Doctor of Philosophy (PhD)
Molecular Genetics & Cell Biology
UBR5 is part of the ubiquitin proteasome system (UPS), most well-known as a degradation and recycling pathway in which proteins are tagged with a chain of ubiquitin protein and degraded by the proteasome. However, the UPS is multifunctional and other important roles include tagging protein with ubiquitin for cell signaling purposes. UBR5 is a homologous to the E6AP C-terminus (HECT) E3 ubiquitin ligase, meaning ubiquitin is transferred to UBR5 on a cysteine residue within the HECT domain and then onto a target substrate. UBR5 has mutations in many types of cancers: breast, ovarian, liver, and lymphomas. Recently published data shows that in 18% of all of the B cell non-Hodgkin’s lymphoma (NHL) Mantle Cell Lymphoma (MCL), patients have a mutation UBR5. These mutations are specific to MCL and truncate UBR5 within the HECT domain, cutting of the cysteine residue needed for ubiquitination. MCL is a small percentage of NHL, but lack of sustained successful therapeutic treatments translates into the poorest NHL survival rate.
Due to a high rate of MCL relapsing, it is important to identify potential targets for treatment. UBR5 is unique in that it contains a Mademoiselle (MLLE) domain, which is only found in some PABP proteins, like PABPC1 and PABPC4, and RRM4. This MLLE domain allows for binding to the PAM2 protein motif, which is found on proteins involved in RNA splicing. To study the impact of truncated UBR5 in B cell development for the identification of novel therapeutic targets, we made a novel mouse model mimicking the UBR5 mutations (Ubr5∆HECT) found in MCL patients and recently published our data in Blood. The mature naïve B cell population, follicular B cells, the MCL initiating population, have decreased numbers and an abnormal phenotype similar to what is seen in MCL. Proteomic analysis with multiple mass spectrometry (MS) experiments and others involving flow cytometry show higher UBR5 expression in Ubr5∆HECT mice compared to Ubr5WT mice and have identified five novel interacting proteins that are components of the spliceosome (PRPF8, SNRNP200, EFTUD2, SF3B3, and DHX15) as well as other proteins that UBR5 can interact with during B cell receptor (BCR) stimulation or important for B cell differentiation into plasma cell. Pathways related to RNA splicing are the most impacted in our MS analysis. We then isolated RNA from follicular B cells and sent it off for sequencing. We found that Ubr5∆HECT mice contain less RNA and have changes in alternative splicing compared to Ubr5WT mice. In one class of alternative splicing, the skipped exon class, one motif was present in almost every single preceding intron of the skipped exon. Many skipped exons caused changes in genes involved in transcription in some capacity.
In our analysis, our two most striking initial findings were that there is a block in B cell differentiation into plasma cells in Ubr5∆HECT mice and IgD is the most downregulated protein in Ubr5∆HECT B cells. When performing in vitro assays to assess Ubr5∆HECT B cell responses to various stimuli to rescue those downregulated phenotypes, we demonstrated that Ubr5∆HECT B cells are able to differentiate into plasma cells in similar number compared to their Ubr5WT B cell counterparts. This shows that Ubr5∆HECT B cells are able to differentiate into plasma cells in vitro but not in vivo. Additionally, when treated with stimuli to activate the non-canonical NF-kB pathway with BAFF, IgD levels were rescued in Ubr5∆HECT B cells and were not significantly different than the Ubr5WT B cell counterparts.
We attempted to make a MCL-like mouse model using overexpression of CCND1, which is common in MCL, and Ubr5∆HECT but we were unsuccessful. Altogether, we identified a novel role of UBR5∆HECT affecting RNA abundances and hope that the continual studying of UBR5 in MCL could lead to novel therapies for MCL and other cancers.
Gilbreath, Tyler J., "Role of E3 Ubiquitin Ligase UBR5 in B Cell Development and Mantle Cell Lymphoma" (2022). Theses & Dissertations. 693.
Available for download on Thursday, November 28, 2024