Why study genome maintenance?

The genetic material is what defines all living organisms; thus, cells have evolved sophisticated mechanisms to ensure it is replicated and segregated faithfully during cell division. This is especially true for humans, considering an astronomical number of cell division must occur from one cell embryo to a grown adult, with each cell division requiring faithful replication and segregation of over 3 billion base pairs of DNA. How do cells accomplish this amazing feat? How mistakes in doing so cause disease? We are studying the roles of protein phosphorylation, sumoylation and ubiquitination in protecting genome integrity. A major thrust of our approaches is to develop and apply mass spectrometry (MS) based quantitative proteomics to address these questions.

Mechanisms of the SUMO and Ubiquitin Pathways

The ubiquitin system has a universal role in regulating protein turnover. Small Ubiquitin-like Modifier (SUMO) is a member of the ubiquitin-like proteins. Using the model organism Saccharomyces cerevisiae, we have developed powerful quantitative proteomics technology and applied it to discover that SUMO E3 ligases have substantial overlapping and distinct substrates (Albuquerque et al, 2013). On the other hand, the Ulp2 SUMO protease specifically desumoylates three protein complexes at the centromeres, the ribosomal DNA (rDNA) and the origins of DNA replication, while Ulp1 suppresses the bulk of intracellular sumoylation (Albuquerque 2016).

Ulp2-Csm1-structure.png

To understand the exquisite substrate specificity of Ulp2, we collaborated with the Corbett lab to solve the crystal structure of the Ulp2-Csm1 complex (Liang et al, 2017), demonstrating that Csm1 directly recruits Ulp2 to the nucleolus. Once recruited to the nucleolus, Ulp2 employs a negative feedback mechanism to target hyper-sumoylated substrates (Albuquerque, 2018). We continue to investigate the regulation, specificity and function of the SUMO proteases in yeast and human.

Preventing Genome Instability via SUMO and Ubiquitin

Genome instability, including chromosomal translocations and aneuploidy, is a hallmark of cancer and many other diseases. We are interested in understanding how post-translational protein modifications prevent genome instability.

Chromosome translocation. Human genetic studies have shown that mutation of NSMCE2, the human ortholog of Mms21 in yeast, causes genome instability syndrome and primordial dwarfism. We first discovered that inactivation of Mms21 caused substantial chromosomal translocations (Albuquerque et al, 2013). In collaboration with the Kolodner lab (Liang et al, 2018), we showed that the primary function of Mms21 is to prevent the accumulation of spontaneous DNA breaks, most likely occurred during DNA replication, which are then processed to generate chromosomal translocations via break-induced replication (BIR). We are studying how Mms21 dependent sumoylation regulates DNA replication to prevent the accumulation of DNA breaks from causing chromosomal translocations and other genome rearrangements.

Aneuploidy, the presence of an abnormal number of chromosomes in cells, is caused by errors in chromosome segregation. The kinetochore is the central molecular machine that controls chromosome segregation in all eukaryotes. Earlier yeast genetic study identified a major role of SMT3 and SMT4, two genes encoding SUMO and Ulp2, respectively; in regulating kinetochore assembly; however, the mechanism has been elusive for over 20 years. Recently, we found that Ulp2 specifically desumoylates the inner kinetochore CCAN complex (Albuquerque, 2016), which triggered our investigation to understand how protein sumoylation prevents aneuploidy, a major source of birth defect, mental retardation, and a hallmark of the cancer genome.

Mass Spectrometry based Proteomics

New technologies enable biological discovery and expand the frontier of research; therefore, we have a long standing interest in developing MS-based proteomics technology to analyze proteins. MS is arguably the most powerful analytical tool for protein analysis, owing to its ability to detect and sequence peptides in milliseconds with amazing sensitivity and unmatched accuracy. Through the incorporation of stable isotopes into proteins or peptides by metabolic or chemical methods, MS is capable of quantifying proteins with an accuracy and sensitivity unrivaled by few other techniques. We are particularly interested in applying MS to analyze protein phosphorylation, sumoylation and ubiquitination. To detect them, we have developed a number of tools by combining synthetic chemistry, analytical chemistry and biochemistry approaches; and using these tools we have performed proteome-wide analyses of protein phosphorylation, protein sumoylation and ubiquitination. However, a number of major challenges remain; and to address them, we continue to develop new tools by further exploiting the awesome power of MS and inter-disciplinary approaches.