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 core machinery of eukaryotic DNA replication, how its activity is regulated in cells remains poorly understood. We have discovered a key function of protein sumoylation in regulating eukaryotic DNA replication through a series of studies, showing that: 1) inactivation of Mms21, an E3 ligase that catalyzes sumoylation, caused substantial accumulations of chromosomal translocation (Albuquerque et al, 2013), 2) Mms21 specifically targets the Mini-Chromosome Maintenance (MCM) complex, the ATPase core of the replicative DNA helicase (Albuquerque, 2016), and 3) the chromosomal rearrangement defect of mms21 mutant lies in the accumulation of spontaneous DNA breaks, likely as a result of defective DNA replication (Liang et al, 2018). We are investigating the mechanisms by which protein sumoylation regulates eukaryotic DNA replication further.

Aneuploidy, the presence of an abnormal number of chromosomes in cells, is a major cause of birth defect and a hallmark of cancer. Previous genetic studies have suggested an important function of protein sumoylation in preventing aneuploidy; however, the mechanism has been elusive. Our recent study showed that the Ulp2 SUMO-specific protease, which plays an evolutionarily conserved role in preventing aneuploidy, specifically desumoylates the inner kinetochore complex (Albuquerque, 2016). Because the kinetochore plays a central role in controlling chromosome segregation, we are investigating how sumoylation of the inner kinetochore may regulate kinetochore remodeling to prevent the accumulation of aneuploidy.

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 our proteomic approach, and uncovered the key concept of substrate-directed feedback that allows a few enzymes to dynamically regulate hundreds of substrates with exquisite selectivity in space and time.

To address the unmet challenges in analyzing protein modifications, we are taking a directed enzyme evolution approach to engineer novel proteins as tools, which are then used to study how cells use protein modification as signal to orchestrate diverse cellular processes to produce specific biological responses.