Mucolipidosis type IV (ML-IV) is a human lysosomal storage disorder affecting critical developmental milestones. The clinical manifestation of ML-IV includes severe neuro-motor, opthalmic and gastric abnormalities. ML-IV is caused by abnormal function of the mucolipin-1 (TRPML1) protein. TRPML1 belongs to the mucolipin (TRPML) subfamily of the transient receptor potential (TRP) superfamily of ion channels. The TRPML subfamily consists of TRPML1, -2 and -3 proteins. The TRPML proteins are believed to play a role in intracellular trafficking, endosomal-lysosomal biogenesis, and in autophagy. Heteromeric subunit interactions and functional redundancy between the three TRPML protein subunits have been reported. Indeed, we have recently shown that TRPML1 and TRPML2 share a very similar electro-physiological profile, while others have shown that loss of TRPML2 or TRPML3 produces a phenotype typically seen in ML-IV cells. We also found that loss of TRPML1 contributes to the tissue-specific down-regulated expression of TRPML2 transcripts. Interestingly, phorbol 12- myristate 13-acetate (PMA;a potent protein kinase C [PKC] activator) and Ionomycin (intracellular calcium mobilizer) significantly up-regulate TRPML2 mRNA expression.
The first aim of this proposal is to determine and map the core promoter region involved in the transcriptional activation of TRPML2 using a dual-luciferase gene reporter and gel mobility shift assays. Secondly, we will determine the potential role of micro-RNAs in its potential role in the post-transcriptional regulation of TRPML2 expression. Finally, we will determine the transcription factor protein involved in regulating TRPML2 transcript levels using chromatin immunoprecipitation assays and mass spectrometry. We will also assess TRPML2 protein levels and correlate it with our genetic reporter data using Western blot and fluorescence imaging techniques, since changes in mRNA transcript levels do not necessarily correspond directly to protein levels. This proposal will open many avenues for research to advance our knowledge on the transcriptional activation and regulation of the TRPML2 ion channel, and at the same time, it will create research opportunities for under-represented undergraduate students. In essence, the knowledge gained from the proposed studies will be the first step to our long-term goal of a proof-of-concept approach to exploit functional redundancy and substitute TRPML2 or TRPML3 protein for the loss of TRPML1 function in ML-IV patients.
Mucolipidosis IV (ML-IV) is a human genetic disorder caused by the dysfunction of the mucolipin-1 (TRPML1) protein. ML-IV is manifested during early child development, and produces debilitating symptoms that include, but are not limited to, abnormal psychomotor milestones, digestive problems, cataract formation, and blindness. This proposal will study the activation process of a closely related member, TRPML2. Knowledge gained from the study could allow us to potentially replace the loss of TRPML1 protein as therapeutic approach for ML-IV.
