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Joazeiro Lab Research

research

Studying the ubiquitin-proteasome system as a way to understand normal cellular function and to develop novel therapeutic approaches for diseases

Ubiquitin is a small protein that is used as a tag either to mark other intracellular proteins for destruction, or to alter their function. We undertake various approaches to uncover pathways and mechanisms involving ubiquitin, and convert this information into tools to study biology and interfere with disease. Our current focus is on elucidating the function of E3 ubiquitin ligases, the components of the ubiquitin system that are responsible for making ubiquitination a specific and controlled process. The relevance of E3 ligases is underscored by the fact that, when mutated, they can cause human disease's as illustrated by mutations of BRCA1 in breast cancer and of Parkin in Parkinson's Disease.

Our laboratory utilizes molecular genetic, genomic, biochemistry, cell biology and molecular biology approaches with mammalian cells, mice and yeast. Our expertise is complemented by close collaborations on structural biology, bioinformatics, mass spectrometry, next-generation sequencing and fly models. We also develop high-throughput assays and use those to carry out screens aimed at identifying small molecules that can be used as research tools and can also be developed as pharmaceutical drug candidates. Finally, we collaborate with clinicians to translate our basic research findings into therapeutically relevant discoveries.

The laboratory's current projects are:

Project I. Development of functional genomic tools to assign function to E3 ligases

E3 ligases are the enzymes that recognize specific target proteins (substrates) and promote their modification with ubiquitin. E3s are essential for most cellular processes that have been examined, including cell division, programmed cell death, signaling and DNA repair. Despite the importance of E3s, little is known about the biological functions and mechanism of action of most of the members of this protein family in humans. This gap in knowledge prompted us to carry out bioinformatic analyses that mapped over 600 human genes encoding E3s, as a starting point to characterizing the family; with this information in hand, we generated cDNA and siRNA libraries targeting all known and "orphan"� E3s, to be used as functional discovery tools (Li et al. 2008). These collections are plate-arrayed and ready to be used in multiple screen formats.

As an example of a screen utilizing this toolbox, we set out to identify E3 ligases that control mitochondrial morphology or distribution, processes whose malfunctioning can lead to cancer or neurodegeneration. With this approach, we found a novel E3 that localizes to mitochondria and regulates the organelle's dynamics and signaling (see Project II below).

 

 Tools to assign function to E3 ligases

 

Project II. Role of the mitochondrial ubiquitin ligase, MULAN, in stress signaling and cancer

Using a functional genomic approach with a toolbox we built in-house (see Project I above), we discovered an E3 ligase that is anchored to mitochondria, with a domain facing the interior of the organelle and the catalytic E3 "RING domain" facing the cytoplasm. This E3 regulates mitochondrial fission/fusion and also mediates communication between the organelle and the nucleus by activating NF-kappaB, a factor that turns on genes involved in stress responses and inflammation. Accordingly, we named it MULAN, for Mitochondrial Ubiquitin Ligase Activator of NF-kappaB. Ongoing work aims at understanding MULAN's physiological role and mechanism of action. As in the Chinese tale of a girl disguised as a man to join the army, MULAN is turning out to be (and do) things that we never expected. For example, we recently found a non-canonical mechanism of NF-kappaB activation by MULAN (Ulbrich et al., manuscript in preparation). Since many bio-pharmaceutical companies are developing NF-kappaB inhibitors to treat cancer and inflammation, our discovery may allow better prediction of those drugs' effects as well.

MULAN

 

Project III. Listerin/Ltn1 and protein quality control in aging and neurodegeneration

Neurodegenerative diseases are incurable and debilitating conditions whose increasing prevalence poses a great public health problem. However, and despite recent advances on the identification of several genes involved, the molecular bases of these diseases remain poorly understood. To shed light on this problem, we are studying a new mouse model of neurodegeneration caused by mutation of Listerin, a previously unknown E3 ligase (Chu et al. 2009).

The focus of our most recent work has been on elucidating this E3's biological role and determining how defects in its function lead to the disease. To our advantage, Listerin is conserved in all eukaryotes. This not only suggests an important biological function, but also provides us with superior tools to investigate what they might be. For example, the budding yeast Saccharomyces cerevisiae represents a terrific genetic and cell biology model to study molecular mechanisms. In addition to an array of tools that have been developed over the decades allowing its efficient genetic manipulation and biochemical analyses, the protein composition of yeast is simpler than that of a mammal, its life cycle is significantly shorter, and the organism is smaller and much less expensive to maintain and work with.

