Enzyme’s Stability in Cells Should Be Considered When Developing New Replacement Therapies, Study Suggests

Enzyme’s Stability in Cells Should Be Considered When Developing New Replacement Therapies, Study Suggests

The stability of lysosomal proteins when tested in a laboratory setting is not equivalent to that observed in live cells, a study has found. This information should be considered when designing new protein versions to improve the effectiveness of enzyme replacement therapy for Batten disease.

Results of the study,”Lysosomal protein thermal stability does not correlate with cellular half-life: Global observations and a case study of tripeptidyl-peptidase 1,” were published in the Biochemical Journal.

Batten disease, also known as neuronal ceroid lipofuscinosis, refers to a group of rare inherited neurological conditions that includes 14 known forms, from CLN1 to CLN14. Each form varies in age at symptom onset (at birth, during childhood or adulthood) and is caused by distinct genetic mutations.

CLN2 disease, also known as late infantile neuronal ceroid lipofuscinosis (LINCL), typically starts during early childhood, from ages 2 to 4, and is characterized by seizures, loss of motor skills and cognitive ability, and a reduced life expectancy.

It is caused by mutations in the TPP1 gene, which result in severe reductions in the activity of an enzyme called tripeptidyl peptidase 1 (TPP1). This enzyme is found in cell structures called lysosomes, which digest and recycle different types of molecules, clearing cells of waste and damaged products.

TPP1 specifically breaks down protein fragments, known as peptides, into their individual building blocks (amino acids).

In people with CLN2 disease, a reduction in functional TPP1 enzyme results in the incomplete breakdown of certain peptides leading to their buildup inside cells. This causes damage to tissues throughout the body. Nerve cells, in particular, seem especially vulnerable to such effects.

Progression of CLN2 disease can be slowed or halted by enzyme replacement therapy, which delivers a recombinant (lab-made) functional TPP1 enzyme to patients.

Now, researchers investigated whether it was possible to increase the stability of recombinant TPP1 as a way of prolonging its durability and activity inside lysosomes and potentially increase the therapy’s potency.

A long-lasting enzyme “could potentially provide the basis for a more effective therapy, decreasing the amount of protein required per treatment, and/or increasing the interval between doses, leading to an improved quality of life,” the researchers wrote.

Using several protein engineering methods, the team designed more than 70 different TPP1 versions. Most of the changes were made with the goal of increasing the thermostability of the enzyme, which refers to a protein’s ability to resist irreversible change at high temperatures.

Among the versions created, the best one (R465G) was significantly more resistant to temperature. This version, which consisted in mutating a specific amino acid position known as R465,  had a melting temperature of 64.4°C (147°F) compared to 55.6°C (132°F) for normal TPP1. This means that the enzyme only started to turn over and lose its shape at temperatures of 64.4°C.

The new version also had an extended half-life at a temperature of 60°C (140°F), which means the time it took for its amount to go down by half was of 21.9 minutes, compared to only 5.4 minutes in the original TPP1 enzyme.

It is important to note that all these measurements were done in test tubes, in the absence of cells, commonly called in vitro tests. However, the properties and fate of biological molecules may vary when they are working in vivo, meaning inside cells.

In fact, when the promising version of TPP1 was tested in cells derived from a patient with CLN2 disease, the results were quite different.

The lifetime of R465G and all other variants tested was no longer higher, but rather similar to the original TPP1.

According to researchers, this indicates that “improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability.”

Further experiments provided a likely explanation for such an “intriguing” observation. Proteins working in the lysosome, which include TPP1, withstand greater temperatures, but also appear to be more rapidly renewed and replaced by cells.

The work brings attention to the importance of evaluating new engineered proteins under their natural or physiological settings, in addition to in vitro tests that are typically done. This may be especially significant for lysosomal proteins like those underlying Batten disease.

Ana is a molecular biologist with a passion for discovery and communication. As a science writer, she looks for connecting the public, in particular patients and healthcare providers, with clear and quality information about the latest medical advances. Ana holds a Ph.D. in Biomedical Sciences from the University of Lisbon, Portugal, where she specialized in infectious diseases, epigenetics, and gene expression.
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Ana holds a PhD in Immunology from the University of Lisbon and worked as a postdoctoral researcher at Instituto de Medicina Molecular (iMM) in Lisbon, Portugal. She graduated with a BSc in Genetics from the University of Newcastle and received a Masters in Biomolecular Archaeology from the University of Manchester, England. After leaving the lab to pursue a career in Science Communication, she served as the Director of Science Communication at iMM.
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Ana is a molecular biologist with a passion for discovery and communication. As a science writer, she looks for connecting the public, in particular patients and healthcare providers, with clear and quality information about the latest medical advances. Ana holds a Ph.D. in Biomedical Sciences from the University of Lisbon, Portugal, where she specialized in infectious diseases, epigenetics, and gene expression.
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