Research Proposal Paper Sample: Determining the Role of the DPP9 Enzyme

Proposed Mode of Research

The research will be done in a laboratory and will also involve literature research on the various studies done in that past surrounding this area of investigations.

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Aims and Objectives

There are no clear physiological roles that have been established to date in regards to DPP9 even though it has been noted in the body immune system, the survival of cells and their behavior, pathogenesis of diseases and body inflammation. For this reason, there is need to further the research on the functions and biological properties associated with DPP9. As such, the aim of this research proposal includes.

A demonstration of the importance played by the DPP9 enzyme activities when the other DPP4 family proteins are lacking.

To explain clearly the biological roles of DPP9 in GKO adult mice lacking

To determine the role played by DPP9 during regeneration of the immune system of a mouse

Synopsis

When a GKO mouse lacking the other DPP4 family proteins was created, the results showed that the mice were healthy which was a clear indication that the absence of DPP9 proteins had a protective effect on the immune system. The inactivation of the DPP9 Enzymatic activities needs to be investigated to determine whether it has a protective impact on the immune system of the mice

As of to date, there has not been conclusive research or study done to examine the role played by dpp9 in the regeneration of the immune system even though DPP9 has in numerous times being detected in the immune system both in vitro and in vivo. As such, the study will revolve around the creation of primary and secondary chimeras to help investigate the role played by DPP9 enzymatic activity when the immune systems of the mice are being developed. The chimeras will be produced by irradiating mice that have been injected with WT and DPP9-GKI mouse lethal liver cells that will involve the primary chimeras and then generate the secondary chimeras by injecting with the bone marrow cells from the primary chimera mice

Background

According to Deacon (2011) and Kirby (2010), the success gained during researches on the oral DPP4 inhibitors for the type 2 diabetes therapy drove the DPP4 family of enzymes to have more importance in research work (Deacon, 2011, Kirby et al., 2010). The family members that belong to this enzyme (DPP4, DPP8, DPP9, and FAP) are known to hydrolyze the post-proline bonds two residual that are found on the terminus of a substrate. In this case, FAP, which is closely related to DPP4, is known to be a potential target in the treatment of cancerous cells (Yu et al., 2010, Brennen et al., 2012, Keane et al., 2012, Christiansen et al., 2013). However the other close relatives (DPP9 and DPP8) are still under extensive study in cell biology, the biology of the immune system and for a tumor (Zhang et al., 2013). There have been inhibitors for DPP4 and FAP that have been developed in the modern medicinal chemistry but so far non has been developed for both DPP8 and DPP9. Some researchers have shown that there exist some compound which can inhibit DPP8 and DPP9 and at the same time not be inhibiting DPP4 and FAP (Van Goethem et al., 2011, Wu et al., 2012).

According to Yu (20016), the over expression of DPP9 enzyme can impair the coordination of cells and can cause less Akt phosphorylation due to the stimulation of EGF (Yao et al., 2011). Research has been done on the role played by DPP9 during antigen presentation whereby DPP9 is used for rate limiting during the degradation of the antigenic proline which is known to contain peptides that include even the tumor (Geiss-Friedlander et al., 2009). DPP9 can also be found in B cells and both CD4+ and CD8+ lymphocytes there are regulated by the mitogen stimulation that occurs in the lymphocytes (Yu et al., 2009, Chowdhury et al., 2013). The inhibiting activity of both the DPP9 and DPP8 enzymatic activity are known to dampen the proliferation of lymphocytes according to Lankas et al. (2005) and (Reinhold et al.) 2009, and thus this makes DPP9 very important in immunobiology.

