Scientists investigate how cells regulatethe proteins that are on the outside of their membranes. This research couldprovide important clues to help develop treatments for Alzheimers disease.

Proteins are the complex molecules that play a critical role in cell biology. They are made up of a chain of amino acids that fold up to form a 3D structure and shape. Each protein has a unique structure that determines its function. Think of proteins like tools, hammers and screwdrivers have different shapes but they are each designed to perform a very specific task. Proteins are made in the cell and sometimes they dont fold correctly or their structural integrity is damaged due to stresses like high temperatures and oxidative stress. This can be dangerous as the abnormal proteins can aggregate and cause damage in the cell. This can lead to a range of illnesses called protein deposition diseases such as Alzheimers disease, Huntingtons disease, and Parkinsons disease. In the case of Alzheimers disease, beta-amyloid peptides are toxic because they stick together and form aggregates. These are often secreted outside the cell and the aggregates then stick to extracellular molecules and form plaques.

Protein quality control systems

In healthy cells, there are protein quality control systems in place to make sure proteins fold correctly. One of these systems uses chaperones, these are proteins whose sole job is to ensure that other proteins are the correct shape. If the target proteins are incorrectly folded one of two things happens, either the chaperone uses energy (in the form of ATP) to refold the protein or the entire protein is degraded.

What happens to the proteins that are secreted and function outside of the cell?

Not all proteins are designed to workwithin the cell. These proteins can be susceptible to damage from stressorssuch as pathological conditions, shear stress, and acidosis and alkalosis (incorrectpH). So what happens when these proteins undergo structural changes? Somechaperone proteins are also secreted, such as clusterin. The problem is thatthere is a thousand times less ATP (energy) outside the cell, meaning that thesechaperones cant refold misfolded proteins. The exact mechanism of how thechaperons work outside the cell is not well understood.

How does clusterin work?

In a recently published article in the Journal of Cell Biology, scientists fromJapan wanted to know how clusterin worked. They speculated that clusterinworked by binding to misfolded proteins and bringing them into contact with thecell. The cell would then engulf the clusterin and misfolded protein complexand degrade it.

New internalisation test developed

The first question the researchers askedwas if clusterin that is bound to a misfolded protein would be able to getinside the cell. They wanted to know if these proteins would be broken downinside a cell structure called the lysosome. Lysosomes are another proteinquality control system and they are responsible for degrading proteins with anacidic environment and digestive enzymes.

The scientists designed a newinternalisation test to help them answer these questions. They used geneticengineering to attach two fluorescent proteins to clusterin (one green and theother red). The red fluorescent protein is resistant to acidic conditions anddigestive enzymes. So if the scientists saw red under the microscope they couldconclude clusterin was indeed internalised and degraded but if they saw onlygreen they could conclude that the clusterin was internalised but not degraded.The scientists combine their internalisation assay with flow cytometry andfluorescent microscopy to show that the Clusterin-misfolded protein complex waspreferentially internalised and degraded with a lysosome. The team used variouscell types (kidney, ovary, lung, bone, liver, and colon) to test their assayand showed this internalisation occurred generally. They then went to show thatthe beta-amyloid peptide was able to bind to clusterin and was subsequentlydegraded in human embryonic kidney cells.

What is the clusterin receptor?

The scientists wanted to know if there wasa receptor on the cell surface that bound to clusterin and triggered theinternalisation of this complex. The team used a genome-wide CRISPR screen tofind out which genes were important for clusterin uptake. They identified 20different genes important for uptake of clusterin and many of them wereimplicated in heparan sulphate (HS) synthesis. To test if HS was the receptorfor clusterin the scientists used genetic engineering to prevent the expressionof various genes involved in the HS pathway. They found that when these geneswere knocked out there was reduced uptake of the clusterin-misfolded proteincomplex. When they restored the gene expression they found this restoredcomplex uptake. This data showed that HS pathway disruption preventedinternalisation of only the clusterin complex and not of endocytosis ingeneral. The scientists then used a pull-down assay to confirm that clusterindirectly binds to HS. These data strongly suggested that HS is the receptor forthe uptake of clusterin-misfolded protein complexes. The group went on to show that HS was theclusterin receptor independent of the misfolded proteins bound. They testedbeta-amyloid peptide and a variety of misfolded red blood cell proteins.

The researchers were able to show anentirely novel mechanism for regulating extracellular proteins called thechaperone- and receptor-mediated extracellular protein degradation (CRED)pathway. Although this is an exciting discovery the involvement of the pathwayin Alzheimers disease requires further investigation. The tests onbeta-amyloid peptides were conducted in kidney cells and not neuronal celllines. There was also no investigation into whether or not this mechanism worksin animals. It seems unlikely that increasing clusterin expression will resultin a treatment for Alzheimers disease as overexpression of this protein hasbeen linked to cancer pathogenesis. The work present is an excitingcontribution to our basic understanding of protein regulation outside the celland is promising progress toward understanding many of the protein depositiondiseases.

Written by Tarryn Bourhill MSc, PhD Candidate.

References:

1 Yerbury, J. J., Stewart, E. M.,Wyatt, A. R. & Wilson, M. R. Quality control of protein folding inextracellular space. EMBO reports 6, 1131-1136 (2005).

2 Jones, S. E.& Jomary, C. Clusterin. Theinternational journal of biochemistry & cell biology 34, 427-431 (2002).

3 Nuutinen, T.,Suuronen, T., Kauppinen, A. & Salminen, A. Clusterin: a forgotten player inAlzheimers disease. Brain researchreviews 61, 89-104 (2009).

4 Wyatt, A. R.,Yerbury, J. J., Ecroyd, H. & Wilson, M. R. Extracellular chaperones andproteostasis. Annual review ofbiochemistry 82, 295-322 (2013).

5 Itakura, E., Chiba, M., Murata, T. & Matsuura, A. Heparan sulfate is a clearance receptor for aberrant extracellular proteins. Journal of Cell Biology 219 (2020).

Image byKonstantin KolosovfromPixabay

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How do cells regulate proteins that are on the outside of their membranes? - Medical News Bulletin