A Brand New MULAN Makes Her Debut

By Eric Sauter and Mika Ono

Claudio Joazeiro and his colleagues have been studying a protein named MULAN. Like the Chinese myth of the young girl disguising herself as a man to take her father's place in the army, MULAN (the whole mouthful: M itochondrial U biquitin L igase A ctivator of N F- k B) turns out to be something that no one suspected.

Joazeiro, an assistant professor of cell biology who hails from Brazil and who joined The Scripps Research Institute a little more than a year ago, has just published a new paper about his work with MULAN in the journal PLoS ONE .

MULAN is one of the E3 ubiquitin ligases (components in the ubiquitin-proteasome system) that determine which proteins will be destined for degradation. MULAN was discovered using a new screening tool that Joazeiro and colleagues developed.

"The study really has two separate subjects," Joazeiro said. "The first is the genome-wide mapping and the screening tool that can be used in functional genomic studies to assign function to ligases. The other is our discovery of a novel ligase that represents a new and decisive link between mitochondria and the cell nucleus."

Marvelous Mitochondria

About the size of bacteria, the mitochondria are the cell's primary energy supply—energy to move, divide, etc. Mitochondria are also involved in intracellular signaling, particularly in apoptosis or programmed cell death. In human cells, mitochondria signal stress to the nucleus through little understood pathways and various transcription factors, one of those being NF- k B (a protein involved in immunity, cell growth, apoptosis, inflammation, cancer, asthma, neurodegenerative disease, and more).

In the study, the team showed that abnormal expression and knockdown (elimination) of MULAN/FLJ12875 both interfere with mitochondrial dynamics and signaling.

"That we were able to confirm the fact that MULAN is an NF- k B activator gives us an important new link between the mitochondria and NF- k B activation and an opportunity to help decipher how the mitochondria become integrated into the cellular environment," Joazeiro said.

MULAN is located in the mitochondrial outer membrane (MOM), the one place no one ever thought to look until recently.

"Even though the ubiquitin-proteasome system is known to be involved in a number of cellular processes, the role of E3s in the regulation and degradation of mitochondrial proteins and in mitochondrial biology is only just starting to emerge," Joazeiro said. "But anyone who had been interested in mitochondrial protein degradation had focused on the interior of the organelle rather than the outer membrane. This was a serendipitous discovery."

Not that serendipitous. The tool that Joazeiro and his colleagues developed to find MULAN involved developing an in-house library of E3 clones carefully culled from various sources—which narrowed the field down to approximately 600 E3s.

E3s are an important part of the ubiquitin-proteasome system, the process by which specific cells are targeted for destruction. Ubiquitin, a regulatory protein identified in 1975, is so named because it is, well, ubiquitous in cells.

During the protein degradation process, E1 activates ubiquitin and binds to it; the ubiquitin is then sent to E2; then E3 binds to E2 and the hapless protein; E3 attaches a whole host of ubiquitins to the protein—strapping the victim to the chair, so to speak--and putting out the signal to destroy the protein. Like the unfortunate barber chair victims in Sweeney Todd, the protein is unceremoniously dispatched to the basement (the proteasome, a garbage can with teeth) to be ground up.

Wide Open

Poor protein degradation has been implicated in a number of diseases including such well known neurodegenerative disorders Alzheimer's, Parkinson's, and Huntington's disease. So far, o nly a small fraction of all E3s has been functionally characterized, which Joazeiro noticed left the future wide open.

"We've known for years that there are hundreds of E3s in the human genome but no one took the time to annotate them carefully," he said. "We realized there was nothing more important than developing tools to help systematically investigate on the function of E3s. As a result, our representation of the E3 family is significant, user-friendly, and rapid. You can do an assay on the bench-top without the need for robotics in one afternoon and have your target a few days after that.

With the new tool, investigators can test for gain or loss of function, changes in biochemical pathways or changes in specific proteins.

"Our tools have flexibility to look at individual proteins or signaling pathways or whole cell phenotypes," Joazeiro said. "We fully expect that other researchers will use this to discover novel mechanisms of ubiquitin regulation and even new drug targets. "

Joazeiro points out that recent studies have begun to elucidate the process of mitochondrial dynamics—the fusion, fission, and movement of the organelle, which are essential for normal cellular function and development. MULAN plays a role there, although the exact nature of that role remains something of a mystery.

"Even though we're not entirely certain how MULAN functions vis a vis the mitochondria, the evidence we found strongly suggests that the regulatory role is very specific," Joazeiro said. "MULAN's role in mitochondrial dynamics requires both its ligase activity and its localization to the organelle. If you target MULAN any other place in the cell it won't function. We know that MULAN regulates mitochondrial dynamics and plays an active role in signaling pathways to the cell nucleus, but we don't know if there is a connection between the two or whether they are independent of one another. Right now, the missing link is MULAN's targets, and we're going after those."  

As for the name of the ligase, Wei Li, the first author of the study, came up with the acronym MULAN.

"The name manages to synthesize important known properties of the ligase," Joazeiro said. "I thought it was very creative."

In addition to Joazeiro, authors of the new study Genome-Wide and Functional Annotation of Human E3 Ubiquitin Ligases Identifies MULAN, a Mitochondrial E3 that Regulates the Organelle's Dynamics and Signaling, include, Wei Li, Mario H. Bengtson, and Axel Ulbrich of The Scripps Research Institute; Akio Matsuda of the Institute for Life Science Research, ASAHI KASEI Corporation, Japan; Venkateshwar A. Reddy, Anthony Orth, Sumit K. Chanda, and Serge Batalov of The Genomics Institute of the Novartis Research Foundation. See http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0001487.

The study was supported by Scripps Research and the Genomics Institute of the Novartis Research Foundation.

Send comments to: mikaono[at]scripps.edu

Claudio Joazeiro and his team recently published a new paper on the protein MULAN in PLoS ONE.