Investigating Mechanisms of Protein Degradation During Stem Cell Differentiation

When stem cells differentiate into other cell types, they must perform a radical change to the types of proteins that they express. How the stem cell labels proteins for degradation during this process is a matter of debate. Our lab has identified an enzyme called KMT5b that may be an important factor in labeling proteins for degradation. We hypothesize that knocking down, or silencing, the expression of this enzyme would prevent cellular differentiation. Results from this study could identify a novel protein degradation system and provide valuable information for future potential clinical therapies. To test our hypothesis, we will knockdown KMT5b using shRNA as a vector. I will treat cells with retinoic acid for either one day or seven days, which makes cells stop dividing to become neuron-like. Lastly, I will perform a western blot for the control groups (no knockdown) and KMT5b knockdown groups to observe the protein level of tyrosine hydroxylase, a neuronal differentiation marker. We expect that the group with enzyme knockdown and 7-day retinoic acid treatment will show the lowest amount of tyrosine hydroxylase protein expression, which indicates unsuccessful differentiation. In conclusion, this experiment will assess whether KMT5b controls stem cell differentiation