New & Noteworthy
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.
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).
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.
October 10, 2013
Cheap and easy genome sequencing has been both a blessing and a curse. We are able to find an incredible wealth of variation, but for the most part we have no easy way to tell whether a difference might contribute to a disease or not.
The poster child for this problem is autism. Lots of genome wide association studies (GWAS) have been done and lots of rare variants in lots of different genes have been found – unfortunately, way too many to pick out the ones that really matter.
Luckily our friend yeast can help. Various researchers have identified a number of variants in the human cation/proton antiporter gene NHE9 that associate with autism. In a new study, Kondapalli and coworkers used the NHE9 ortholog NHX1 from S. cerevisiae as an initial screen to identify which variants impact the activity of the NHE9 protein. They found that two of the three mutations they looked at compromised the activity of yeast Nhx1p.
They then set out to confirm these results in mammalian cells. When they looked at protein activity in glial cells, they found that all three mutations compromised the activity of NHE9. This is obviously different from what they found in yeast.
Now this doesn’t mean that yeast is useless for this approach (God forbid!). No, instead it means that it is probably only useful for a subset of autism mutations. Kondapalli and coworkers had suspected this, but apparently the subset is smaller than they initially thought.
The first thing they did was to generate a rough three dimensional map of the NHE9 protein in order to see which parts the two proteins shared. The idea is that they could then do a quick screen in yeast with mutations that affect the shared structure.
While the structure of NHE9 has not been solved, we do have the structure of its distant bacterial relative, NhaA. Kondapalli and coworkers aligned the two along with the yeast ortholog Nhx1p and identified conserved regions.
Three of the NHE9 mutations associated with autism—V176I, L236S, and S438P—were all predicted to be in shared, membrane-spanning parts of the protein. The researchers introduced the equivalent mutations into NHX1—V167I, I222S, and A438P.
A yeast deleted for NHX1 grows poorly in high salt and low pH and also has increased sensitivity to hygromycin B, as compared to a yeast with a functioning NHX1. Two of the mutant genes, carrying A438P or I222S, failed to rescue these growth defects. The other mutant gene, with the V167I change, worked as well as wild type NHX1 at rescuing the yeast. So at least in yeast, two of the three mutations appear to impact protein activity.
The next step was to see if the same was true in mammals. Easier said than done! Ideally they would want to investigate whether these mutations affected the protein in the cells where NHE9 is usually active. Too bad no one knows this protein’s natural habitat. This is why the researchers starting slicing mouse brains to figure out when and where the protein is expressed.
While we don’t have time or space to go into all the details here, Kondapalli and coworkers found that when and where in the brain NHE9 was expressed made sense as far as a possible contribution to autism. They also found that glial cells had about 1.2 fold more NHE9 transcripts than did neuronal cells. They therefore did their assays of protein activity in a type of glial cells called astrocytes.
While they couldn’t completely knock out NHE9 in mouse astrocytes, they were able to knock down its expression by over 80%. When they added back the mutant NHE9 genes, they found that all three failed to mimic the effect of adding back wild type NHE9 to these cells. This is different than what they found in yeast, where only two of the mutations impacted protein activity.
When they went back to their 3D model, they saw that the mutation that differed, V167I, affected a less defined part of the structure. This points to the fact that for the quick yeast screen to work, they need to be looking at parts of the protein where the structure is shared between the yeast and the human version. In a perfect world they would have had crystal structures of each to work off of instead of having to kludge together a model.
In any event, this is the first step towards validating yeast as a quick screen for identifying mutations that can impact protein activity and so are good candidates for being involved in disease. Yeast may help scientists separate the wheat from the chaff of GWAS and so help figure out how diseases happen and maybe help find treatments or even cures. Well done yeast.
October 7, 2013
Congratulations to Randy Schekman, James Rothman, and Thomas Südhof, who have been awarded the 2013 Nobel Prize in Physiology or Medicine for their work in understanding how the cell organizes its transport system. Randy Schekman used the awesome power of yeast to identify and characterize genes required for vesicle traffic. James Rothman characterized these and other proteins in mammalian cells, and Thomas Südhof showed the critical role of vesicle trafficking in nerve cells. You can read summaries of their Nobel winning work at Nature, The Scientist, and The New York Times, or search SGD to see how each of these researchers has used our model organism in their research: Randy Schekman, James Rothman, Thomas Südhof.