Projects in Molecular Cell Biology
- Project 1
- Project 2
- Project 3
Project 1 - Regulation of autophagy by deubiquitinating enzymes (DUBs)
Autophagy selectively delivers cytoplasmic components to lysosomes for degradation and recycling. Increasing evidence suggests that protein ubiquitination is directly involved in the regulation of autophagy by (a) controlling stability of upstream regulators or components of the autophagy machinery, and (b) facilitating the recruitment of autophagy adaptors. While a variety of ubiquitin E3 ligases were already demonstrated to govern autophagic substrate degradation, the role of DUBs in this context remains largely elusive. DUBs are essential for cells to rescue proteins from ubiquitin-mediated degradation, and to tightly control the dynamics of ubiquitin-mediated signaling events. Many DUBs associate with E3 ligase complexes to fine-tune the ubiquitination status of a common substrate.
Our project aims to characterize DUBs that are indispensable for the correct course of autophagy. A mass spectrometry-based interactome analysis revealed interactions between several DUBs and regulators or constituents of the autophagy machinery. We are characterizing the impact of these enzymes on autophagy induction, autophagosome biogenesis, and subsequent substrate clearance. These studies will allow a better understanding of how ubiquitination modulates the autophagy pathway by the combined action of E3 ligases and DUBs.
Project 2 - Control of deubiquitinating enzymes (DUBs)
Ubiquitination plays an essential role in modulating protein functions and deregulation of the ubiquitin system leads to the development of multiple human diseases. Ubiquitin (Ub) is covalently attached via its C-terminus to substrate lysine residues by a well-orchestrated enzymatic cascade including an E1 Ub-activating enzyme, an E2 Ub-conjugating enzyme and E3 ligases. Ubiquitin itself contains seven lysine residues and an N-terminal amino group, all of which can be linked to another ubiquitin to form polymers of eight different linkage types. It was shown that differently linked ubiquitin chains trigger distinct cellular responses, suggesting that ubiquitin can act as a code to store and transmit information.
Given the large complexity of possible ubiquitin modifications, the appropriate removal of ubiquitin by DUBs presents a significant problem to the cell. DUBs are essential to maintain free ubiquitin levels, rescue proteins from ubiquitin-mediated degradation, and control the dynamics of ubiquitin-mediated signaling events. Thus, the activity of DUBs must be tightly regulated in order to recognize both the correct substrate and the correct context in which to deubiquitinate target proteins. We are investigating cellular mechanisms of how the catalytic activity of these proteases is controlled (e.g. by PTMs, protein binding partners, or reactive oxygen species (ROS), especially in cellular stress responses to hypoxia.
Project 3 - Functional role of atypical protein ubiquitination in hypoxia signaling
Humans have evolved elaborate systems to ensure that oxygen levels are precisely maintained, since an excess or deficiency leads to cellular dysfunction and cell death. The transcription factor HIF (composed of an oxygen-labile alpha-subunit and a stable alpha-subunit) plays a key role in the cellular adaptation to hypoxia by regulating the expression of genes that control glucose uptake, metabolism, angiogenesis, erythropoiesis, cell proliferation, and apoptosis. HIF-1alpha is constitutively expressed and immediately degraded by the Ub-proteasome system in normoxic conditions, but is stabilised in hypoxia. Turnover of HIF-1alpha in normoxia is mediated by a well-characterised enzymatic cascade involving prolyl hydroxylases (PHDs) and a specialised cullin RING E3 Ub ligase complex (CRL2VHL). Oxygen-dependent hydroxylation of HIF-1alpha by PHDs results in its recognition by pVHL, ubiquitination, and rapid degradation by the proteasome. In hypoxia, oxygen-dependent hydroxylases are gradually inhibited, and HIF-1alpha protein levels rise dramatically. The importance of HIF-1alpha control is illustrated for example in von Hippl-Lindau disease, in which deregulated HIF-1alpha levels caused by inactivating VHL mutations predispose humans to a variety of cancers.
We have shown that the OTU family deubiquitinase Cezanne (OTUD7B) regulates HIF-1alpha homeostasis in a proteasome-independent manner (Bremm et al. EMBO Rep. 2014). Currently, we are investigating how Cezanne and atypical ubiquitination control HIF-1alpha degradation via chaperone-mediated autophagy (CMA).