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The stability of lysosomal proteins when tested in the lab is not equivalent to that seen in living cells, according to one study. This information should be taken into account when designing new versions of proteins to improve the effectiveness of enzyme replacement therapy for Batten disease.

Study results ”,Thermal stability of lysosomal proteins is not correlated with cell half-life: overall observations and 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 diseases that includes 14 known forms, from CLN1 to CLN14. Each form varies with age at onset of symptoms (birth, childhood, or adulthood) and is caused by distinct genetic mutations.

CLN2 The disease, also known as late infantile neuronal ceroid lipofuscinosis (LINCL), usually begins in infancy, between 2 and 4 years of age, and is characterized by seizures, loss of motor and cognitive skills, and life expectancy. scaled down.

It is caused by mutations in the TPP1 uncomfortable, which cause 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, ridding cells of wastes and damaged products.

TPP1 specifically breaks down fragments of proteins, called peptides, into their individual building blocks (amino acids).

In people with CLN2 disease, a reduction in the functional enzyme TPP1 results in the incomplete degradation of some peptides, resulting in their accumulation inside cells. This causes tissue damage throughout the body. Nerve cells, in particular, seem particularly vulnerable to such effects.

The progression of CLN2 disease can be slowed or stopped by enzyme replacement therapy, which delivers a functional recombinant TPP1 enzyme (made in the laboratory) to patients.

Now, researchers are investigating whether it is possible to increase the stability of recombinant TPP1 in order to prolong its durability and activity inside lysosomes and potentially increase the potency of the therapy.

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

Using multiple protein engineering methods, the team designed over 70 different versions of TPP1. Most of the changes were made in an effort to increase the thermostability of the enzyme, which refers to a protein’s ability to resist irreversible changes at high temperatures.

Among the versions created, the best (R465G) was significantly more resistant to temperature. This version, which involved mutating a specific amino acid position known as R465, had a melting temperature of 64.4 ° C (147 ° F) versus 55.6 ° C (132 ° F) for the 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), meaning the time it took for its quantity to halve was 21.9 minutes, compared to just 5.4 minutes in the original. Enzyme TPP1.

It is important to note that all these measurements were carried out in test tubes, in the absence of cells, commonly called in vitro testing. However, the properties and fate of biological molecules can vary when they work. in vivo, that is, inside the 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 lifespan of the R465G and all other variants tested was no longer longer, but rather similar to that of the original TPP1.

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

Other experiments have provided a probable explanation for such an “intriguing” sighting. Proteins acting in the lysosome, including TPP1, resist higher temperatures, but also seem to renew themselves more quickly and be replaced by cells.

The work draws attention to the importance of evaluating new proteins modified in their natural or physiological context, in addition to in vitro tests that are usually performed. This can be especially important for lysosomal proteins like those that underlie Batten disease.

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