Results and Future Direction

Undergraduate Research Initiative – Blog 4

 

Over the past few months, Dr. Nigel Yarlett and I have been working on characterizing the substrate specificity of Spermidine:spermine N1 – Acetyltransferase (SSAT). In this time we have isolated and expressed the C. parvum SSAT for kinetic studies in order to classify the enzyme either as a true SSAT or a general N-acetyltransferase (GNAT). Previous research has shown that the polyamines: putrescine, spermidine, and spermine have all been found to be essential for the growth, development, and metabolism of C. parvum. SSAT is a major regulator for polyamine synthesis, particularly spermine, and without it the parasite will surely die. Applying chemical kinetic studies allowed us to gauge and study the rates of chemical processes. We can also investigate how different experimental conditions (different substrates) can influence the speed of a chemical reaction and then construct mathematical models, in the form of graphs, describing this chemical reaction based on how much of a certain substrate and varying concentrations are reacting. Initial results indicate that spermine is the preferred substrate for SSAT with minor activity being demonstrated for spermidine, histone 2A, para-amino benzoic acid (PABA), and dapsone. This information was collected through both kinectic analysis of spermine, spermidine, and histone 2A and high–performance liquid chromatography analysis (HPLC) using PABA and dapsone. Upon completion of our kinetic studies and HPLC analysis we may be able to confirm the true substrate specificity for SSAT. If spermine is shown to have the highest activity out of all the substrates tested we can then exploit these biochemical characteristic for rational drug design. This project was very beneficial for me because it has allowed me to use and understand advanced techniques in the science field. My future goal for this project is to synthesize a polyamine analogue structurally similar to spermine. Considering SSAT is substrate specific for spermine, this analogue may be able to act as inhibitor for SSAT, prohibiting parasitic growth and promoting death of C. parvum. These findings will generate a manuscript and will be published in the Journal of Molecular and Biochemical Parasitology. I am glad that I was able to receive so much support from the undergraduate research initiative team in my pursuit of greater knowledge of scientific research. I have learned so many different techniques, how they work, and how I can analyze and interpret raw data. My mentor, Dr. Nigel Yarlett, was definitely inspiring and very supportive in times of frustrations and failures. Overall, I truly enjoyed this experience and thank Dr. Nigel Yarlett and the rest of Haskins Laboratories for this experience.

Blog Post#3

Cryptosporidium parvum is an intracellular parasitic protozoan that causes cryptosporidiosis, a diarrheal disease occurring in humans and animals. Not only does C. parvum remain as one of the top organisms responsible for debilitating diarrheal infections in developing countries, it is also known to cause outbreaks in industrialized countries. The main way C. parvum spreads is via contaminated water. Due to their small size and high resistance to chlorination and other disinfectants, water filtration systems have had little success in the removal of the parasite from water sources and early detection measures are still being researched. Individuals with healthy immune systems who contract this disease will experience symptoms of unrelenting diarrhea, fever, dehydration, vomiting, and nausea, which may last up to two or three weeks. Treatment for these individuals involves drinking lots of fluids and rest. However, for immunocompromised individuals characterized by AIDS or cancer, the onset of the disease is relentless and very severe, often fatal. Currently, there is no effective treatment for cryptosporidiosis and a new chemotherapeutic drug is needed.

Previous research has shown that the polyamines: putrescine, spermidine and spermine, have all been found to be essential for the growth, development, and metabolism of C. parvum. Spermidine: Spermine N1- Acetyltransferase (SSAT) is a major regulator for polyamine synthesis, particularly spermine, and without it the parasite will surely die. As a result, last semester at Haskins Laboratories I focused on cloning and purifying the SSAT protein so that I could perform several assays on different substrates to develop a substrate curve. Due to Hurricane Sandy, my research was significantly affected and I had to restart the cloning and purification process. However, I have had a breakthrough and have successfully cloned and purified the SSAT protein needed to proceed. I am currently in the process of running multiple assays with substrates such as spermine, spermidine, histone, dapsone, and tris-HCl. The data obtained from these assays will generate substrate curves that will enable us to differentiate if SSAT is a general N1-acetyltransferase (GNAT) or if is a specific for spermine. If the GNAT is more active with other substrates, rather than with spermine, then it is probably not a true spermidine:spermine acetyltransferase. This is troublesome because we want to develop drugs that can inhibit the major polyamine spermine in order to inhibit the growth, development, and metabolism of C. parvum, thus potentially being able to cure cryptosporidiosis.

If these results indicate SSAT is truthfully the regulator of spermine then the process in synthesizing a drug with inhibitory abilities could be the sole determinant factor impeding a cure. With the assistance of Dr. Jamielee Rizzo and many more months of research our overall goal may come to fruition. Throughout the academic year, Dr. Nigel Yarlett has been an excellent mentor and role model. With his expertise and wisdom I have become fascinated with my research project and I have also learned to look past initial failures and reflect on my successes as a researcher.