Taking advantage of yeast to study Listerin, we recently found that its budding yeast homolog (Ltn1/Rkr1/yMR247c) is critical for the ubiquitin modification and degradation of aberrant proteins encoded by mRNA lacking stop codons ("non-stop mRNA"). We also found that ubiquitination of these aberrant proteins appears to be triggered by their stalling in ribosomes upon translation through the poly(A) tail, and that the E3 is predominantly ribosome-associated. Moreover, those aberrant proteins cannot be corrected by the chaperones responsible for protein quality control, and their increased production was toxic to cells lacking Ltn1.

These discoveries are important because although it has been known for over 15 years that the products of "non-stop mRNA" are degraded in bacteria by a specialized system (known as tmRNA or SsrA), how eukaryotic cells eliminate those proteins had remained unknown. Moreover, our results now also raise the possibility that a defective role of Listerin in protein quality control underlies the neurodegenerative phenotype of Listerin-mutant mice.

Press releases:

Science Blog: http://scienceblog.com/38418/scripps-research-scientists-solve-long-standing-mystery-of-protein-quality-control-mechanism/
Nature: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature09371.html

 Model for the degradation of eukaryotic nonstop proteins

Figure legend:
Model for the degradation of eukaryotic nonstop proteins, based on our work. The illustration depicts a ribosome translating eukaryotic non-stop mRNA through the poly(A) tail (represented in cross-section). Translation of poly(A) results in stalling of the nascent polypeptide and recruitment of the Ltn1 E3 ligase (in green), which in turn recruits ubiquitin-charged E2s via its RING domain. The 60S-bound Ltn1-E2-ubiquitin complex is properly positioned to mark the stalled nascent nonstop protein with ubiquitin (Ub) for degradation.

 

Project IV. Discovery of E3 ligase small molecule inhibitors

E3 ligases have been linked in many ways to various human diseases. For example, the Mdm2 E3 ligase is amplified/overexpressed in 7% of all cancers. Despite the potential of some E3s to become the next blockbuster drug targets, the discovery of inhibitors that target these enzymes remains an unmet challenge. Ongoing research in the lab is aimed at identifying small molecule E3 inhibitors that can be used as tools both for the development of new therapies and for understanding biology.
Prior to joining Scripps, the Joazeiro laboratory was for six years at the Genomics Institute of Novartis (GNF) in San Diego. At GNF, we identified and validated new drug targets and carried out drug discovery activities. This Industry experience places us in a competitive position among our colleagues with a more traditional Academic career, and is enhanced by our ongoing efforts on elucidating biochemical mechanisms and structural biology of E3 ligases.

 

Further reading

Bengtson, M.H. and Joazeiro, C.A.P. 2010. Role of a ribosome-associated E3 ubiquitin ligase in protein quality control. Nature 467: 470-473.

Chu, J., Hong, N., Masuda, C., Jenkins, B., Nelms, K., Goodnow, C., Glynne, R., Wu, H., Masliah, E., Joazeiro, C.A.P. and Kay, S.A. 2009. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration. Proceedings of the National Academy of Sciences of the USA vol. 106: 2097-2103

Li, W., Bengtson, M., Ulbrich, A., Matsuda, A., Orth, A., Chanda, S., Batalov, S. and Joazeiro, C.A.P. 2008. Genome-wide and Functional Annotation of Human E3 Ubiquitin Ligases Identifies MULAN, a Mitochondrial E3 that Regulates the Organelle's Dynamics and Signaling. PLoS ONE vol. 3:e1487

Zhang, Q., Liu, Y., Gao, F., Ding, Q., Cho, C., Hur, W.-Y., Jin, Y., Uno, T., Joazeiro, C.A.P. and Gray, N. 2006. Discovery of EGFR-Selective 4,6-Disubstituted Pyrimidines from a Combinatorial Kinase-Directed Heterocycle Library. Journal of the American Chemical Society vol. 128:2182-2183

Joazeiro, C.A.P., Anderson, K.C. and Hunter, T. 2006. Proteasome inhibitor drugs on the rise.
Cancer Research vol. 66:7840-7842

 

Financial support

National Institutes of Health Logo

http://www.nih.gov/

National Institute of General Medical Sciences

http://www.nigms.nih.gov/

NINDS

http://www.ninds.nih.gov/

NCI

http://cancer.gov/

American Cancer Society

http://www.cancer.org/