Yeast screens explore the therapeutic potential of chemical rescue.

Anyone who’s worked in a lab knows that sinking feeling of discovering that the temperature of an incubator, carefully set the night before, has crept up high enough to ruin the experiment. While such a mishap usually spells disaster, occasionally, it can lead to an unexpected discovery.

One such revelation was prompted by an uncooperative incubator in the lab of Michael McMurray, a cell biologist at University of Colorado’s Anschutz Medical Campus. McMurray studies the septin family of cytoskeletal proteins, and inside the incubator were plates of yeast with temperature-sensitive septin mutations. The mutant yeast could survive only at mild temperatures, so the incubator was set to a comfortable 27°C.

For this experiment, a chemical called guanidine hydrochloride had been added to some of the plates, to test whether it would stop the mutants from growing at the permissive temperature. When the incubator was found roasting away at more than 30°C, however, all of the yeast should have been dead.

“Amazingly, one of the mutants actually grew,” says McMurray. “The guanidine restored its viability.”

That discovery launched an investigation of how, exactly, guanidine had protected the mutant from normally lethal conditions. In a paper in the September issue of G3: Genes|Genomes|Genetics, Hassell et al. report several mutants that can be rescued by guanidine. They also show that another naturally occurring small molecule can correct an even broader range of mutants.

Guanidine can stand in for a lost arginine

Guanidine’s molecular structure mimics the side chain of the amino acid arginine. Researchers had previously shown that guanidine could restore function to an enzyme that had been mutated to lack an arginine in its active site. But all of this work had been done in vitro. This piqued McMurray’s interest even more. “Arginine is the most commonly mutated amino acid in human disease,” he says. “If guanidine can restore function to arginine mutant proteins, why has no one explored this in living cells?”

McMurray’s team began by testing enzymes in which a single arginine mutation disabled the enzyme enough to cause disease, such as ornithine transcarbamylase (OTC). OTC deficiency is an inherited metabolic disease that leads to a buildup of toxic ammonia in the body. The researchers created yeast with the same OTC arginine mutation that causes the human disease, making the yeast unable to grow without nutritional supplementation. Adding guanidine hydrochloride to the growth media restored some of the lost enzyme function.

“The effect was pretty small,” McMurray says. “It wasn’t a full rescue, but it was something.”

Next, the researchers decided to broaden their investigation. Instead of testing candidate enzymes, they screened hundreds of yeast mutants to see if guanidine restored function to any of them. “We decided to let the cells tell us what would work best,” McMurray says. “That’s when things started to get interesting.”

The screen uncovered 11 new candidates, the most interesting of which was an arginine mutant of actin, another cytoskeletal protein. “It just so happens that arginine is also mutated in human cardiac beta actin, and that mutation causes disease,” McMurray says.

As an ATPase, actin is technically an enzyme, but the arginine mutation is far from the active site, and guanidine isn’t restoring catalytic activity per se. Instead, McMurray says, it’s helping the protein fold into its proper 3D shape. “All proteins have to fold,” McMurray says. Protein folding results from chemical interactions between the side chains of various amino acids. “To rescue the mutant, the guanidine just has to be able to fix what’s missing and restore the folding.”

The idea of rescuing mutants by restoring proper protein folding led them to investigate other chemicals that can influence protein folding. “From a biological perspective, what are other cases in nature in which organisms have to deal with alterations in protein folding?” McMurray says. “Then we thought of sea creatures — sharks and rays.”

Moving beyond guanidine

Because they live in saltwater, sharks maintain high concentrations of urea in their bodies to keep from losing water through osmosis. Urea, however, is toxic to proteins, and causes them to unfold. To counteract the urea, these animals also have high levels of a chemical called trimethylamine oxide (TMAO), which promotes protein folding.

Does the shark’s protein protection trick work in other contexts? To follow up, research assistant Daniel Hassell screened yeast mutants using TMAO. He turned up hundreds of mutants that were rescued by the molecule. The genes and mutant types were all very different from each other, suggesting that TMAO has a more general stabilizing effect rather than specifically replacing a particular amino acid. This broad effect suggests a potential role for the molecule in synthetic biology, as a way to design proteins with an on/off switch system.

For its part, guanidine is already FDA-approved as a treatment for an inherited autoimmune disorder called Lambert-Eaton myasthenic syndrome. McMurray remains curious about whether it has the potential to treat other genetic diseases.

“That would be my ultimate hope, that someone would be inspired by our work to try it in an animal model or the clinic,” McMurray says.

CITATION

Chemical rescue of mutant proteins in living Saccharomyces cerevisiae cells by naturally occurring small molecules
Daniel S Hassell, Marc G Steingeisser, Ashley S Denney, Courtney R Johnson, Michael A McMurray
G3 Genes|Genomes|Genetics 2021; jkab252
https://doi.org/10.1093/g3journal/jkab252

An evolutionary approach outperforms a design approach in modeling protein sequence variation.

