mechanism

Sheets of cells in our bodies called epithelial tissues coat our organs, create wall-like barriers that protect us from bacteria, viruses and other disease-causing invaders. And when potentially harmful gaps between these cells emerge, a molecular switch gets flipped to call the repair crew and fix the leaks.

Using a novel live-imaging technique, University of Michigan researchers have achieved the first direct detection of short-lived leaks in epithelial tissues as they occur. Their microscopy barrier assay also allowed them to discover that the repair mechanism involves local activation of a protein called Rho.

The developed assay could help provide insights into the mechanisms of diseases that target the epithelial barrier – ailments caused by microbes and allergens, as well as various inflammatory states, immune disorders, diabetes and even cancers. And the assay could potentially be used to screen drugs to treat those afflictions, according to the researchers.

The first author is Dr Rachel Stephenson, a research scientist in Professor Ann Miller’s lab who carried out the project for her doctoral dissertation.

“An important unanswered question about epithelial tissues is: How are the junctions between cells able to maintain the biological barrier function even as cells change shape?” Prof Miller said.

In the study, Prof Miller’s team used epithelial cells in live frog embryos, which have cell-cell junctions similar in structure and protein composition to those in human epithelial tissues.

During embryonic development, many epithelial cells work together to bend and fold tissues. Using their new microscopy assay – which is known as ZnUMBA for Zinc-based Ultrasensitive Microscopic Barrier Assay – the researchers studied what happens at the cellular level when epithelial cell-cell junctions are stretched.

They showed that leaks in barrier function happened when cell-cell junctions elongate. But the leaks are short-lived, suggesting there is an active repair mechanism.

On further investigation, the researchers discovered that the repair mechanism involves local activation of the protein Rho, in a sudden burst of activity they dubbed a Rho flare. Rho then activates proteins that contract the junction, repairing it.

“We discovered that cells are normally very proactive when it comes to maintaining the barrier,” Stephenson said. “This repair mechanism happens quickly and is carried out very locally, affecting only a small part of the cell junction, rather than multiple cells or the whole tissue.

“We think that this proactive approach is what gives our cells the flexibility to move and change shape without compromising the barrier function of the tissue. Diseases involving a leaky barrier might be due to a faulty repair mechanism or the cells’ inability to detect leaks and flip the switch.”

Dr Stephenson and other members of Prof Miller’s Lab are now working to determine how the switch gets flipped to turn on Rho at the right time and place and to identify other proteins that are part of the cellular repair crew to plug leaky biological barriers.

The results of the study were published in the journal Developmental Cell.

mRNA

Thousands of databases that include biological data are now publicly available, and include data on gene and protein sequences and detailed measurements of different cellular parameters, such as the exact quantities of all proteins produced and degraded by a given cell in various experimental conditions. 

Brazilian researchers explored mRNA and protein public databases and found out how gene sequence choice can predict different aspects of protein synthesis, such as protein production efficiency.

The genetic information contained in the cell nucleus in the form of DNA is copied in messenger RNAs (mRNAs). Different from the DNA, mRNAs are dynamic and unstable molecules that leave the nucleus and are translated by the ribosomes, the molecular machines able to convert a sequence of nucleotides that make RNA (and DNA) into a sequence of amino acids that form proteins. Each amino acid corresponds to one or more combinations of 3 nucleotides – or codon. Because the same amino acid can be translated from different codons, the genetic code is described as degenerate (or redundant).

Scientists already know that even though the same protein can be produced from alternative gene sequences, some combinations result in higher protein yields. They also know that optimal codons and non-optimal codons can decrease or enhance mRNA degradation, respectively. Different groups have measured mRNA production and degradation rates, but, surprisingly, there are many deviations in the data.

The team of scientists synthesised apparently disparate pieces of data and extended our knowledge of how gene sequence choice can predict different aspects of protein synthesis, such as mRNA stability and production efficiency.

A research group led by Dr Fernando Palhano and Dr Tatiana Domitrovic at the Federal University of Rio de Janeiro used a metric derived from mRNA codon composition to compare the existing data to different cellular parameters. They found that this metric correlated well with protein abundance and protein production efficiency, indicating the most coherent mRNA decay datasets. Their work reiterated that mRNA degradation is somehow connected to protein production efficiency.

“Even proteins needed in high levels under specific conditions, such as stress response, have their gene sequence optimised for efficient translation”, said Dr Palhano.

The researchers identified a group of low abundance proteins coded by a non-optimal subset of codons. The team showed how codon choice is vital not only to guarantee high protein production but also to tune down the output of proteins that should be produced in minimum amounts, such as regulatory proteins.

The amount of protein produced in a cell is crucial to maintaining the organism function – “Many human diseases are caused by inefficient or unbalanced protein production, such as cystic fibrosis and cancer”, said Dr Tatiana. She added that “from a practical perspective, understanding the relationship between the genetic sequence and protein production can have a profound effect both on medicine and bioengineering”.

The authors note that many ‘silent’ DNA mutations, that is, mutations that alter the codon sequence, but not the coded amino acid, can lead to significant modifications on protein production rates, which could lead to disease. By carefully selecting the gene sequence one can finely tune the protein production and boost biotechnological applications of genes and proteins.

The study, published in Nucleic Acids Research, could help the development of new biotechnological applications of genes and proteins.

protein

Genetically modified chickens could produce human proteins in their eggs, offering a cost-effective method of producing certain drugs.

Researchers at University of Edinburgh’s Roslin Institute and Roslin Technologies initially focused on the production of high quality proteins so they could be used in research, but found that the drugs produced worked as well as the proteins produced during existing methods. Using a simple purification method, high quantities of the protein could be recovered from the eggs, with no adverse effect on the chickens themselves.

The team focused on two proteins that were essential to the immune system and also had therapeutic potential, IFNalpha2a and macrophage-CSF. The human protein IFNalpha2a, has powerful anticancer and antiviral effects, while the human and pig versions of the protein called macrophage-CSF, is being developed as a therapy that stimulates damaged tissues to repair themselves.

The team mentioned how this could be a cheap method of producing high quality drugs to be used in research studies, and in the future, possibly in patients. Only three eggs were needed to produce a clinically relevant dose of the drug, and because chicken can lay up to 300 eggs a year, the approach could be more cost effective than methods currently used to produce important drugs.

Professor Helen Sang, of the University of Edinburgh’s Roslin Institute, said: “We are not yet producing medicines for people, but this study shows that chickens are commercially viable for producing proteins suitable for drug discovery studies and other applications in biotechnology.”

Currently, eggs are used to grow viruses that are used as vaccines, such as the flu vaccine. This approach is different in that it encodes the therapeutic proteins in the chicken’s DNA which is then produced as part of the egg white.

Whilst the researchers have not yet produced medicines for use in patients, the study could be adapted to produce many other therapeutic proteins for protein based drugs, such as avastin and herceptin, which are used to treat cancer.

Dr Lissa Herron, Head of the Avian Biopharming Business Unit at Roslin Technologies, said: “We are excited to develop this technology to its full potential, not just for human therapeutics in the future but also in the fields of research and animal health.”

For most of these protein drugs, the best method of producing them with sufficient quality uses mammalian cell culture techniques that are usually expensive with low yields. Other methods with better results often have raised costs, due to the complex purifying systems and processing techniques required.

Dr Ceri Lyn-Adams, Head of Science Strategy, Bioscience for Health with BBSRC, said: “These recent findings provide a promising proof of concept for future drug discovery and potential for developing more economical protein-based drugs.”

The study was published in the journal BMC Technology.