Vol 7. Issue 25 / September 10, 2007

New Protein Family Provides Unexpected Link Between Two Regulatory Pathways

By Matthew Busse

The groups of Nick Boddy, Clare McGowan, and John Tainer, biologists at The Scripps Research Institute, have found an unexpected link between SUMO and ubiquitin, two classically separate regulatory modifications that govern essential cellular processes. This discovery has important implications for basic cell biology, and could lead to new therapies for cancer and neurodegenerative diseases.

The research, which was published in an advance online issue of the journal of the European Molecular Biology Organization (EMBO) on August 30, 2007, reveals a never-before-seen protein complex, Slx8-Rfp1. This represents a new family of proteins, dubbed SUMO-Targeted Ubiquitin Ligases (STUbL), which enable communication between the sumoylation and ubiquitination pathways.

"By utilizing the powerful genetic methods afforded by the fission yeast model organism, we're working out what this family of proteins can do," says Boddy, an assistant professor in the Scripps Research Department of Molecular Biology. "Our belief is that the SUMO-tagged proteins have a specific function, and if you fail to down-regulate that function, then you start to see genome instability associated with replication defects and DNA damage."

In the study, the Scripps Research team found the new family of proteins link SUMO and ubiquitin by recognizing proteins marked with a SUMO tag, and then attaching a ubiquitin tag.

Ubiquitin, first discovered in 1980 and recognized with the 2004 Nobel Prize in Chemistry (to Aaron Ciechanover, Avram Hershko, and Irwin Rose), has emerged as a preeminent regulator of cell function. The small protein tag serves mainly as a "kiss of death"; when attached to other proteins, it targets them for degradation. In this manner, the ubiquitin pathway clears proteins out of the cell after they have fulfilled their function.

SUMO is a related protein tag that serves a different function. Sumoylation marks proteins for transport to different cellular compartments, and plays a key role in regulating DNA repair, replication, and recombination.

Notably, Boddy was one of the first to characterize SUMO (he called it PIC1), in 1996, but didn't work on the protein tag again until his current research on chromosome structure led him back to SUMO.

While investigating Nse5, a novel DNA repair protein Boddy's team discovered in the fission yeast, S. pombe, the scientists found it interacted with an unidentified protein containing a RING finger domain, which they called Rfp1. Rfp1 caught their attention because RING finger domains are often found in proteins that catalyze the addition of ubiquitin to target proteins. Boddy's interest was further piqued when his team found Rfp1 interacts with another RING finger protein named Slx8 to form a protein complex similar in structure to that of BRCA1-BARD1. Mutations in BRCA1 cause a strong predisposition to breast cancer.

When the team deleted the genes coding for Rfp1 and Slx8 to investigate their function, the effect was lethal, indicating the proteins are essential. When the scientists used mutated forms of the proteins with reduced function, the cells exhibited increased sensitivity to DNA-damaging chemicals, more replication defects, and high levels of spontaneous DNA damage—phenotypes often associated with mutations in the SUMO pathway.

An inspection of the sequence of these new proteins revealed that Rfp1, but not Slx8, harbored a SUMO–interacting motif (SIM). Additional experiments confirmed that Rfp1 directly binds to SUMO. Further tests indicated that Slx8, but not Rfp1, can act as an E3 ubiquitin ligase, catalyzing the attachment of ubiquitin to other proteins.

This new protein pair, Slx8-Rfp1, represents a never-before-seen protein complex that binds to sumoylated proteins and ubiquitinates them, hence the name SUMO-Targeted Ubiquitin Ligase.

Exploring the function of this new complex further, Boddy and colleagues found that reducing the activity of Slx8-Rfp1 caused an excess of sumoylated proteins to accumulate within the cell. Without the Slx8-Rfp1 complex, cells were unable to tag sumoylated proteins for degradation. Importantly, these phenotypes were largely suppressed when the researchers also removed the ligase responsible for the majority of sumoylation, indicating that the observed effect is specific to SUMO.

Having established that the function of these SUMO-Targeted Ubiquitin Ligase proteins in yeast is to regulate the balance of sumoylated proteins, Boddy's group looked for similar proteins in other species, and found that all higher eukaryotes have these proteins. In a remarkable display of functional conservation, the human SUMO-Targeted Ubiquitin Ligase family protein, RNF4, can substitute for the Slx8-Rfp1 complex in fission yeast, illustrating how critical this protein family is for cellular life.

"RNF4 has been studied in mammalian cells for the last ten years, and no one knew exactly what it was doing," says Boddy. "Now, we can analyze RNF4 function from a totally new perspective."

Boddy points to recent evidence that SUMO plays an important role in controlling cancer metastasis, suggesting that RNF4, as a regulator of the SUMO pathway, could present an exciting new therapeutic target for inhibiting cancer growth.

The article, "SUMO-Targeted Ubiquitin Ligases (STUbLs) in Genomic Stability," is authored by John Prudden, Stephanie Pebernard, Grazia Raffa, Daniela A. Slavin, J, Jefferson P. Perry, John A. Tainer, Clare H. McGowan, and Michael N. Boddy. See EMBO Journal at http://www.nature.com/emboj/index.html.

The research was funded by the National Institutes of Health.


Send comments to: mikaono[at]scripps.edu











"By utilizing the powerful genetic methods afforded by the fission yeast model organism, we're working out what this family of proteins can do."

—Nick Boddy