August 21, 2014
Say you want to send a letter to your friend on the other side of the country. First off you’ll need to put the right address and postage on the envelope. Then you’ll need the U.S. Postal Service (USPS) to take your letter and deliver it to the right person. The stamp tells the USPS to deliver the letter, and the address indicates where it should be delivered (unimpeded by snow nor rain nor heat nor gloom of night, of course!).
It turns out something similar happens in human cells with aggregated proteins. Aggregated proteins are “stamped” by attachment of the small protein ubiquitin and “addressed” to the Atg8 protein. Atg8p triggers the aggregated proteins’ incorporation into autophagosomes for eventual degradation in the lysosome.
And just as it can be devastating if your mail doesn’t get to where it needs to go, so too can it be devastating for these aggregates to accumulate instead of being properly delivered. A buildup of these aggregates is a big factor in Alzheimer’s and Huntington’s diseases.
Enter the cellular USPS. Just as is the case for a prepared letter, the human cell has a service that delivers the ubiquinated proteins to the autophagosome, in the form of the protein adaptor p62 (SQSTM1) and its relative, NBR1.
These adaptor proteins can act as a postal service because they recognize both the aggregated proteins’ stamp (ubiquitin) and their addressee (Atg8p). Specifically, they each possess an ubiquitin-conjugate binding domain (UBA) and an Atg8-interacting motif (AIM). The protein p62 in particular has been shown to associate with protein aggregates linked to neurodegenerative diseases like Huntington’s disease.
In a new paper published in Cell, Lu et al. asked whether there is a link between the ubiquitin and autophagy systems in yeast. If so, yeast might provide some clues about diseases like Huntington’s. Proteins stamped with ubiquitin are known to be addressed to the proteasome for degradation in yeast, but no link between ubiquitination and autophagy had previously been seen, even though many central components of autophagy were actually first described in yeast.
Indeed, the authors showed that cells specifically deficient in the autophagy pathway (atg8∆, atg1∆, or atg7∆), accumulated ubiquitin conjugates under autophagy-inducing conditions. This suggests that the ubiquitin and autophagy pathways are connected in yeast, as is the case for humans.
Next, the researchers looked to see if there is an adaptor in yeast analogous to p62 in humans. When they pulled down proteins that bind yeast Atg8p under starvation conditions, they found ubiquitin conjugates and, using mass spectrometry, further identified peptides from a few other proteins – one of which was Cue5p.
Could Cue5p, like p62 in humans, be the postal service that recognizes both stamped ubiquitin conjugates and the addressee Atg8p in yeast? Strikingly, Cue5p had both a CUE domain that binds ubiquitin and an Atg8p-interacting motif (AIM). The authors confirmed in vivo that Cue5p binds ubiquitin conjugates and Atg8p using these domains, particularly under starvation conditions. They also showed that it acts specifically at the stage of ubiquitin-conjugate recognition and on aggregated proteins, without affecting the process of autophagy itself.
Given that Cue5p functions similarly to p62 and p62 is known to associate with protein aggregates involved in neurodegenerative disease, Lu et al. were quick to look for Cue5p substrates. Analyzing ubiquitin-conjugated proteins that accumulated in cue5 mutant cells, they identified 24 different proteins. Although these 24 Cue5p substrates had diverse functions, the common thread was that many had a tendency to aggregate under certain conditions such as high temperature.
Could Cue5p then actually facilitate removal of cytotoxic protein aggregates in neurodegenerative diseases? Indeed, the authors showed that CUE5 helped clear cytotoxic variants of the human huntingtin protein (Htt-96Q) when it was expressed in yeast, and that Htt-96Q is ubiquitinated in yeast.
These experiments started with an observation in human cells that prompted discovery of an analogous system and adaptor protein in yeast. Now the authors turned the tables and used yeast to look for new adaptor proteins in human cells. Using bioinformatics, they identified a human CUE-domain protein, Tollip, which, although different in its domain organization from Cue5p, contains 2 AIM motifs.
To make a long story (and a lot of work!) short, they showed that Tollip binds both human Atg8p and ubiquitin conjugates and clears cytotoxic variants of huntingtin in human cells. Expressed in yeast, it similarly binds ubiquitin conjugates and Atg8p and suppresses the hypersensitivity of cue5∆ cells to the variant huntingtin protein Htt-96Q. So Tollip is a newly defined adaptor protein and functional homolog of Cue5p!
Letter carriers of one sort or another have been around for as long as human civilization has existed, from homing pigeons to FedEx. Now we know that for even longer, cells from yeast to human have been using similar ways to recognize stamped proteins and deliver them to the right address. And once again, yeast has helped us understand the inner secrets of human cells.
by Selina Dwight, Ph.D., Senior Biocurator, SGD
January 17, 2013
(Please click the musical note and listen to the music while reading.) The music you’re listening to starts off with a marimba. Then a flute joins in and as the marimba fades, in comes a shamisen. The piece progresses similarly with a harp, and then ends with the reappearance of the marimba. A nice, jaunty little piece of music.
This song is actually a tool for learning about autophagy in the yeast S. cerevisiae. Autophagy is a way to break down damaged or no longer useful proteins and recycle their components for later use. It is a very important pathway in keeping starving yeast alive. Many of the proteins involved in autophagy are highly conserved, and autophagy defects are implicated in several kinds of human disease.
As described in a recent paper, Takahashi and coworkers converted the sequences of four proteins involved in a step in autophagy – Atg9, Sso1, Sec9, and Sec22 – into pieces of music using UCLA’s Gene2Music program. Each protein was then assigned a musical instrument. Atg9 was played with the marimba, Sso1 with the flute, Sec9 with the shamisen, and Sec22 with the harp. The orchestrated piece of music reflects how each protein interacts with the others in the autophagy pathway.
Atg9 is a transmembrane protein that is key to making the vesicles that carry the damaged or unused proteins to the lysosome for destruction. But it, like the marimba, is not enough. Atg9 is recruited into service by at least three other proteins, Sso1, Sec9, and Sec22. These appear in succession in the musical piece as a flute, shamisen, and harp. Just like all four are needed for the orchestral piece, all four are also needed for successful autophagy.
Now listen to the music again. With this background, did you find the piece more illuminating? If you didn’t, it may simply be because it doesn’t fit your learning style, or match the type of intelligence that is your strength. Some people may respond to music better than they do to pictures of pathways or memorizing the steps involved. It may be that these people’s understanding of complicated pathways is enhanced with a musical component.
There will need to be more research on musical representation of complex pathways to see if they actually help students and even the public better understand science. If they do, I am looking forward to hearing the Krebs Cycle put to music. Or the assembly of the RNA polymerase II preinitiation complex. Which pathways do you want put to music?
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight