New & Noteworthy

Identifying the Unstable

November 08, 2012

One of the many stumbling blocks in finding better treatments for genetic diseases is figuring out the cause of the disease.  These days, this doesn’t necessarily mean simply identifying the gene with the mutation.  No, nowadays it can mean figuring out what each specific mutation does to the gene it damages.

See, many genetic diseases are not caused by single mutations.  Instead, lots of different mutations can all damage the same gene in different ways.  And each class of mutation may require different treatments.

It’s easy to tell this bridge is unstable. It is a lot harder to tell with proteins.

Cystic fibrosis (CF) is a great example of this.  While most cases of this ultimately fatal disease are caused by mutations in the CFTR gene, not every mutation does the same thing to the CFTR protein.  Because of this, scientists have found different drugs to treat people with different classes of CFTR mutations.

So one drug, Ivacaftor, targets CFTR proteins that can’t open up as well as they should, while another investigative drug, PTC124, targets prematurely stopped CFTR proteins.  Each only treats a specific subset of CF patients who have the correct CFTR mutation.

All of this screams out for a quick and easy assay to figure out how a mutation actually disables a certain protein.  And this is where a new study by Pittman and coworkers just published in the journal GENETICS can help.

The authors have come up with a sensitive in vivo assay in S. cerevisiae that allows scientists to quickly identify mutations that lead to unstable proteins.  This kind of instability isn’t rare in human disease either.  Some of the more famous examples include a kidney disease called primary hyperoxaluria type 1 (PH1), Lou Gehrig’s disease (ALS), Parkinson’s disease, spinal muscular atrophy (SMA), and even some forms of cancer.

The assay basically inserts wild type and mutant versions of the gene of interest into the middle of the mouse dihydrofolate reductase (DHFR) gene, individually adds these chimeric genes to yeast lacking DHFR, and then measures growth rates.  The idea is that if the mutation leads to instability, the DHFR chimeric protein will be unstable too and the yeast will show growth defects under certain conditions.  This is just what they found.

Initially they focused on a gene involved in PH1, the AGT gene encoding alanine: glyoxylate aminotransferase.  They were able to show that disease causing mutations known to affect protein stability affected growth in this assay.  Not only that, but there was a strong correlation between growth and level of protein stability.  In other words, the more unstable the protein, the more severe the growth defect.

They then expanded their assay beyond known AGT mutations.  First they were able to identify a subset of disease-causing AGT mutations as affecting the stability of the AGT protein.  But the assay ran into trouble when they switched to the more stable SOD1 protein.  This protein, which is involved in most cases of ALS, is so stable that mutations that destabilized it were invisible in the assay.  The authors solved this problem by introducing a mutation into DHFR that destabilized it.  Now they could identify mutants that destabilized SOD1.

As a final step, they used their assay to screen a library of stabilizing compounds to identify those that specifically stabilized their mutant proteins.  Unfortunately, in this first attempt they only found compounds that stabilize DHFR, but the assay has the potential to find drugs that stabilize disease-related proteins as well.

Whether or not that potential is realized, this technique should still be a very useful way to determine whether a mutation affects protein stability.  Then, when drugs that stabilize the protein have been found, using this or other screens, doctors will know which patients can be helped by these compounds.  And this will be a boon for scientists and patients alike.

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight Yeast and Human Disease

Tags: yeast model for human disease , protein stability , Saccharomyces cerevisiae