Stem Cell Biochemistry Unit: Research

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Ubiquitin-Dependent Cell-Fate Decisions During Human Development and Disease

Mammalian development relies on the precise execution of highly coordinated cell-fate decisions by stem cells, which can undergo self-renewal, reversibly exit into a quiescent state, or terminally commit to a cell differentiation program (Figure 1).

Figure 1. Our lab uses human embryonic stem cells as model system. Stem cells can reversibly exit a quiescent state to become proliferative and undergo cell division to give rise to more stem cells (self-renewal), or they can decide to undergo differentiation to give rise to the ~200 different cell types of our human body.
Figure 1. Our lab uses human embryonic stem cells as model system. Stem cells can reversibly exit a quiescent state to become proliferative and undergo cell division to give rise to more stem cells (self-renewal), or they can decide to undergo differentiation to give rise to the ~200 different cell types of our human body.

To orchestrate these decisions, stem cells make frequent use of ubiquitylation, an essential post-translational modification that alters the stability, activity, localization, or interaction landscape of target proteins (Figure 2). Ubiquitin is attached to target proteins by ubiquitin E1 activating, E2 conjugating, and E3 ligating enzymes and cleaved off by deubiquitylases (DUBs). We study the molecular mechanisms of how these ubiquitylation enzymes determine cell-fate decisions during differentiation. We focus on those enzymes linked to congenital diseases of impaired brain and craniofacial development or autoinflammation.

Figure 2. Schematic overview of protein ubiquitylation. Ubiquitin is activated and transferred to tens of E2 conjugating enzymes by one of two E1 enzymes.
Figure 2. Schematic overview of protein ubiquitylation. Ubiquitin is activated and transferred to tens of E2 conjugating enzymes by one of two E1 enzymes. In large part, the specificity of ubiquitylation is mediated by ~600 human ubiquitin E3 ligases, which interact with substrates and ubiquitin-loaded E2 enzymes to catalyze the transfer of ubiquitin to substrates. Ubiquitin modification can alter the stability, activity, localization, or interaction landscape of a substrate in question. Ubiquitylation can be reversed by ~100 human deubiquitylation enzymes, which counteract E3 ligases.

For our studies we leverage the unique clinical environment of the NIH intramural program and its large research hospital, and closely collaborate with human geneticists. This way, we combine our lab’s core expertise in biochemistry, proteomics, and human pluripotent stem cell culture with human genetics to determine the molecular mechanism of how ubiquitylation controls embryonic cell-fate choices. Current projects in our lab center around CUL3-based E3 ligases, a large family of multi-subunit E3s that uses one of ~90 BTB proteins as substrate adaptors. Dysregulation of or mutations in many of these CUL3-BTB complexes lead to human diseases (Figure 3, Asmar, et al., ECR, 2020) and we are eager to elucidate the molecular underpinnings of how this occurs. Examples include the E3 ligase complex CUL3-KBTBD8, which we have shown to regulate signaling pathways that promote neural crest specification (Werner, et al., Nature, 2015; Werner, et al., eLife, 2018) and when dysregulated results in the craniofacial development disease Treacher Collins Syndrome. Similarly, we recently found CUL3-KLHL4 to regulate cytoskeletal signaling networks to control early steps in the development of the brain, face, and skin (Asmar, et al., Nat Commun, 2023). In addition, we are interested in understanding how the DUB OTUD5 regulates chromatin dynamics during early stages of human development and if misregulated leads to a multiple congenital anomaly disease we have named LINKED (LINKage-specific-deubiquitylation-deficiency-induced Embryonic Defects) syndrome (Beck, et al., Sci Adv 2021). Finally, we aim to determine how the ubiquitin activating enzyme UBA1 and regulated ubiquitin activation controls hematopoietic cell-fate decisions and if misregulated leads to an autoinflammatory disease we have termed VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome (Beck, et al., 2020, N Engl J Med;Ferrada, et al., 2022, Blood).

 

Figure 3. CUL3-BTB E3 ligases are important regulators of development and frequently mutated in human diseases CUL3 BTB E3 ligases are multi-subunit E3s that consist of a stable catalytic core given by CUL3 and RBX1 and one of ~90 human interchangeable BTB proteins that serve as substrate adaptor. Many of these BTB proteins as well as CUL3 itself are mutated in diverse human diseases.
Figure 3. CUL3-BTB E3 ligases are important regulators of development and frequently mutated in human diseases. CUL3-BTB E3 ligases are multi-subunit E3s that consist of a stable catalytic core given by CUL3 and RBX1 and one of ~90 human interchangeable BTB proteins that serve as substrate adaptor. Many of these BTB proteins as well as CUL3 itself are mutated in diverse human diseases.

 

We hope that the results of our studies will provide mechanistic insights into important aspects of human development and into the molecular origin of human diseases, which will be useful for developing novel therapeutic approaches.

Principal Investigator
Last Reviewed
October 2023