Ubiquitin networks in cell death regulation and non-apoptotic signalling

The posttranslational modification with ubiquitin (Ub), a process referred to as ubiquitylation, controls important aspects of cell death and survival. Ub can be attached to pro- and anti-apoptotic proteins as a single moiety or in the form of polymeric chains in which successive ubiquitin molecules are connected through specific isopeptide bonds. Reminiscent of a code, the various ubiquitin modifications adopt distinct conformations and lead to different outcomes in cells. To prevent ubiquitylation from being constitutively on, modifications are reversed by de-ubiquitylating enzymes (DUB) that cleave off the Ub adduct. While the consequence of Ub-attachment is intensely studied, little is known with regards to the effects of deconjugating enzymes that remove the Ub-adduct. The aim of the proposed programme is to unravel how the ubiquitin-signal is conjugated and edited to modulate cellular processes that are required during normal development and tissue homeostasis. We will use a series of interlocking genetic and biochemical approaches to identify and characterise DUBs that regulate caspase-mediated cell death and non-apoptotic signalling. In a pilot study we identified several DUBs that, when removed, suppress cell death in vivo. Here we propose to investigate the physiological role and molecular mechanism of five of these DUBs. Particularly, we will assess whether they function as integral and evolutionarily conserved components of the tissue repair process. Importantly, a growing body of evidence indicate that caspase-mediated signalling generates the release of signals that communicate with the cellular environment to coordinate compensatory proliferation, tissue regeneration and wound healing. Hence, we will also assess whether loss of the identified DUBs also affects caspase activation required for adaptation to tissue stress. Taken together, we propose to investigate the dynamics and specificity of Ub networks and study how Ub conjugation and deconjugation impact on signaling outcomes. Unravelling how DUBs regulate cell death and non-apoptotic signalling is critically important as these processes play fundamental physiological roles in animal development and tissue homeostasis.

The conjugation and de-conjugation of Ubiquitin (Ub) to target proteins influence diverse biological processes that can contribute to tumour formation when deregulated. Protein levels and activity of many pro- and anti-apoptotic molecules are controlled by E3-mediated conjugation of Ub. While the consequence of Ub-attachment is intensely studied, little is known about the deconjugating enzymes that remove the Ub-adduct. Here, we propose to investigate the dynamics and specificity of Ub networks and study how Ub conjugation and deconjugation impact on cell survival. We will use a series of interlocking genetic and biochemical approaches to identify and characterize deubiquitylating enzymes (DUBs) that regulate cell death. In a pilot study we identified several DUBs that, when knocked-down, suppress cell death in vivo. Here we propose to investigate the physiological role and molecular mechanism of four of these DUBs. Particularly, we will assess whether they function as integral and evolutionarily conserved components of the apoptosis machinery. Since cell death signalling components also execute nonapoptotic functions, we will assess whether loss of these DUBs also affects caspase activation required for cell migration and cell-fate decisions. Our preliminary data on one of these DUBs indicate that it is required for caspase activation following genotoxic stress, most likely because it removes Ub chains from the apoptosome. Intriguingly, we also found that genotoxic stress results in phosphorylation of this DUB, which appears to change its cleavage preference towards distinct Ub chain types. Moreover, we will also identify DUBs that regulate the Ripoptosome, a novel mammalian cell death-inducing platform that is negatively regulated in an Ub-dependent manner. Unravelling how DUBs regulate cell death and non-apoptotic signalling is critically important as these processes play fundamental roles in animal development and tissue homeostasis.

Impact and potential beneficiaries of this research:

-Scientific community:
While naturally occurring cell death was already observed 170 years ago, it was long considered a passive phenomenon and viewed as an inevitable end point of biological systems. This view began to change with studies of developmentally timed cell death in the silkworm and tadpole. These early studies showed that cell death can be delayed with inhibitors of protein or RNA synthesis and that neuronal cell survival requires extracellular survival factors termed neurotrophins. A breakthrough in elucidating the mechanism by which cells undergo programmed cell death came from genetic studies in the nematode C. elegans. It is now recognized that proper regulation of cell death plays a fundamental role in animal development and tissue homeostasis. Abnormal regulation of this process is associated with a wide variety of human diseases, including immunological and developmental disorders, neurodegeneration, and cancer. Nevertheless, some aspects of cell death regulation remain obscure and limit our understanding of how tissue homeostasis is achieved and how organisms adapt to tissue malfunction. The successful conclusion of the proposed project is expected to provide a better understanding of the molecular mechanisms that control Ub-dependent regulation of signal transduction pathways that influence the ability of multi-cellular organisms to adapt to perturbations. As such our research addresses fundamental issues relevant to normal animal physiology and aging. Further, As ubiquitin-mediated regulation of cell death and tissue repair is involved in almost every aspect of life, the proposed project will also impact the health-care community. Of particular interest is the notion that cell death regulatory proteins also fulfill nonlethal functions in differentiation and tissue remodeling. It is now clear that the cell death machinery is involved in releasing signals to communicate with their cellular environment, to promote cell division, tissue regeneration, and wound healing. Unraveling the molecular details governing this process could lead to a better understanding of aging, tissue regeneration and cancer.

- Cancer patients:
One of the key problems in cancer is the adaptive nature of tumours. It is now recognised that the ubiquitin system modulates key signal transduction pathways that have direct implications in the 'evolvability' of tumours. Hence, cancer treatments would be significantly more successful if it was possible to target ubiquitin-dependent signalling events. Our research addresses a fundamental aspect of ubiquitin-dependent tissue repair. Ultimately, this work will provide new insights into the mechanisms through which ubiquitin-dependent signalling contributes to cancer, and how we can translate this information for patient benefit.

- Academia/Pharma based drug development teams:
Due to the growing interest in ubiquitin-dependent signalling events, and the notion that the conjugation and de-conjugation of ubiquitin to target proteins influence diverse biological processes that can contribute to aging as well as tumour formation when deregulated, it is likely that the results of this project will be of interest not only for academic but also private drug development groups. The identification of the molecular mechanisms through which adaptation to tissue stress and repair are co-ordinated in a Ub-dependent manner will reveal new signalling nodes and signal transduction pathways that could be targeted by treatment with inhibitors that block deubiquitylating enzymes.