Serotonergic dysfunctions have been linked to many neuropsychiatric illnesses. Medications to treat these disorders aim to stabilize the levels of serotonin in the synaptic cleft. While current treatments have provided relief to millions of patients, they present tolerance and efficacy problems. Owing to this, there is a need for conceptually novel therapies capable of safely treat a high proportion of patients. Our long-term objective is to understand Tryptophan Hydroxylase 2 (TPH2). TPH2 catalyzes the first and rate-limiting step in the transformation of tryptophan into serotonin in the brain. TPH2 has been found to be phosphorylated;this modification has been reported to result in increased TPH2 stability and enhanced activity. We hypothesize that post-translational modification (PTM) of TPH2 to play an important role in the in vivo regulation of this key enzyme. To corroborate this hypothesis, I plan to map in vivo PTMs on TPH2 through mass spectrometry (MS) based proteomics. Furthermore, through the use of affinity purification in combination with MS, I aim to characterize binding partners for TPH2. The proposed work is innovative, because it utilizes modern techniques to solve questions inaccessible through conventional biochemical experiments. The results of this study can provide important information about the physiological control of TPH2 and can open the door to a new, more selective generation of antidepressants with fewer side effects, able to increase serotonin synthesis through the enhancement of brain-specific TPH2 activity. We will pursue these studies in two specific aims:
Specific Aim #1 : Explore the in vivo post-translational modification of TPH2 through MS -based proteomics.
We aim to explore the post-translational regulation of TPH2 in physiologically relevant settings through mass spectrometry (MS) proteomics. To do this, I will stably transform 6XHis-tagged TPH2 into mammalian (PC12) cells. Alternatively, I plan to extract TPH2 from rat brain raphe. I will analyze PTMs of TPH2 from these two sources through MS based proteomics.
Specific Aim #2 : Identify protein-protein interactions involving TPH2 through affinity purification in combination with MS.
We aim to identify novel TPH2 binding partners. Interacting proteins can regulate TPH2's function. To date, 14-3-3 proteins are the only known TPH2 binding partner. In these experiments, I will extract tagged TPH2 from PC12 cells, and identify co-purifying proteins through MS. Again, co-purifying (interacting) proteins will be identified through MS.
Low levels of the neurotransmitter serotonin are linked to various psychiatric disorders such as depression, obsessive compulsive disorder, schizophrenia and autism among many others. In the brain, Tryptophan hydroxylase 2 (TPH2) catalyzes the first and rate-limiting step in the transformation of tryptophan into serotonin. The regulation of this protein is poorly understood;
we aim to comprehend how it is controlled. The results of this study can ultimately open the door to a new, more selective generation of antidepressants with fewer side effects.
|Torrente, Mariana P; Gelenberg, Alan J; Vrana, Kent E (2012) Boosting serotonin in the brain: is it time to revamp the treatment of depression? J Psychopharmacol 26:629-35|