Exposure of living cells to a synthetic protein

This unique field constitutes a completely new space for synthetic biology and may, in the end, lead to the development of new drugs.

One of the ways to research components in living organisms is the attempt to create their artificial substitutes, using principles of chemistry, engineering and genetics. A collection of particularly effective methods - collectively known as synthetic biology - is currently used to create molecules capable of self-replication, artificial pathways in living systems and organisms that include synthetic genes.

Researchers from Arizona State University were able to produce an artificial protein in the laboratory and test the reaction of living cells to it. "If we take a protein created in a test tube and put it into a living cell, will it still function?" asks the lead researcher. "Will the cell recognize him? Will the cell digest it and expel it from it?" This unique field constitutes a completely new space for synthetic biology and may, in the end, lead to the development of new drugs.

The research findings, published in the scientific journal ACS Chemical Biology, describe an extraordinary ability of Escherichia coli cells to adapt to a synthetic protein, known as DX. Inside the cell, DX proteins bind to molecules of adenosine triphosphate (ATP) - the unit of energy used in all biological systems known to us. "Adenosine triphosphate is the energy "currency" of life," explains the researcher. In the bonds of this material is stored the energy required to activate the reactions that occur in living systems, energy that is released with the dissolution of these bonds. The reduction in the amount of ATP available within the cell caused by binding to the DX proteins disrupts normal metabolic activity within the cell, preventing the cells from dividing (even though they are still developing). Following the exposure to the DX proteins, the Escherichia coli bacterium, which is normally spherical in shape, changes into elongated filaments. Inside the elongated bacteria are compressed intracellular lipid structures that divide the cell to create regular intervals along its length. These unusual structures, which the researchers called "endoliposomes" are an unprecedented phenomenon in cells of this kind.

"Somewhere along these elongated forms, processes begin to occur that we have not been able to fully understand at the genetic level, even though we can see the results of these processes," explains the researcher. "These compressed lipid structures are formed in highly ordered areas along the elongated cells and seem to constitute a type of defense mechanism that allows the cell to create internal substructures." This particular adaptation has never been observed in bacterial cells and appears to be unique to unicellular organisms.

Creating synthetic proteins such as DX, which are able to mimic the sophisticated folding properties of natural proteins and bind to an important metabolite such as ATP, is not an easy task. The researchers used a sophisticated method called mRNA display with which it was possible to produce, adjust and increase the amount of synthetic proteins capable of binding to ATP with high levels of selectivity and affinity, similar to natural proteins that bind to ATP.

In the first step, large libraries of random peptide sequences are created from the four nucleic acids that make up DNA, with each of the sequences being 80 nucleotides long. These sequences are then transcribed into RNA sequences with the help of an enzyme - RNA polymerase. If a natural ribosome (the "factory" for creating proteins in the living cell) is introduced into this mixture, it binds to these sequences and "reads" the random RNA sequence as if it were natural RNA, while creating a synthetic protein. With this method, it is possible to create synthetic proteins based on random RNA sequences. Says the main researcher: "The big question is how can the genetic information be restored? A protein cannot be traced back to the DNA sequence from which it was created. Therefore, we must use all these biological tricks." During the current study, Escherichia coli cells exposed to DX changed their shape to elongated filaments, a shape that occurs naturally when such cells are subjected to stress conditions. The cells exhibit low metabolic activity and little cell division, this is due to the lack of ATP.

The researchers say that there is still much to learn about the bacteria's behavior and the different reaction options following the exposure of such cells to new situations, similar to an unfamiliar synthetic protein. The study also notes that many pathogens rely on a dormant state (similar to the VBNC state observed in Escherichia coli exposed to DX) to avoid exposure to antibiotics. A better understanding of the mechanisms that generate this behavior could provide a new approach to attack such pathogens. The relative safety of Escherichia coli as a model organism for biological studies may provide a useful means for more in-depth research into VBNC states in disease-causing organisms. Moreover, in view of the central importance of ATP for living organisms, damaging it may provide a new route to fight various diseases. One possibility would be an engineered bacterium that could transfer DX genes into disease-causing organisms.

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