Genome instabilities: from basic mechanism to diagnosis
Genome instabilities, including chromosomal rearrangements and aneuploidy, are caused by problems in DNA replication and chromosome segregation, respectively. Genetic evidences point to a major role of multiple signaling pathways that regulate these processes. To study them, our work falls into three interconnected areas: chromosomal translocation, aneuploidy, and proteomics technology development.
In the first area, we discovered a major function of protein sumoylation in preventing chromosomal rearrangements. Our previous studies showed: 1) that inactivation of Mms21, a SUMO E3 ligase, causes over 100-fold increase in the rate of chromosomal translocation (Albuquerque et al, 2013), 2) that Mms21 specifically targets the Mini-Chromosome Maintenance (MCM) complex, the ATPase core of replicative DNA helicase (Albuquerque, 2016), and 3) that spontaneous DNA breaks occurred during DNA replication likely caused chromosomal translocations in the mms21 mutant (Liang et al, 2018). These findings raised a key question: Is MCM-SUMO modification responsible for preventing chromosomal rearrangements and if so, how? Our latest study (Quan, et al, 2022) identified a site-specific SUMO modification of the Mcm3 subunit and showed that this modification is needed to prevent chromosomal translocation through facilitating MCM loading. This exciting finding opens a new direction in studying regulated MCM loading, a critical step in ensuring chromosomal DNA replication and preventing chromosomal rearrangements.
In the second area, we investigate how mis-regulated kinetochore assembly can cause aneuploidy. Kinetochores control chromosome segregation by connecting chromosomal centromeres to the spindle microtubules and serving as a signaling hub to prevent mis-segregation. During mitosis, kinetochores are firmly attached to microtubules to segregate sister chromatids. However, such a tight connection is incompatible with the fleeting passage of DNA polymerase to replicate the centromere in the S phase. To meet these different demands, kinetochores must disassemble and re-assembly once during each cell cycle. Post-translational modification (PTM) pathways play a major role in regulating dynamic kinetochore assembly. For instance, we found that the Ulp2 SUMO protease specifically targets the inner kinetochore (Albuquerque, 2016), and it does so through a dual substrate recognition mechanism including direct contact with the inner kinetochore (Quan, et al, 2021, Suhandynata et al, 2019). We have also identified a phosphorylation regulation of the yeast CENP-C (Mif2) in promoting inner kinetochore assembly (Hinshaw, et al, 2023). Current work is directed towards understanding how dynamic kinetochore assembly is controlled during the cell cycle.
Finally, in the third area, we aim to develop mass spectrometry (MS) based proteomics technology for diverse basic research and clinical applications. MS is unquestionably the most powerful protein analytical tool, owing to its ability to sequence peptides in milliseconds with unmatched sensitivity, throughput and accuracy. For over two decades, we have developed many MS based technologies and used them to detect protein-protein interactions and post-translational protein modifications. However, there is a pressing and unmet need to ensure accurate protein identification and quantification in the MS analysis of complex biological samples. To address this, we, in collaboration with the Suhandynata lab, are developing a targeted proteomics technology platform that combines targeted and high resolution MS and a suite of tools to generate synthetic protein standards. By applying this new technology to study basic and clinical research questions, we seek to develop targeted MS based proteomics as the gold standard technology that will be broadly used in all areas of biomedical research.