The transforming growth factor-ß (TGFß) pathway, including the bone morphogenetic protein (BMP), plays critical roles during embryogenesis and in adult tissue homeostasis. Hence, malfunctions in TGFß/BMP signalling result in several diseases. Signalling is initiated by ligand binding to cell surface receptor kinases, which phosphorylate and activate the R-SMAD transcription factors. R-SMADs translocate to the nucleus and regulate the transcription of hundreds of genes. The cellular responses to TGFß/BMP signals are tightly controlled and highly regulated. TGFß/BMP receptors and R-SMADs, as the intracellular mediators of TGFß/BMP ligands, are key targets for regulation to control duration and potency of signalling. Reversible ubiquitylation of R-SMADs and TGFß/BMP receptors is a key mechanism to control TGFß/BMP signalling. Several E3 ubiquitin ligases have been reported to regulate the turnover and activity of TGFß/BMP receptors and R-SMADs, however little is known about their cognate deubiquitylating enzymes (DUBs). A proteomic screen identified the DUBs OTUB1 and USP15 as potential novel regulators of the TGFß and BMP pathways respectively. Endogenous OTUB1 was recruited to the active phospho-SMAD2/3 complex only upon TGFß induction and OTUB1 had a crucial role in TGFß-mediated gene transcription and cellular migration. OTUB1 inhibited the ubiquitylation of phospho-SMAD2/3 by binding to and inhibiting the E2 ubiquitin-conjugating enzymes independently of its catalytic activity. Consequently, the depletion of OTUB1 in cells caused a rapid loss in levels of TGFß-induced phospho-SMAD2/3, which was rescued by the proteasomal inhibitor Bortezomib. These findings demonstrated a novel signal-induced phosphorylation-dependent recruitment of OTUB1 to its target. Hence, OTUB1 could be exploited as a target to intervene against diseases that are provoked by an imbalance in TGFß signalling. DUBs are highly regulated enzymes and recent reports have shed light into the molecular regulation OTUB1. The N-terminal region of OTUB1 harbours an ubiquitin binding domain, which is critical for its function to inhibit ubiquitylation. While investigating the role of OTUB1 in TGFß signalling, it became apparent that OTUB1 itself could be post-translationally modified by phosphorylation. Two phosphorylation sites at the OTUB1 N-terminal region have been identified by mass spectrometry. S18 of OTUB1 was phosphorylated in vitro by the type I TGFß receptor (ALK5), whereas S16 was phosphorylated by the constitutively active kinase CK2 in vitro and in vivo. Phosphorylation of the OTUB1 N-terminal region could affect its physiological function and requires further investigation. Although much is known about DUBs that target the type I TGFß receptor, no DUBs that target the type I BMP receptors had been identified. USP15 was identified in a proteomic screen as an interactor of SMAD6, which is a negative regulator of the BMP pathway. USP15 also binds to and deubiquitylates the type I BMP receptor (ALK3), thereby enhancing BMP signalling. Consequently, USP15 impacts BMP-induced SMAD1 phosphorylation, mouse osteoblastic differentiation and Xenopus embryogenesis. A proteomic approach identified O-GlcNAc transferase (OGT) as an interactor of SMAD2. SMADs have not been associated with O-GlcNAc modifications and the regulation of TGFß/BMP signalling by O-GlcNAcylation has not been investigated. Endogenous SMADs1-3 bound OGT and pulled down potential O-GlcNAc modified proteins. Furthermore, SMAD4 was possibly O-GlcNAcylated, which implies that O-GlcNAc modification could regulate TGFß/BMP signalling. Further investigation is needed to decipher the precise molecular mechanisms of this potential regulation.

<p>The Deubiquitinating (DUB) enzymes, LotA and UCHL1 R178Q, were examined biochemically and also structurally in the case of UCHL1 R178Q. LotA is a bacterial effector of <i>Legionella pneumophila</i> that enables the pathogen to establish a replicative niche. LotA has two Deubiquitinase (DUB) domains specific to different substrates. Here, I report biochemical examinations the first DUB domain that is specific to Lys6-linked di-ubiquitin. Michaelis- Menten kinetic parameters were determined for this domain. Through activity assays of various truncations a series of residues were discerned that contribute to interaction of the distal binding site of ubiquitin chain.</p> UCHL1 mutant R178Q displays enhanced activity when compared to wild type (WT). The mutant was crystallized for structural analysis to gain insights into the higher catalytic activity of the mutant. The structure revealed that the catalytic histidine maintains a misaligned orientation similar to the WT enzyme. Biochemical analysis was done to ascertain the role of key residues that interact with the catalytic histidine. The residue type at position 178 in the structure plays a key role in enhancing the enzyme activity.