Alpha-1 antitrypsin (AAT) is protein synthesized in the liver, which is secreted into the blood. A naturally occurring point mutation (E342K) in the protein known as ATZ results in a conformational disease defined as alpha1-antitrypsin deficiency. The mutation results abnormal folding of the protein and predisposes it to self-association, leading to the formation of stable polymers that are retained as aggregates within hepatocytes. The histopathological standard in hepatocytes of ATZ homozygous individuals is the appearance of endoplasmic reticulum (ER)-derived, PAS positive, diastase negative, intrahepatic globules containing ATZ aggregates. This is also associated with the appearance of ATZ-derived mitochondrial damage. My studies show that transient trasfection with ATZ in murine Hepa1-6 cells recapitulates features of ATZ-containing globules in the liver samples from individuals homozygous for ATZ and transgenic mice expressing ATZ.
Formation of ATZ-containing globules may also be why the unfolded protein response (UPR) fails to be activated in response to ATZ. The UPR functions as a cytoprotective mechanism aimed at decreasing the levels of unfolded proteins and to enhance the functional capacity of the ER. The UPR consists of three branches: protein kinase-like endoplasmic reticulum kinase (PERK), inositol-requiring protein 1(IRE1) and activating transcription factor 6 (ATF6). I hypothesized that selective activation of a branch of the UPR might accelerate ATZ disposal. Here, I find that overexpression of active ATF6 results in the reduction of ATZ levels by approximately 75%, a loss of globule formation, and increased ATZ degradation via ERAD in cells at steady state. My results indicate that most of ATZ is degraded by HRD1-dependent ER-Associated Degradation (ERAD) early after protein synthesis in the presence or absence of active ATF6. Selective activation of ATF6 does not result in apoptosis and reduces ATZ-dependent mitochondrial damage. Additional, experiments also show that treatment with molecular chaperones or protein chimeras shown to increase ERAD or inhibit polymerization are also capable of reducing ATZ. We foresee that this research will help identify new and novel therapeutic approaches in treating AAT deficiency.
|Commitee:||Diekman, Alan B., Raney, Kevin D., Storrie, Brian, Tackett, Alan J.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 73/10(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Biochemistry|
|Keywords:||Activating transcription factors, Antitrypsin, Endoplasmic reticulum-associated degradation,, Proteasomal degradation, Ubiquitin ligase, Ufolded protein response|
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