New & Noteworthy

(Almost) All Hands on Deck for Calcineurin

July 31, 2018

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If there was a World Cup soccer championship for cellular proteins, it’s a pretty sure bet that calcineurin wouldn’t make the team. That’s because this protein is one of those players that just can’t help but use their hands! And as pretty much everyone knows, that’s a big no-no for soccer players (except goalies, of course).

Conserved across virtually all eukaryotic organisms — from plants and protozoa to fungi and humans — calcineurin is a very abundant calcium-binding protein. In fact, it’s so abundant that it makes up 1% of the total protein content in a cow’s brain!

But what does this ubiquitous protein do? Well, it’s “merely” responsible for regulating many diverse, fundamental life processes… things such as fertilization, development, behavior, life span, responses to environmental cues, immune responses, cell death, and so on.

For eukaryotes, certain environmental and developmental cues, such as hormones or nutrient availability, can initially signal their presence by causing a change in the cell’s internal calcium levels. Calcineurin helps detect these calcium level changes and then passes the signal on in a chain of events.

Calcineurin is a calcium-regulated protein phosphatase, meaning that when it is activated by calcium level changes, it removes a phosphate group(s) from other proteins, particularly transcription factors. When these transcription factors have their phosphate group(s) chopped off by calcineurin, they travel into the cell’s nucleus and turn on a specific set of genes needed to make the cell respond appropriately to the original environmental or developmental cue.

Calcineurin is actually made up of two different proteins that bind together. One is a catalytic protein subunit (called CNA) and the other is a regulatory subunit (CNB). In our yeast friend Saccharomyces cerevisiae, the regulatory CNB protein subunit of the calcineurin complex is encoded by the CNB1 gene.

The Cnb1 protein contains a set of four almost-identical short amino acid domains that are conserved in the CNB proteins of all other organisms. These motifs are called “EF hand” domains because each of them looks like a spread thumb and forefinger of a human hand. And crucially, each “EF hand” can grab and hold onto calcium ions (Ca²⁺). The four EF hands of the Cnb1 protein are called EF1, EF2, EF3 and EF4, respectively.

But besides holding onto calcium ions, what else do these hands do? Why are there 4 of them? Are each of the hands doing the same thing? Does the right hand know what the left (or the middle, or the other middle) hand is doing?

Well, in the July issue of GENETICS, Connolly and co-workers describe experiments they’ve performed that help figure out some of the answers to these questions for the yeast Cnb1 protein.

The authors made a series of 4 different mutant CNB1 genes, each one having a disabling mutation in one of the 4 EF hands so that the hand can’t grab calcium ions any more. They put these mutant-handed CNB1 genes into yeast cells that had their normal CNB1 gene completely removed. The yeast cell thus ends up depending on a Cnb1 protein with one mutant hand and three functional hands.  In a way, they are making Cnb1p have one of its 4 hands tied behind its back, and then seeing how well it can do its job!

How did they test how well each of the mutant-handed proteins works? Remember that the Cnb1 protein is the regulatory subunit of calcineurin, and calcineurin activates a transcription factor by dephosphorylating it. In yeast, the transcription factor regulated by calcineurin is encoded by the CRZ1 gene. The Crz1 protein recognizes and binds a certain DNA sequence (called CDRE) located just upstream of each one of its target genes and turns these genes on, ultimately changing the yeast cell’s behavior during calcium signaling. The authors put a special reporter gene into their yeast strains; this reporter gene has the special CDRE DNA sequence fused to an often-used bacterial gene called lacZ. The amount of lacZ protein produced by the yeast cells (which positively correlates with calcineurin function) can be sensitively monitored by an easy test tube assay.