According to research by Lu et al. (2011), both DPP8 and DPP9 enzymatic activity are used to protect Ewing sarcoma cells from self-destruction or death caused by neuropeptide Y. There has been progress in the importance of DPP9 in tumor biology, whereby the inhibition by both DPP8 and DPP9 has been found to enhance the anti-leukemia activity in both the acute leukemia cell lines (Spagnuolo et al., 2013). An adjuvant effect that is usually triggered by the inhibition of DPP9 has been found to be the process used by the compound Val-boro-Pro to control the regression of tumors. Many cancer patients are known to express an unregulated DPP9 expression (Yu et al., 2009, Olsen and Wagtmann, 2002, Ajami et al., 2004, Stremenova et al., 2010).

DPP9 and DPP8 are known to be intracellular enzymes and are usually expressed in both the tissues and most cell lines (Yu et al., 2010, Yu et al., 2009). However, DPP4 and FAP are mostly expressed on the cell surface. The body organs that are well known to express DPP9 and D998 include lymphocytes and epithelial cells which include the lymph nodes, liver, brain, and muscles (Yu et al., 2009, Chowdhury et al., 2013, Schade et al., 2008, Harstad et al., 2013). However, there is not much known about the composition of the natural substrate that constitutes DPP9 except the glucagon-like peptide that is found in both DPP4 and FAP (Lu et al., 2011). In recent researches, there have been some substrates that have been identified to be found in DPP9. The substrate includes adenylate kinase 2 and calreticulin which could only indicate that there is a role played by DPP in energy homeostasis (Wilson et al., 2013). As such, we can only conclude that DPP9 has many diverse roles in the body and it can be very elusive to determine the primary role it plays.

Yu (2010) states that up to date, no mouse has a GKO for a protein that has the DPP activity has been known to exist. An experiment done showed that DPP4-GKO and FAP-GKO were all healthy (Yu et al., 2009, Niedermeyer et al., 2001, Marguet et al., 2000). The DPP4-GKO mice over a period showed tolerance to glucose after an experiment was done on glucose challenge and resist diet-induced obesity (Marguet et al., 2000, Conarello et al., 2003). As a result, the knockout mice have been of great importance and help during studies on the therapeutic potential that exist in both DPP4 and FAP inhibitors. Since these mice have complete ablation of all function s in a protein, extreme care is necessary during these experiments (Yu et al., 2010, Keane et al., 2012).

Expected Research Contribution

By the end of this research, I should have achieved my three aims and objectives as shown above. I should be in a position to demonstrate that DPP9 enzymatic activities play a vital role in the absence of the other DPP4 family proteins. Also, I should be in a position to show clearly the biological role of DPP9 in a gene knockout adult mouse. Lastly, I should be in a position to clearly show the exact roles played by DPP9 during the regeneration of the immune system of a mouse.

The Proposed Methodology

Materials/equipment

1 x PBS- this should be made from 15 Dulbecco's Phosphate which is a buffered saline.

FACS wash- 15 ml FACS (10% conc.), 1000 l of 0.8 M ethylenediaminetetraacetic acid (5mM) and 300 l of 5% Sodium azide (0.05%) which makes the volume to be 300 ml.

50 x TAE buffers- prepared using 245 g of Tris-base, 57.5 ml of acetic acid (glacial), 200 ml of 1 M ethylenediaminetetraacetic acid (pH 7.0) was made up to 1.2 L with triple distilled water. A working solution of 1.2 x Sodium azide was made by diluting 30 ml of 60 x Sodium azide buffer in 1000 ml of triple distilled water.

0.8 M ethylenediaminetetraacetic acid (pH 7.0)- 187.2 g of ethylenediaminetetraacetic acid. Heavy water was dissolved in 900 ml of water and stirred vigorously. An adjustment is done to the PH to 7.0 with Sodium hydroxide and water being added and the final volume of 1 L obtained.

(RBC) buffer - 8.30 g (NH4Cl), 1.1 g (KHCO3) and 0.137 g of

Ethylenediaminetetraacetic acid was dissolved in 1.1 L of water.

Hydroxyproline assay reagents:

Liver cell buffer

Cell freezing solution -15 ml Dimethyl sulfoxide added to 100 ml Fetal Calf Serum

Antibodies

Source of specialty mouse supplies

Methodology

Generate a DPP9 enzyme-inactive mouse.

