Preventing Genome Instability via Phosphorylation, SUMO and Ubiquitin Pathways

Genome instability, including chromosomal rearrangements and aneuploidy, are often caused by errors occurred during DNA replication. Although much has been learned about the basic machinery of eukaryotic DNA replication, how it is regulated and interfaces with other chromosome-associated processes, including chromosome segregation and gene transcription, remain poorly understood. Through the use of yeast genetics, we discovered that inactivation of Mms21, an E3 ligase that catalyzes protein sumoylation, caused substantial accumulations of chromosomal translocation (Albuquerque et al, 2013). We have further identified the substrates of Mms21 in the DNA replication fork (Albuquerque, 2016), and determined the genetic defect of mms21 as a failure in DNA replication (Liang et al, 2018). Together, these studies revealed that protein sumoylation and ubiquitination play a major, yet poorly understood, role in regulating DNA replication, which we are investigating further.

Aneuploidy, the presence of an abnormal number of chromosomes in cells, is a major cause of birth defect and cancer. Again, genetics provided the clue pointing to a major role of protein sumoylation in preventing aneuploidy; however, the mechanism has been elusive. Our discovery that the Ulp2 SUMO-specific protease specifically desumoylates the inner kinetochore complex has triggered our investigation into this poorly understood area (Albuquerque, 2016). Through the investigation of these regulatory pathways, we seek to understand how multiple chromosome-associated processes are integrated and coordinated to ensure accurate transmission of the genetic material.

Mass Spectrometry, Proteomics and Systems Biology

New technology enables biological discovery and expands the frontier of research; therefore, we have a long-standing interest in developing mass spectrometry (MS) based proteomic technology. MS is arguably the most powerful analytical instrument for protein analysis, owing to its ability to detect and sequence peptides in milliseconds with unmatched sensitivity, throughput and accuracy; but MS cannot do it alone.

We have taken an interdisciplinary approach to develop tools that enable MS to detect protein phosphorylation, sumoylation and ubiquitination on a proteome-wide scale. As a recent example, we applied our proteomic technology to map the enzyme-substrate relationships in the protein sumoylation pathway (Albuquerque et al, 2013, 2016), and further determined the mechanisms by which the SUMO specific proteases achieve their distinct substrate specificity in vivo (Liang et al, 2017, Albuquerque, 2018). These studies illustrated the power of the proteomic approach, and uncovered the concept of substrate-directed feedback that allows a few enzymes to dynamically regulate hundreds of substrates with exquisite selectivity in space and time.

Going forward, we combine protein engineering, genetics and fluorescence approaches to develop new proteomic tools to address unmet challenges in analyzing protein modifications. Our long-term goal is to apply these tools to determine how cells use protein modifications as signals to orchestrate different cellular processes and generate specific responses, as no single biological process acts alone from the perspective of the cell. To achieve such a systems-biology level understanding, we seek an in-depth knowledge of the regulatory processes involved.