Using this test tube assay, Connolly and co-workers tested how well each of the EF hand mutants worked. First they tested how well each could turn on the CRZ1-regulated genes, and also how well the mutant proteins detected calcium. Then they also tested how well each of the mutant-handed Cnb1 proteins worked in high salt environments, during the mating response, under oxidative stress conditions, and even in the presence of immunosuppressive drugs! Why the latter? Calcineurin is a target of immunosuppressive drugs, which are used when people get organ transplants to stop their own bodies from attacking the “foreign” organ. Yeast calcineurin is so similar to human calcineurin that it too is affected by these drugs!

The results were clear (well, actually yellow in the assay)—in all cases, the Cnb1 protein was able to have its EF4 hand disabled and still function perfectly or almost as well as the intact Cnb1 protein! But whenever one of the other EF hands was disabled, the function of calcineurin suffered, and this was true for each of the many ways it was tested, from salt to mating to immunosuppressive drugs.

It appears that when any of the useful hands (EF1, 2 or 3) were mutated, it causes Cnb1p to improperly change its shape in response to calcium, and this misshapen protein can’t do its job of activating Crz1p and ultimately getting the cell to respond to calcium-mediated signals properly.

And (#APOYG alert!) these yeast genetic results for the 4 EF hands match very closely to what’s been seen for mammalian calcineurin EF hand mutants in test tube (“in vitro“) experiments, giving an even stronger confirmation to these mammalian results. Maybe yeast will help develop new strategies for calcineurin-related diseases!

So now back to soccer… As we’ve found out, calcineurin HAS to use its hands, so it’s not a good pick for a regular soccer position player. But maybe it could be a super-awesome goalie since it has 4 hands! It even seems that you can tie its EF4 hand behind its back and Cnb1p can still guard the goal just fine with its remaining 3 hands – the EF4 hand seems to be a totally useless appendage! But if you disable any of the other hands, then it causes the Cnb1 protein to bend awkwardly and not do its job anymore.

Thanks to the efforts of Connolly and coworkers, we now know that it’s not quite “all hands on deck” for Cnb1p, but rather “EF 1, 2, and 3 hands on deck” in order to carry out its “goal” of regulating cellular responses to calcium!

by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.

Categories: Research Spotlight

Tags: protein structure, signal transduction

Something from Nothing: A New, Structured Protein from Noncoding DNA

November 01, 2017

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In the Rick and Morty episode Mortynight Run, a gaseous life form pulls off what alchemists had been trying to do for centuries. It literally creates gold out of thin air!

Our ever faithful friend, Saccharomyces cerevisiae can’t do that (at least not yet). But what it has done is create a protein, Bsc4p, out of noncoding intergenic DNA. And while not gold, it is a fully functioning protein.

In a new study, Bungard and coworkers show that this recently evolved gene, BSC4, encodes a protein that can fold into at least a partially defined structure. This matters as there was some debate about whether newly generated proteins could attain a defined structure or if they would remain as intrinsically disordered proteins (IDPs). A reasonable debate, given how rare structure is among amino acid sequences and how plentiful IDPs are in a cell.

Bsc4p is a great protein to study in this regard as there is very strong evidence that it has evolved relatively recently in S. cerevisiae, but not in other closely related species. And it definitely does work in a S. cerevisiae cell. While not essential, some genetic studies (including this one) indicate that it plays an important role in DNA damage repair pathways.

Here is an example from S. paradoxus of the noncoding sequence that the S .cerevisiae BSC4 gene almost certainly sprang from:

gtg TCT GTA ATT CTA CGG AAA AGT AAA CAA AAA AAC TGT AAT TGC ATA ACG AGC AAT TTA TAT ACA ATA CAC ATA GAA AGA CTT TCG CTC tga TGT CCG AAC TGC CAT TGT CAT TGG AGA AAA TCC TTA TGT GGA GTG GAG TTC CCT GCA GGT TAT TTT CAG AGA AAA CGT GGT TAC AAA AAG GGA CCA GAT TCG CCC tag CTT ACA ACT CGC TTG AAT CAT CTT TAT GCC AGA CCT TTC AAC GCC GCG ACC CCA AAA ACA taa ATG CTG AGT CAC CAT GGT GCT GGG CGC TGT CGC TGT CGC GCT GTT CCT TTC CGA GAA AAG CAC GGC AAC AAC AAC AAC AGT CCA TAT GAC CAA AAA AAA AAT AAC CGC AAA TGG CAG tga AAT GCA ATT ATC ATT GTA TAC GA?