Over generations, evolution shapes proteins, leading to variation in their amino acid sequences both between and within species. Despite our ever-increasing knowledge of the physical constraints that guide protein structure, advanced modeling techniques don’t capture the site-specific variability observed in natural proteins. Bafflingly, complex models that account for physical influences on the positions of all atoms in a protein often perform worse than elementary models at recapitulating natural proteins’ variability.

In GENETICS, Jiang, Teufel, et al. provide evidence for a possible explanation: advanced modeling techniques don’t take into account the order of the steps by which protein sequences change. A popular protein-design suite called RosettaDesign, for example, deletes the amino acid side chains from a template structure, leaving only the peptide backbone, and then replaces them with new side chains all at once. After this dramatic step, additional changes are made to maximize the protein’s calculated stability.

Evolution works very differently. Sequence changes are usually made one amino acid residue at a time, meaning the effect of each alteration depends on how it fits with the existing sequence. Whether a sequence change will be fixed or lost depends on how it affects fitness, which is partly influenced by how it impacts protein stability—variations that make proteins prone to unfolding are typically not favorable.

When the group tested their new algorithm, which functions more similarly to evolution, on the same natural proteins, they found that its effects were different in several ways from those of RosettaDesign. In almost every case, their evolved sequences resembled natural ones more than designed proteins’ sequences did. This might not, at first, seem surprising, since the designed proteins started with a completely stripped peptide backbone and thus shouldn’t have been influenced as much by the natural starting sequences—but the researchers found that this wasn’t the reason. Even when they used a designed sequence as a template, the evolution-based simulation created sequences that better mimicked natural ones.

In protein design, the ability to build proteins with sequences unlike natural ones could be interpreted as a positive thing since it’s conceivable that these proteins would have a wider range of properties than those of proteins found in the wild. But despite the fact that the designed sequences had diverged more from the starting sequences, their site-specific variability was lower than that of the evolved sequences. This implies that, even though a greater number of sites were altered in the designed sequences, the changes were restricted to a smaller set of amino acid residues.

RosettaDesign and similarly sophisticated software have facilitated major advances in protein design, such as developing new enzymes and previously unseen protein folds, and Jiang, Tuefel, et al.’s findings don’t make these types of software obsolete. Different computational techniques fill different niches, and they evolve just as proteins do, with new variants continuously under development. By tweaking existing methods and studying the effects of new algorithms, we can improve how we use these techniques—and perhaps develop new ones with even better fitness than their ancestors had.

CITATION:

Beyond Thermodynamic Constraints: Evolutionary Sampling Generates Realistic Protein Sequence Variation
Qian Jiang, Ashley I. Teufel, Eleisha L. Jackson, Claus O. Wilke
GENETICS 2018 208: 1387-1395; https://doi.org/10.1534/genetics.118.300699
http://www.genetics.org/content/208/4/1387

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Wild yeast aren’t picky about their mates. For Saccharomyces cerevisiae, setting the mood is as simple as providing an abundant supply of nutrients, which prompts each yeast cell to search for another of the opposite mating type. If a lonesome yeast cell can’t find a suitable partner, it’s no problem—it can alternate between mating types, if needed, each cell division.

In contrast, lab yeast can’t pull off this trick; they carry deletions or other disabling mutations in parts of their genome needed for mating-type switching. Their inability to switch helps prevent the yeast from inconveniently changing types in the middle of an experiment, but it also creates a lot of extra work for geneticists when they’re trying to construct new strains while maintaining strict isogenicity. Now, researchers have developed a CRISPR/Cas9-based method to rapidly and efficiently switch mating types in a single cotransformation.

Mating-type switching is essential for constructing strains that are genetically identical except for the mating-type locus. These isogenic haploid strains are useful because they can mate with each other to form a diploid cell with two identical copies of the genome. And since diploid cells differ in many ways from their haploid equivalents, it’s often important to study genetic phenomena in both.

The ability to induce yeast to switch mating types is a particularly vital tool in the assembly of Sc2.0: a completely synthetic yeast genome. Creating and subsequently studying this genome could answer deep questions about biology—like how far we can go in modifying a eukaryote’s genome while still keeping the organism alive.

Completing this massive project, which is led by some of the authors of this study along with their collaborators around the world, will require many mating-type switches to consolidate the final synthetic chromosomes in a single strain. Switching the mating types of lab strains used to require a process with many steps, including manual dissection of spores using an exceptionally fine needle under a microscope. Having to rely on this classic but laborious process would slow the progress of the Sc 2.0 project, so the faster and easier CRISPR-Cas9-based technique offers a major advantage.

Although construction of the synthetic yeast genome isn’t yet in its final stages, many other yeast geneticists stand to benefit from the group’s speedy new technique, which would also be useful for building isogenic pairs of strains and even building isogenic tetraploids. Plus, freeing up geneticists from tedious tasks gives them more time to dream up schemes for extraordinary advances—like a whole genome built from scratch.

