Sequence alignment should be completed in six months time.

Future directions. With respect to the long term goals of our proposal, we will have reached a vantage point upon realization of the specific aims stated above. Completion of Aim 1 will equip us with knowledge about the cellular localization of fad158. Completion of Aim 2 will open up a panoramic view about the signaling processes and binding networks that drive adipocyte differentiation.

Next, we will determine the effects of fad158 knockdown on the expression and function of some of its binding partners that would be tied to signaling networks impinging on adipogenesis. . If any of the binding partners of fad158, that we find, is implicated in signaling networks, we will examine the effects of anti-sense knockdown of fad158 on that signaling network in preadipocytes.

Specific information about proteins that interact with fad158 could be used in a two-pronged approach: peptide inhibition of fad158 and its partners could be used to slow down adipogenesis; alternatively, fad158 and some of its newly discovered partners could be used to transfect cells in areas in dire need of fatty tissues, such as mammary glands after mastectomy and areas around regenerating intervertebral discs.

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