In order for this sequence to become a gene coding a protein, at a minimum the first lowercase, dark orange codon needs to be mutated to ATG, a start codon, and the rest of the lowercase, dark orange codons need to be mutated away from being stop codons.

This seems to be part of what happened in S. cerevisiae:

ATG TCT ATT GTG CTA CGG AAG AGT AAC AAA AAA AAC AAA AAC TGC ATA ACA AGC AAG TTT TAT ACA ATA CAC ATT ATA AAA ATT TCT ACT CCG GTG TTC CGA GCT CCC ATT GCC ATT GGA GAA AGC CCT TAT GTG GAG TGG AGC TGC CTA CAG GTT GTT TTC AGG AAA GAC ATG GTT ACA AAA AAG ACG ACA TTC GCC CAA CTT ATC ACT CGC TTG AAC CAC TTT TTA TGC CAA GCC CTT AAA CGC CGC GAC TCA AAA ACA TAC ATA CTG TGC CGC ACG GCA GTT TTT GGC GCT ATG ACA CCC TTT TCC CCA AGA AAA TCG CAT ATT AAC AAC AAA TTA CCC ATG CAA CCC AGG AAA AAA AAA ATA GTC ATT ATA TAC GTA GTG CGC TTT CAT TGA

Through a few small changes, we now have a 131 amino acid polypeptide where before we had some noncoding DNA between LYP1 and ALP1.

Bungard and coworkers use a variety of techniques to show that this newly evolved protein has structure. Not as much structure as many proteins that have been around longer, but more than many of those IDPs.

Consistent with protein structure, Bsc4p forms compact oligomers under native conditions that are partially resistant to proteolysis, has a far UV circular dichroism (CD) spectra consistent with beta sheets, has a buried tryptophan, Trp47, that becomes solvent accessible under denaturing conditions, as measured by tryptophan fluorescence, and has a near UV spectra consistent with a hydrophobic core. However, they found no evidence of any significant interactions between the secondary structures to form a single three dimensional shape, in the protein.  In other words, no evidence of a tertiary structure.

And that wasn’t the only sign that Bsc4p wasn’t a mature, fully structured protein. For example, that near UV CD that showed a hydrophobic core, was weak in intensity, which is consistent with at least “partially molten character.” And Bsc4p bound certain dyes: Congo red, Thioflavin T, and ANS, in a way consistent with some molten globule and/or amyloid character.

So we have a bit of a mixed bag with Bsc4p. One way to think about it is as a young protein still developing its ultimate three dimensional structure. Or, it could be that for the job it does, this is all the structure it needs.

In any event, it is definitely a newly evolved protein with at least some structure which shows that this can indeed happen. Sometimes Mother Nature can make structured proteins from noncoding DNA. Like that gaseous being on Rick and Morty, producing gold out of thin air.

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

Categories: Research Spotlight

Tags: BSC4, newly evolved gene, noncoding DNA, protein structure

A Lot Hinges on Ndc80

December 04, 2014

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A collapsible protein complex makes sure that dividing cells get the right number of chromosomes:

The invention of collapsible tent poles was a boon to backpackers everywhere. These long, rigid poles provide strong support for a tent, but when it’s time to pack up and go, they fold up into a short package. The secret? Flexible regions in between the rigid sections.

Although the inventors of these poles couldn’t have known it, something similar already existed in nature. In a new paper in GENETICS, Tien and colleagues used the awesome power of yeast genetics, along with some cool biochemistry, to look at the shape of the S. cerevisiae Ndc80 complex in vivo. They found that, just like a tent pole, it bends sharply at a flexible region to fold two rigid sections next to each other. And this ability to fold is critical for accurate chromosome segregation.

