New & Noteworthy

How Yeast May One Day Help Michael J. Fox

October 31, 2013

Folks, yeast has been on a roll lately with regard to helping to understand and finding treatments for human disease. Last week we talked about how synthetic lethal screens may find new, previously unrecognized druggable targets for cancer. And this week it is Parkinson’s disease.

Now of course yeast can’t get the traditional sort of Parkinson’s disease …it doesn’t have a brain.  But it shares enough biology with us that when it expresses a mutant version of α-synuclein (α-syn) that is known to greatly increase a person’s risk for developing Parkinson’s disease, the yeast cell shows many of the same phenotypes as a diseased neuron.  The yeast acts as a stand-in for the neuron.

In a new study out in Science, Tardiff and coworkers use this yeast model to identify a heretofore unknown target for Parkinson’s disease in a sort of reverse engineering process. They screened around 190,000 compounds and looked for those that rescued toxicity in this yeast model. They found one significant hit, an N-aryl-benzimidazole (NAB) compound. Working backwards from this hit they identified its target as Rsp5p, a Nedd4 E3 ubiquitin ligase. 

The authors then went on to confirm this finding in C. elegans and rat neuron models where this compound halted and even managed to reverse neuronal damage. And for the coup de grace, Chung and coworkers showed in a companion paper that the compound worked in human neurons too. But not just any human neurons.

The authors used two sets of neurons derived from induced pluripotent cells from a single patient.  One set of neurons had a mutation in the α-syn gene which is known to put patients at a high risk of Parkinson’s disease-induced dementia.  The other set had the mutation corrected.  The compound they identified in yeast reversed some of the effects in the neurons with the α-syn mutation without significantly affecting the corrected neurons.  Wow.

What makes this even more exciting is that many people thought you couldn’t target α-syn with a small molecule. But as the studies here show, you can target an E3 ubiquitin ligase that can overcome the effects of mutant α-syn.  It took an unbiased screen in yeast to reveal a target that would have taken much, much longer to find in human cells. 

The mutant α-syn protein ends up in inclusion bodies that disrupt endosomal traffic in the cell.  The NAB compound that the authors discovered restored endosomal transport and greatly decreased the numbers of these inclusion bodies.  Juicing up Rsp5 seemed to clear out the mutant protein.

The next steps are those usually associated with finding a lead compound—chemical modification to make it safer and more effective, testing in clinical trial and then, if everything goes well, helping patients with Parkinson’s disease.  And that may not be all.

The α-syn protein isn’t just involved in Parkinson’s disease.  The dementia associated with this protein is part of a larger group of disorders called dementia with Lewy bodies that affects around 1.3 million people in the US.  If everything goes according to plan, many of these patients may one day thank yeast for their treatment.

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

Using Yeast to Find Better Cancer Treatments

October 24, 2013

Current cancer treatments are a lot like trying to destroy a particular red plate by letting a bull loose in a china shop.  Yes, the plate is eventually smashed, but the collateral damage is pretty severe.

Ideally we would want something a bit more discriminating than an enraged bull.  We might want an assassin that can fire a single bullet that destroys that red plate. 

One way to identify the assassin that can selectively find and destroy cancer cells is by taking advantage of the idea of synthetic lethal mutations.  “Synthetic lethal” is a genetic term that sounds a lot more complicated than it really is.  Basically the idea is that mutating certain pairs of genes kills a cell, although mutating each gene by itself has little or no effect.

A synthetic lethal strategy seems tailor made for cancer treatments.  After all, a big part of what happens when a cell becomes cancerous is that it undergoes a series of mutations.  If scientists can find and target these mutated genes’ synthetically lethal partners, then the cancer cell will die but normal cells will not.

This is just what Deshpande and coworkers set out to do in a new study in the journal Cancer Research.  They first scanned a previous screen that looked at 5.4 million pairwise interactions in the yeast S. cerevisiae to find the best synthetic lethal pairs. They found 116,000 pairs that significantly affected cell growth only if both genes in the pair were mutated.

A deeper look into the data revealed that 24,000 of these pairs had human orthologs for both genes. In 500 of these pairs, at least one of the partner genes had been shown to be mutated in certain cancers. Using a strict set of criteria (such as the strength and reproducibility of the synthetic lethal effect, and the presence of clear one-to-one orthology between yeast and human), the authors narrowed these 500 down to 21 pairs that they decided to study in mammalian cell lines.

When the authors knocked down the expression of both genes in these 21 gene pairs in a mammalian cell line, they found six that significantly affected growth.  They focused the rest of the work on the strongest two pairs, SMARCB1/PMSA4 and ASPSCR1/PSMC2.  These mammalian gene pairs correspond to the yeast orthologs SNF5/PRE9 and UBX4/RPT1, respectively.

