IDENTIFICATION OF HUMAN PGC-1α-b ISOFORMS USING A NOVEL PGC-1α-b SPECIFIC ANTIBODY

摘要

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is known as the master regulator of mitochondrial biogenesis. PGC-1α holds this role by acting as a transcriptional coactivator for an array of transcription factors and nuclear hormone receptors, such as NRF-1/2 and ERRα/γ, whose downstream targets function in mitochondrial biogenesis and oxidative phosphorylation. PGC-1α is regulated both at the transcriptional and post-translational level in several signaling pathways, including p38 MAPK and AMPK. This regulation affects which transcription factor binding events can occur in a given tissue, and thus affects regulation of PGC-1α target genes. PGC-1α is downregulated in many neurodegenerative disorders as well as in muscular dystrophies, diabetes, and aging. Therefore, PGC-1α is prized as a potential therapeutic target to create novel treatments for these various diseases.However, details governing the spatio-temporal regulation of PGC-1α are not completely understood, and overexpression of PGC-1α throughout the body or even in certain tissues or subsets of cells have had detrimental effects in animal and cell models. Therefore, it is necessary to gain knowledge of how to modulate PGC-1α in a tissue-specific manner utilizing these different levels of regulation in order to develop novel therapies. In order to further understand all the functions that have been attributed to PGC-1α, the PGC-1α isoforms need to be accounted for and understood in human tissues. Several murine isoforms have been published, as well as several human brain and muscle isoforms. However, most of these isoforms have only been validated as mature transcripts, and it is not known whether they produce functional protein. Our lab has identified the isoform b transcript in human brain tissue via 5’ RACE and have developed an isoform b specific antibody. This project aimed to characterize the isoform b transcripts and also to validate and optimize this antibody for immunoblotting conditions for detection of further PGC-1α-b isoform protein variants in human tissues. Preliminary studies in our lab have shown that in postmortem frontal cortex from age-matched PD and healthy patients, isoform a transcript levels were 10-15 times more abundant than that of isoform b. These differences in regulation could be partially attributed to the isoform b promoter region being heavily methylated, as shown in this thesis through bisulphite cloning and sequencing as well as 454 bisulphite sequence analysis. The high degree of methylation, correlated with the low level of isoform b transcript in brain and it is not known whether this transcript would be translated into protein in this tissue. In order to probe for isoform b protein expression using human cell lines and tissues, however, it was necessary to create a recombinant protein in order to have a positive control with which to optimize our novel antibody. In our previous 5’ RACE studies, an alternatively spliced PGC-1α-b transcript was found which coded for an early stop codon. This truncated isoform was called PGC-1α-b-3T1, and mature transcript was found in both human skeletal muscle and brain. For this project, PGC-1a-b-3T1 was cloned from human skeletal muscle into a bacterial expression vector to create a recombinant GST fusion protein. This protein was used to validate and optimize our PGC-1α-b specific antibody as well as to determine sensitivity and specificity. The purified recombinant protein contained 3 bands of lower molecular weight that were detected via western blot with both GST and the PGC-1α-b specific antibody. These bands were trypsin cleaved and subjected to mass spectrometry analysis, which verified that all bands detected by the PGC-1a-b specific antibody contained the epitope sequence, and thus binding was specific. This protein was then used to determine western blotting conditions and sensitivity, which is 10 ng using a 1:100 dilution of the antibody. This antibody was then used to probe SH-SY5Y WCL, a human neuroblastoma cell line. Peptide competition assay confirmed 5 PGC-1α-b specific proteins in these lysates. The sizes of these proteins matched to several murine PGC-1α-b isoforms as well as putative PGC-1α-b versions of PGC-1a-a isoforms. These findings provided the putative identities of several endogenous functional human PGC-1α-b isoforms. Mammalian overexpression vectors of these isoforms are still in development. By using this antibody and these expression vectors to further characterize these isoforms, including determining tissue specificity, more knowledge of PGC-1α will be gained. This information could then be used to develop novel, tissue specific treatments for pharmacological intervention of diseases characterized by PGC-1α misregulation.