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The Potts lab is interested in understanding the biochemical and molecular mechanisms behind fundamental cellular processes that when deregulated result in cancer. Recently, we have addressed a long-standing question in cancer biology regarding the function of cancer-testis antigen proteins (CTAs). CTAs are genes whose expression is typically restricted to cells of the germline, but becomes aberrantly activated in a wide variety of human tumors. The melanoma antigen (MAGE) family of proteins is the largest (>50) and original family of CTAs. MAGE proteins are divided into two types based on their tissue expression pattern. The type I MAGE cancer-testis antigens show restricted expression to the germline and trophoblast lineages and are reactivated in cancer cells. The type II MAGE proteins are more ubiquitously expressed in a variety of somatic cells. Importantly, both type I and type II MAGE protein family members share a well conserved MAGE homology domain that can be traced back to protozoa, but was of unknown structure and function. Additionally, expression of type I MAGE CTAs in tumors correlates with advanced disease and poor patient prognosis in several cancer types. However, the biochemical and cellular function of MAGEs in tumor, somatic, and germ cells had been enigmatic.

Through biochemical, structural, proteomic, and cellular studies, we have discovered that MAGE proteins form a novel family of E3 ubiquitin ligases. We have shown that a conserved property of MAGE proteins is their ability to bind E3 RING ubiquitin ligases. Furthermore, specific MAGE proteins bind specific E3 RING proteins through a conserved MAGE homology domain (MHD) that we have structurally defined to consist of two winged-helix motifs. Importantly, MAGEs not only bind E3 RING proteins, but also enhance their ubiquitin ligase activity in vitro and in cells. Utilizing this biochemical definition of the MAGE protein family, we have started to molecularly define the oncogenic activity of MAGE-RING ligases in cancer. For example, we have shown that several MAGE-TRIM28 E3 ubiquitin ligases ubiquitinate and degrade the critical tumor suppressor p53 in cancer cells.

ubiquitin cascade
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Our goal is to elucidate the biochemical, cellular, and physiological function of MAGE proteins in cancer and in normal somatic and germ cells.

We are currently pursuing several avenues of research into questions spanning molecular, cellular, and organismal biology:

1) Mechanistic insights into how MAGEs enhance the activity of E3 RING ubiquitin ligases
We have shown that MAGEs bind to and enhance the activity of E3 RING ubiquitin ligases. We are currently examining the mechanism(s) behind this observation by examining the effects of MAGE proteins on the various steps in the ubiquitination enzymatic cascade.

mage ring model
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2) Investigate the tumorigenic function of MAGE-RING E3 ubiquitin ligases
Several lines of evidence that some MAGE cancer-testis antigens promote tumorigenesis in mice and correlate with advanced disease and poor prognosis in humans. However, it is unclear how many MAGE proteins contribute to tumorigenesis and what cellular processes they regulate. We are examining the tumorigenic properties of all MAGEs, including their role in promoting anchorage independent growth, invasion, chemoresistance, in vivo tumor growth and metastasis.

bli calipers
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3) Identification of MAGE-RING E3 ubiquitin ligase substrates
Given the identification of MAGE-RING ligase and their importance in various tumorigenic processes, discovering the substrates for these MAGE-RING ligases is of utmost importance. We are currently using a variety of proteomic approaches to identify substrates of MAGE-RING ligases and understand their role in tumor initiation and/or progression.

mass spec
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4) High-throughput screening for novel cancer cell-specific therapeutics targeting MAGEs
The identification of several cancer cell-specific MAGE-TRIM28 E3 ligases that target the critical tumor suppressor p53 provides much excitement for developing MAGE CTA inhibitors. As our biochemical and structural understanding of MAGE-RING ligases progresses, we are developing assays to screen for small molecules that inhibit MAGE-RING ligases. Ultimately, we aim to identify novel, cancer-cell specific therapeutics targeting MAGE-RING ligases.

velcade
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5) Role of the ubiquitous MAGE-G1-NSE1 E3 ubiquitin ligase in DNA damage repair
Through a variety of biochemical and cellular assays, we have identified over 30 unique MAGE-RING ligase complexes. One of these is the ubiquitous MAGE-G1-NSE1 ligase that is part of the larger SMC5/6 complex that we have extensively characterized in the past to facilitate sister-chromatid and telomere homologous recombination (HR). However, the biochemical function of MAGE-G1-NSE1 in DNA repair has been relatively unknown. In collaboration with Maojun Yang at Tsinghua University, we have recently solved the structure of the MAGE-G1-NSE1 complex and we showed that MAGE-G1 enhances the ubiquitin ligase activity of NSE1. We are currently examining the role of MAGE-G1-NSE1 in HR-mediated double-strand break repair and telomere elongation in ALT (alternative lengthening of telomeres) cancer cells.

SMC 5-6
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6) Function of a type II ubiquitous MAGE-RING ligase in endosomal protein trafficking
Upon examining the subcellular localization of several MAGE-RING ligases, we found that MAGE-RING ligases localize to distinct subcellular compartments throughout the cells, such as the nucleus, cytoplasm, nucleolus, PML bodies, and unknown cytoplasmic bodies. Interesting we identified a single type II ubiquitous MAGE-RING ligase that localized to specific endosome structures. Current efforts are ongoing in the lab to understand the function of this MAGE-RING ligase in certain endosomal protein trafficking pathways.

endosome
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7) Physiological function of MAGE cancer-testis antigens in the germline
MAGE genes have undergone a recent evolutionary expansion, going from a single MAGE gene in lower organisms (including protists to early mammals, such as platypus) to more than 60 MAGE genes in humans. A large portion of this expansion is in the type I MAGE cancer-testis antigens that are normally only expressed in the germ cell and trophoblast lineages, but their expression is aberrantly activated in tumors. Therefore, the primary physiological functions of MAGE cancer-testis antigens is within the germline and trophoblast lineages, but their functions in these cell types are completely unknown. We hope to understand the function of MAGE cancer-testis antigens in the germline and determine if these activities are conserved in cancer cells.

Testes Histology Spermatogenesi
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