Developing a circularly permuted variant of Renilla luciferase as a bioluminescent sensor for measuring Caspase-9 activity in the cell-free and cell-based systems.

Biosensors and whole cell biosensors consisting of biological molecules and living cells can sense a special stimulus on a living system and convert it to a measurable signal. A major group of them are the bioluminescent sensors derived from luciferases. This type of biosensors has a broad application in molecular biology and imaging systems. In this project, a luciferase-based biosensor for detecting and measuring caspase-9 activity is designed and constructed using the circular permutation strategy. The spectroscopic method results reveal changes in the biosensor structure. Additionally, its activity is examined in a cell-free coupled assay system. Afterward, the biosensor is utilized for measuring the cellular caspase-9 activity upon apoptosis induction in a cancer cell line. In following the gene of biosensor is sub-cloned into a eukaryotic vector and transfected to HEK293T cell line and then its activity is measured upon apoptosis induction in the presence and absence of a caspase-9 inhibitor. The obtained results show that the designed biosensor detects the caspase-9 activity in the cell-free and cell-based systems.

Caspases interplay with kinases and phosphatases to determine cell fate.

Cellular differentiation is one of the critical processes in the life of multicellular organisms. In this phenomenon, a non-specialized cell is converted to a specialized one with its own specific function and morphology. One of the requirements for specialization is silencing of the pathways involved in cell proliferation in parallel with turning on the molecular mechanisms involved in differentiation. Similar to other biological phenomena, the change in cellular state from the proliferative to the differentiated needs molecular switches to persuade the change in response to the internal or external inducers. The quiddity of these molecular switches has not been identified, yet. However, there exists a growing body of evidence showing that the same agents involved in apoptosis have a broad contribution to differentiation progression. To our knowledge, this evidence is still ambiguous because it has raised fundamental questions that require more proof to be answered. The most important questions are: How can two totally different cellular fates act through a similar pathway? What is the separating edge? What forces a cell to choose one of them (death or differentiation)? To address these issues, we will concentrate on three groups of molecules; caspases as the key players of apoptosis, protein kinases, and phosphatases as the major regulators of many cellular and biochemical processes. The evidence reveals a triangle of caspases, kinases, and phosphatases in which their communication leads to the fine-tuning of caspases and consequently they determine cell fate.