Embryonic and early neonate studies

Histochemical studies

Immunofluorescence Imaging

SDS-PAGE urine analysis

Generation and immortalization of mouse embryonic fibroblasts (MEFs)

Enzyme assays

Immunoblotting

Quantitative real-time PCR (qPCR)

Work Plan

The research will take place over a period of 3 years. During the first six months, I will do a thorough literature review on the topic to get more knowledge on it. The next six months I will spend it repairing for the actual experiments which I will spread them over a period of two years for the three objectives. For the first objective, I will spend 8 months doing the experiment, for the second aim I will also spend eight months doing the experiment and the last aim will also take eight months. However, these timelines can be adjusted after more consultation with my supervisor.

Resources

The experiments will take place at the university laboratories, and some of the equipment I will need for the experiments will be found in the school laboratory. However, I will need financial aid so that I can purchase the resources that cannot be provided by the university laboratory. Additionally, I will also require financial resources in case I may need to travel during the experiments.

References

Brennen, W. N., Isaacs, J. T., & Denmeade, S. R. (2012). Rationale Behind Targeting Fibroblast Activation Protein-Expressing Carcinoma-Associated Fibroblasts as a Novel Chemotherapeutic Strategy. Molecular Cancer Therapeutics, 11(2), 257-266. doi:10.1158/1535-7163.mct-11-0340

Chapter 5. Targeting Dipeptidyl Peptidase-4 (DPP-4) and Fibroblast Activation Protein (FAP) for Diabetes and Cancer Therapy. (n.d.). RSC Drug Discovery, 118-144. doi:10.1039/9781849733151-00118

Chowdhury, S., Chen, Y., Yao, T., Ajami, K., Wang, X. M., Popov, Y., ... Gorrell, M. D. (2013). Regulation of dipeptidyl peptidase 8 and 9 expression in activated lymphocytes and injured liver. World Journal of Gastroenterology, 19(19), 2883-2893. doi:10.3748/wjg.v19.i19.2883

Deacon, C. F. (2010). Dipeptidyl peptidase-4 inhibitors in the treatment of type 2 diabetes: a comparative review. Diabetes, Obesity and Metabolism, 13(1), 7-18. doi:10.1111/j.1463-1326.2010.01306.x

Lankas, G. R., Leiting, B., Roy, R. S., Eiermann, G. J., Beconi, M. G., Biftu, T., ... Thornberry, N. A. (2005). Dipeptidyl Peptidase IV Inhibition for the Treatment of Type 2 Diabetes: Potential Importance of Selectivity Over Dipeptidyl Peptidases 8 and 9. Diabetes, 54(10), 2988-2994. doi:10.2337/diabetes.54.10.2988

Lu, C., Tilan, J. U., Everhart, L., Czarnecka, M., Soldin, S. J., Mendu, D. R., ... Kitlinska, J. (2011). Dipeptidyl Peptidases as Survival Factors in Ewing Sarcoma Family of Tumors. Journal of Biological Chemistry, 286(31), 27494-27505. doi:10.1074/jbc.m111.224089

Van Goethem, S., Matheeussen, V., Joossens, J., Lambeir, A., Chen, X., De Meester, I., ... Van der Veken, P. (2011). Structure-Activity Relationship Studies on Isoindoline Inhibitors of Dipeptidyl Peptidases 8 and 9 (DPP8, DPP9): Is DPP8-Selectivity an Attainable Goal? Journal of Medicinal Chemistry, 54(16), 5737-5746. doi:10.1021/jm200383j

Wu, W., Liu, Y., Milo, L. J., Shu, Y., Zhao, P., Li, Y., ... Lai, J. H. (2012). 4-Substituted boro-proline dipeptides: Synthesis, characterization, and dipeptidyl peptidase IV, 8, and 9 activi...