During cell division, chromosomes must be correctly attached to the mitotic spindle so that the mother and daughter cells each get one, and only one, copy of each. If a yeast cell doesn’t get this process right, it can become sick or even die. And if it happens in an animal cell, it can lead to cancer.

The Ndc80 complex, which is conserved from yeast to humans, is an integral part of this process because it connects the chromosomes to the spindle during mitosis. It consists of four subunits and has an elongated middle and globular parts on each end, sort of like a dumbbell. The Ndc80 protein, one of the subunits, has an unstructured  “loop” region in the middle of its elongated section.

Previous work had shown that the loop region of Ndc80 is flexible in vitro, and in vivo experiments had shown that the whole complex can change its conformation. Tien and colleagues wanted to know whether the Ndc80 loop region was important for the shape of the complex during mitosis, and whether flexibility in this region was important for function.

They started with a genetic approach, and isolated mutations in NDC80 that caused heat sensitivity. One particular allele, ndc80-121, was especially interesting. The mutant protein had two amino acid changes, near each other and near the loop region. The Ndc80 complex containing the mutant protein was just as stable, and bound to microtubules just as tightly, as the wild-type complex. So why did the cells die at higher temperatures?

Tien and colleagues visualized mitosis in the mutant cells using fluorescence microscopy. They could see that when they raised the temperature, dividing mutant cells had lots of aberrant attachments between chromosomes and the spindle. Because of these attachments, proceeding through mitosis caused their spindles to break—a lethal event.

However, if they timed the temperature shift to happen later in the cell cycle, the ndc80-121 mutant cells were fine. If chromosomes had already been lined up correctly on the spindle before the temperature was raised, then the rest of mitosis could go on without a problem.

Tien and coworkers wondered whether the mutation might disrupt the binding of some other protein to the complex at high temperatures. To look for interactions, they selected mutations that suppressed the heat-sensitive phenotype of ndc80-121. But they didn’t find any suppressor mutations in other genes. However, they did find an intragenic suppressor mutation within the ndc80-121 gene.

Interestingly, this mutation affected a residue that was on the other side of the loop relative to the original two changes. If the Ndc80 complex is a dumbell, imagine that the dumbell is collapsible like a two-segment tent pole, with the loop region of Ndc80 as the elastic between the sections. If you folded the complex in this way, the amino acids changed in the ndc80-121 mutant protein would be positioned close to the amino acid that the suppressor mutation affected—an intriguing explanation for how these mutations might affect each other.

Of course, genetic interactions don’t prove a direct physical interaction. So the researchers looked to see whether they could detect physical interactions between these regions. They treated the complex with a reagent that would permanently cross-link amino acids that were close to each other. Then they chopped the complex into smaller peptides using a protease, and analyzed the cross-linked peptides using mass spectrometry to locate the linked residues.

Sure enough, they were able to detect multiple cross-links within the complex, and their locations confirmed that the complex folds much like a tent pole. Based on their mutant phenotypes, the researchers think it’s likely that the original ndc80-121mutation destabilizes folding of the complex and that the intragenic suppressor mutation makes folding tighter. Consistent with this idea, the intragenic suppressor mutation alone confers a slow-growth phenotype, as if it makes the complex fold just a little too tightly to support vigorous growth.

These experiments as a whole establish that the Ndc80 complex folds tightly early in mitosis. So, creative inventors and Mother Nature have arrived at similar solutions for the tent pole and for this important complex. And just as collapsible tent poles have become ubiquitous in the backpacking world, so too has the collapsible Ndc80 complex been conserved throughout evolution: even the specific residues that mediate the folding are highly conserved. Since this work has shown that correct folding of the yeast complex is necessary for its role in helping chromosomes to line up accurately on the spindle, the same is almost certainly true in mammalian cells.

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

Categories: Research Spotlight

Tags: mitosis, Ndc80 complex, protein structure, Saccharomyces cerevisiae

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