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Description
The sphingomyelinase hydrolysis reaction produces two noteworthy molecules; S1P and ceramide. Ceramide helps initiate the apoptic pathway. Sphingosine-1-phosphate (S1P) and is a second messenger in cell proliferation and survival. Therefore, S1P has garnered attention as a possible cancer target. According to the WHO and the American Heart Association, Cardiovascular Disease is the number one killer in the world. Although the pathway is not completely understood, ceramide and Neutral Sphingomyelinase or nSMase (a mammalian SMase) may play a key role in related diseases such as Myocardial Infarction (MI) or Cerebrovascular Accident (CVA). If SMase can be inhibited once blood flow is reestablished, this may reduce damage of an ischemic event, leading to an improved prognosis. Although SMase inhibitors have been identified, their biological activities are not sufficient to be effective drugs. SMase C from B.cereus and SMase D from L.laeta were expressed using an IPTGinducible plasmid. They were expressed in E. coli cells, lysed, and then underwent the initial phase of purification. SMase C was purified by hydroxyapatite chromatography and SMase D (which has a Histidine tag) was purified by using a HisTrap column. The lysate was loaded onto size exclusion columns to obtain our specific proteins. Using Phosphorus Nuclear Magnetic Resonance (P-NMR), we can perform kinetics experiments on SMase C and D. The phosphate on sphingomyelin's head group has a distinct chemical shift of -0.7 ppm. After adding SMase C or D, the byproducts ceramide and ceramide-1-phosphate are generated, each with their own respective chemical shifts. The rate in which the sphingomyelin peak diminishes is used for kinetics studies. The kinetics experiments will be performed using the NMR 500 MHz. We have optimized our purification procedures to obtain roughly 10-15 mg/mL of protein. By testing various inhibitors against SMase C and D, we can create a model through which we can hypothesize what functional groups should be included in inhibitors of nSMase. A sphingomyelin bound protein crystal can guide us into creating more specific inhibitors. We would be able to see which functional groups of sphingomyelin are critical in substrate binding and use it to adjust our plans for synthesis. As we continue to test compounds, we will hopefully build a library of inhibitors that we can take to the next step of drug discovery by testing against mammalian nSMase.
