Regulating the ATP Synthase Activity

Event start date
Event start time
12.00
Place

Arvo building, Jarmo Visakorpi auditorium, address: Lääkärinkatu 1.

Doctoral defence of M.Sc. Heidi Kontro

Regulating the ATP Synthase Activity - Characterization of the F-ATP synthase subunit DAPIT and its over-expression in HEK293T cells

The field of science of the dissertation is Paediatrics.

The opponent is docent Eija Pirinen (University of Helsinki). Professor emeritus Markku Mäki acts as the custos.

The language of the dissertation defence is Finnish.

Effects of impaired activity of an energy producing enzyme in the cell

Type 1 diabetes is a complex metabolic disorder that causes changes in cellular physiology, metabolism, and gene expression. Insulin deficiency is known in mouse tissues to impair gene regulation, for example by decreasing the mRNA levels of the proteins comprising the enzymes of the respiratory chain. On the other hand, the changes in expression levels of proteins involved in ATP synthase, as well as their effects on its activity in diabetes, is not known.

This dissertation study has identified and characterized the ATP synthase Fo complex subunit Diabetes Associated Protein in Insulin-sensitive Tissues, DAPIT. In addition, the effect of its over-expression on cellular physiology has been studied by molecular biology techniques to clarify its meaning in diabetic and other disease states.

DAPIT proved to be a conserved protein in the animal kingdom. By using a custom made antibody, its mitochondrial and lysosomal expression was demonstrated. In histochemical studies of tissues from healthy humans and rats, DAPIT was abundant in insulin-sensitive tissues employing aerobic metabolism and in epithelium actively transporting nutrients and ions. In insulin-sensitive tissues of diabetic rats, DAPIT was over-expressed in myocardium, skeletal muscle and adipose tissue, but down-regulated in the liver compared to healthy rats. Studies on cellular level showed that DAPIT over-expression inactivated ATP synthase, the result in line with other metabolic changes caused by insulin–deficiency. Since the cellular studies were performed in the presence of insulin, the other results associated with this study are not directly proportional to the diabetic condition.

In a cell-line over-expressing DAPIT, it was also shown that an increase at the protein level severely impaired mitochondria by inactivating the ATP-synthase activity regardless of active respiration of the cells. This resulted in increased production of oxygen radicals, which most likely up-regulated the nuclear transcription factors, further promoting the anaerobic metabolism and the cell dedifferentiation. As a result, the epithelial type of cells transformed into mesenchymal-like and decelerated its proliferation rate. The dedifferentiated cells showed several features of cancer stem cells, which are discussed.

In addition to the DAPIT over-expressing cell line and type 1 diabetes, this over-expression, according to literature and databases, was also seen in Parkinson’s disease, multiple sclerosis (MS), high weight gain, cardiac hypertrophy and arsenic-related toxicity. These findings suggest that DAPIT over-expression could be a more common phenomenon in regulating ATP-synthase and inactivity of oxidative phosphorylation in diseases where metabolic disturbances play a significant role, for example diabetes, cancer, degenerative, autoimmune, toxin and epigenetically regulated conditions. The knock-in mouse recognizing the pathologic changes in question would be of help in understanding the consequences of mitochondrial impairments due to DAPIT over-expression. Also other characteristics of DAPIT over-expressing cells resembling cancer stem cells should be elucidated.

In overall, this dissertation study produced the DAPIT protein and provided new information of its location, histology and diabetic expression, as well as the effects of its over-expression on ATP synthase activity, mitochondrial function and cellular behavior. These characteristics will help to better understand the molecular biological and metabolic effects of impaired activity of an energy producing enzyme in contest of diseases where observed.

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The dissertation is published in the publication series of Acta Universitatis Tamperensis; 2275, Tampere University Press, Tampere 2017. The dissertation is also published in the e-series Acta Electronica Universitatis Tamperensis; 1777, Tampere University Press 2017.