 

—Emmanuel Bujans—
Pace University-Pforzheimer Honors College
Biochemistry Class of 2014
Research Assistant at Haskins Laboratories
(NERA) Medprep Scholar

Tel: (239) 961-9238
eb53946n@pace.edu

Characterization of Spermidine:spermine N1- acetyltransferase from the intestinal parasite Cryptosporidium parvum

Blog #2

November 26, 2012

Emmanuel Bujans

 

In my first blog post, I described the many issues as well as actions that need to be taken surrounding the organism Cryptosporidium parvum and the disease it causes, Cryptosporidiosis. Since the beginning of the semester, I have been working on cloning and expressing a new purified SSAT protein, however, the last protein I stored was found to be denatured.

Although I was at the final stages of purifying the SSAT protein, because of Hurricane Sandy all my samples were ruined. This occurred due to the power outage, which affected the -4 °C fridge where my temperature-dependent samples were being stored. Since there was no power, the heat in the fridge slowly rose to room temperature thus denaturing my samples. Denaturation of proteins usually occurs because the bonding interactions responsible for the secondary structure (hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure there are four types of bonding interactions between “side chains” including: hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic interactions. Therefore, a variety of reagents and conditions can cause denaturation. The common condition in this case was heat.

Even though horrible disasters may happen, which may impede one’s research, I have learned that I must continue past all those frustrations if I truly want to be successful. Therefore, for the next few weeks, I will restart the long process of cloning and expressing the SSAT protein. This is done by PCRing the desired known DNA sequence, then choosing one of the PCR samples containing the closest molecular weight size marker in a electrophoresis gel, DNA cleanup before ligation, ligation into a suitable vector, and transformation and screening to identify recombinant clones. This process usually takes about a full three weeks to complete.

Overall, as the semester has progressed, working with Dr. Nigel Yarlett has taught me how to apply knowledge learned in the classroom and relate it to microbial research. The next steps will be to perform experimental assays that measure the SSAT activity of the new purified protein in relation to C. parvum oocysts.

Characterization of Spermidine:spermine N1- acetyltransferase from the intestinal parasite Cryptosporidium parvum

BLOG POST # 1

Hello everyone! My name is Emmanuel Bujans and I am a Biochemistry major, minoring in Latin American Studies. This is my junior year at Pace University (NYC) and I am extremely grateful to have received this grant to pursue research in my desired field. As an ambitious individual, my future aspirations are to enter the medical field and become a physician. In order to become a better physician I have decided to immerse myself in scientific research. What better way to help people, than to learn the most fundamental concepts of research and applying them in order to make a significant discovery? In hopes of discovering something that will help change people’s lives for the better, I began research with Dr. Nigel Yarlett, Department Chair of Chemistry and the Physical Sciences. Together we are studying the characterization of Spermidine: spermine N1- acetyltransferase (SSAT for short) from the intestinal parasite Cryptosporidium parvum.

Cryptosporidium parvum is an intracellular parasitic protozoan that causes cryptosporidiosis, a diarrheal disease occurring in humans and animals. Not only does C. parvum remain as one of the top organisms responsible for debilitating diarrheal infections in developing countries, it is also known to cause outbreaks in industrialized countries. The main way C. parvum spreads is via contaminated water. Due to their small size and high resistance to chlorination and other disinfectants, water filtration systems have had little success in the removal of the parasite from water sources and early detection measures are still being researched.

Individuals with healthy immune systems who contract this disease will experience symptoms of unrelenting diarrhea, fever, dehydration, vomiting, and nausea, which may last up to two or three weeks. However, for immunocompromised individuals characterized by AIDS or cancer, the onset of the disease is very severe and often fatal. Currently, there is no effective treatment for cryptosporidiosis and a new chemotherapeutic drug is needed.

Together with my research mentor, Dr. Nigel Yarlett, we have decided to tackle this issue. Previous research has shown that the polyamines: putrescine, spermidine and spermine, have all been shown to be essential for the growth, development, and metabolism of C. parvum. SSAT is a major regulator for polyamine synthesis, particularly spermine, and without it the parasite will surely die. Currently, we are two-thirds the way into cloning and expressing a new purified protein because the protein we were using was found to be denatured. Our initial analysis of the performed SSAT assays have shown that the protein activity, although active is significantly less than our desired activity. This may have been due to being stored for a long period of time. Generally, this experiment takes about a week or two to complete.

Our overall goal for this project is to develop an effective inhibitor for SSAT that can be used as a target for drug design. The result of these studies will be used to present at national/international conferences and for publication. Altogether, this research has reinforced the material that I have learned in class by enabling me to gain a deeper understanding of why certain experiments are used to get the desired results.