The authors identified two separate cancer cell lines that harbored mutated versions of the SMARCB1 gene.  When this gene’s synthetic lethal partner, PMSA4, was downregulated in these cancer lines, the growth of each cell line was severely compromised. The same was not true for a cell line that had a wild type version of SMARCB1—this cell line was not affected by downregulating PMSA4.  The authors used a synthetic lethal screen in yeast to identify a new cancer target which when downregulated selectively killed the cancer without killing “normal” cells.

This proof of principle set of experiments shows how the humble yeast may one day speed up the process of finding cancer treatments without all those nasty side effects (like vomiting, hair loss, anemia and so on).  Yeast screens can first be used to identify target genes and then perhaps also to find small molecules that affect the activity of those gene products.  Yeast may one day tame the raging bull in a china shop that is current cancer treatments.

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

Memorial Gathering for Dr. Fred Sherman

October 23, 2013

A memorial gathering in memory of Fred Sherman will be held at 10:00 am on Friday, December 6, 2013. The gathering will be held in the Ryan Case Methods Room (Rm #1-9576) of the University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester NY 14642. This event will be a celebration of the life and science of Fred, comprised of reminiscences about Fred by some who knew him well, followed by an opportunity for any guest to say a few words about Fred. For more information, contact Mark Dumont (Mark_Dumont@urmc.rochester.edu), Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642 (Phone 585-275-2466).

A GLAMorous New Role for Prefoldin

October 17, 2013

The prefoldin complex seemed like an ordinary housekeeper. It sat in the cytoplasm and folded protein after protein, just as Cinderella spent her days folding laundry for her stepsisters.

In the old story, the handsome prince searched the kingdom for a girl whose foot would fit the glass slipper. Using this crude screen, he finally found Cinderella and revealed her to be the true princess that she was.

In a new study, Millán-Zambrano and coworkers did essentially the same thing for the prefoldin complex.  They searched the genome of S. cerevisiae for new mutations that would affect transcription elongation. They found the prefoldin complex subunit PFD1 and went on to establish that in addition to its humdrum cytoplasmic role, prefoldin has a surprising and glamorous role in the nucleus facilitating transcriptional elongation.

The researchers decided to cast a wide net in their search for genes with previously undiscovered roles in transcriptional elongation. Their group had already worked out the GLAM assay (Gene Length-dependent Accumulation of mRNA), which can uncover elongation defects.

The assay uses two different reporter gene constructs that both encode Pho5p, an acid phosphatase. One generates an mRNA of average length, while the other generates an unusually long mRNA when fully transcribed. The acid phosphatase activity of Pho5p is simple to measure, and correlates well with abundance of its mRNA. If there is a problem with transcriptional elongation in a particular mutant strain, there will be much less phosphatase activity generated from the longer form than from the shorter one. So the ratio of the two gives a good indication of how well elongation is working in that mutant strain.

Millán-Zambrano and coworkers used this assay to screen the genome-wide collection of viable deletion mutants. They came up with mutations in lots of genes that were already known to affect transcriptional elongation, confirming that the assay was working. They also found some genes that hadn’t been shown to be involved in elongation before.  One of these was PFD1, a gene encoding a subunit of the prefoldin complex. As this deletion had one of the most significant effects on elongation, they decided to investigate it further.

Prefoldin is a non-essential complex made of six subunits that helps to fold proteins in the cytoplasm as they are translated. The authors tested mutants lacking the other subunits and found that most of them also had transcriptional elongation defects in the GLAM assay, although none quite as strong as the pfd1 mutant.

Since prefoldin is important in folding microtubules and actin filaments, the researchers wondered whether the GLAM assay result was the indirect effect of cytoskeletal defects. They were able to rule this out by showing that drugs that destabilize the cytoskeleton didn’t affect the GLAM ratio in wild-type cells, and that mutations in prefoldin subunits didn’t confer strong sensitivity to those drugs.

If prefoldin has a role in transcription, it would obviously need to get inside the nucleus. It had previously been seen in the cytoplasm, but when the authors took another look, they found it in the nucleus as well. Furthermore, Pfd1p was bound to the chromatin of actively transcribed genes! And besides its effect on transcription elongation, the pfd1 mutant has lower levels of RNA polymerase II occupancy and abnormal patterns of histone binding on transcribed genes.

There’s still a lot of work to be done to figure out exactly what prefoldin is doing during transcriptional elongation. Right now, the evidence points to its involvement in evicting histones from genes in order to expose them for transcription. But even before all the details of this story are worked out, this is a good reminder never to assume that an everyday housekeeper is only that.

With the right screen we can find new and exciting things about the most humdrum of characters. A glass slipper screen revealed the princess under that apron and chimney soot. And a GLAM assay revealed the sexy, exciting transcription elongation factor that is prefoldin.

by Maria Costanzo, Ph.D., Senior Biocurator, SGD