CITATION:

Xie, Z.; Mitchell, L.; Liu, H.; Li, B.; Liu, D.; Agmon, N.; Wu, Y.; Li, X.; Zhou, X.; Li, B.; Xiao, W.; Ding, M.; Wang, Y.; Yuan, Y.; Boeke, J. Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of Saccharomyces cerevisiae.
G3, 8(1), 173-183.
DOI: 10.1534/g3.117.300347
http://www.g3journal.org/content/8/1/173

While the science behind the synthetic yeast genome project is cutting edge, the ethical questions surrounding it aren’t new.

The scientists of the Sc2.0 project have a goal that sounds akin to science fiction – they’re working toward building a completely synthetic yeast genome. This new strain of Saccharomyces cerevisiae, affectionately named Sc2.0, will be used to study fundamental properties of chromosomes, genome organization, gene content, function of RNA splicing, the extent to which small RNAs play a role in yeast biology, the distinction between prokaryotes and eukaryotes, and questions relating to genome structure and evolution. In addition to the hard science, the project faces a series of challenges in setting ethical boundaries, educating policy makers and the public, and building a governance plan.

Debra Mathews

Genes to Genomes spoke with Debra J. H. Mathews, PhD, MA, a geneticist and ethicist at the Johns Hopkins Berman Institute of Bioethics. She played an important role in developing the Sc2.0 Statement of Ethics and Governance published in August in GENETICS. We asked about her views on the education, governance, and scientific goals of the project. She asserts that the ethical questions facing synthetic biology scientists are not new issues; they’re new combinations of existing questions.

Genes to Genomes: What is your role in the Sc2.0 project?

Debra Mathews: I’m a geneticist by training, and I do ethics and science policy research now. My role on the project is as an ethics person.

I worked on developing the statement of principles with a terrific graduate student, Anna Sliva, and in the current phase of the project, I’m working on developing a massive open online course (MOOC) on ethics and policy in biology. Colleagues of mine who collaborate on the project are putting together workshops on ethics and policy related to synthetic biology and the yeast project.

G2G: What, in your mind, are the most important goals for educating the public on the Sc2.0 project?

DM: I tend to think more about what are the conversations we can have with the public rather than what we can tell them. I think transparency is critically important. We need to be out there talking about work in language that normal people can understand – translating jargon into words everyone knows.

I think having conversations with the public is critically important. It shouldn’t be a one-way street. We need to hear from them; what they’re thinking about, what they want science to do, what risks and benefits interest them.

G2G: What would your ideal role of government oversight for synthetic DNA work be?

DM: There are lots of kinds of governance that aren’t government; governance of science isn’t necessarily driven by government regulation.

Synthetic biology is really interesting because it is so diverse. I don’t think Sc2.0 is working towards a governance model per se. I think that’s something many people in the field are thinking about, but it’s not something any individual project can take on. With any very new area of science not captured by existing oversight mechanisms, we have to figure it out. This is another case where we have to figure it out, like we did in the 1970s with Asilomar*.

It’s highly unlikely that that we get to a place where there’s one committee or agency in charge of synthetic biology.  It’s more likely to be a complicated and complementary set of governance approaches with government, professional societies, and institutions all working together.

G2G: What are your most exciting goals for synthetic biology?

DM: I think the vast majority of the benefits are going to be basic science benefits. It’s going to dramatically improve our understanding of how simple organisms and gene networks work. That will help us answer other questions. Those are the things I’m more interested in; it’s that on-the-ground work, paying dividends now, furthering basic science now. It’s the small day-to-day work that’ll eventually help us address the bigger things like disease and the environment.

G2G: So it’s more a part of the ongoing conversation with the public about the importance of investing time and effort into basic science research?

DM: It’s absolutely part of that conversation, and it’s a conversation we tend to have very poorly. A more important conversation is “this is how science works.” Science is incremental; it’s slow. We occasionally have leaps, but it takes time because we’re learning. Every time we do an experiment, we’re learning, and we build and build and build. Eventually we get to a result that everyone can see is worthwhile.

It’s hard to have the “how science works” conversation with the public in a way that isn’t deadly boring. I don’t understand why that’s the case. If you get any scientist talking about their science, they get so excited and into it and passionate. Somehow we’re not able to translate that excitement into a conversation with the public. We need a Neil deGrasse Tyson for biology!

*The Asilomar Conference on Recombinant DNA was organized by Paul Berg in 1975. Scientists came together to discuss the progress and potential biohazards of emerging recombinant DNA work.

CITATIONS

Berg, P., Baltimore, D., Brenner, S., Roblin, R.O. III, Singer, M.F. 1975. Summary Statement of the Asilomar Conference on Recombinant DNA Molecules. PNAS, 72(6): 1981-4. http://www.pnas.org/content/72/6/1981.full.pdf

Silva, A., Yang, H., Boeke, J.D., Mathews, D.J.H. 2015. Freedom and Responsibility in Synthetic Genomics: The Synthetic Yeast Project. Genetics, 200(4): 1021-8. doi:10.1534/genetics.115.176370 http://www.genetics.org/content/200/4/1021.full