The Role of BCA2 in the Endocytic Trafficking of EGFR and Significance as a Prognostic Biomarker in Cancer

Breast Cancer Associated gene 2 (BCA2) is an E3 ubiquitin ligase that is over-expressed in >50% of primary breast cancers, and has been shown to increase in vitro cell proliferation and invasion. The protein has been linked to alterations in EGFR degradation, however there is some dispute as to its role and influence on the biology of this receptor. Our work aimed to ascertain the role of BCA2 in EGFR endocytosis and down-regulation and to examine its links with breast cancer outcome. Data generated with the online expression analysis tool KM-Plotter showed that high BCA2 levels are associated with poor prognosis in ovarian, gastric and breast cancer, particularly HER2 over-expressing breast cancers. Experimentally, we demonstrate that over-expression of BCA2 induced a reduction in total EGFR levels. BCA2 over-expressing cells stimulated with EGF exhibited reduced lysosomal degradation of both this ligand and its receptor. Signalling downstream of EGFR in BCA2 over-expressing cells was characterized by a lower magnitude but increased duration. Our findings support a role for BCA2 in receptor endocytosis. Consistent with this we show that BCA2 over-expression reduces the level of vesicle-associated Rab7, a regulator of late endocytosis and documented interaction partner of BCA2. Levels of transferrin receptor and the uptake of transferrin were unaltered by over-expression of BCA2 indicating that trafficking changes may be limited to late endocytic sorting events. This report offers a thorough exploration of BCA2 biology and suggests a context-dependent role for the protein in the endocytic regulation of EGFR and as a prognostic biomarker in cancer

The centrosomal Deubiquitylase USP21 regulates Gli1 transcriptional activity and stability

USP21 is a centrosome-associated deubiquitylase (DUB) that has been implicated in the formation of primary cilia – crucial organelles for the regulation of the Hedgehog (Hh) signaling pathway in vertebrates. Here, we identify KCTD6 – a cullin-3 E3-ligase substrate adapter that has been previously linked to Hh signaling – as well as Gli1, the key transcription factor responsible for Hh signal amplification, as new interacting partners of USP21. We identify a cryptic structured protein interaction domain in KCTD6, which is predicted to have a similar fold to Smr domains. Importantly, we show that both depletion and overexpression of catalytically active USP21 suppress Gli1-dependent transcription. Gli proteins are negatively regulated through protein kinase A (PKA)-dependent phosphorylation. We provide evidence that USP21 recruits and stabilises Gli1 at the centrosome where it promotes its phosphorylation by PKA. By revealing an intriguing functional pairing between a spatially restricted deubiquitylase and a kinase, our study highlights the centrosome as an important hub for signal coordination

Quantitative proteomic analysis of Parkin substrates in Drosophila neurons.

Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson's Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings.Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells.We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells.Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation

A chlamydia effector combining deubiquitination and acetylation activities induces Golgi fragmentation

Pathogenic bacteria are armed with potent effector proteins that subvert host signalling processes during infection1. The activities of bacterial effectors and their associated roles within the host cell are often poorly understood, particularly for Chlamydia trachomatis2, a World Health Organization designated neglected disease pathogen. We identify and explain remarkable dual Lys63-deubiquitinase (DUB) and Lys-acetyltransferase activities in the Chlamydia effector ChlaDUB1. Crystal structures capturing intermediate stages of each reaction reveal how the same catalytic centre of ChlaDUB1 can facilitate such distinct processes, and enable the generation of mutations that uncouple the two activities. Targeted Chlamydia mutant strains allow us to link the DUB activity of ChlaDUB1 and the related, dedicated DUB ChlaDUB2 to fragmentation of the host Golgi apparatus, a key process in Chlamydia infection for which effectors have remained elusive. Our work illustrates the incredible versatility of bacterial effector proteins, and provides important insights towards understanding Chlamydia pathogenesis

Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs

Regulation of ErbB2 Receptor Status by the Proteasomal DUB POH1

Understanding the factors, which control ErbB2 and EGF receptor (EGFR) status in cells is likely to inform future therapeutic approaches directed at these potent oncogenes. ErbB2 is resistant to stimulus-induced degradation and high levels of over-expression can inhibit EGF receptor down-regulation. We now show that for HeLa cells expressing similar numbers of EGFR and ErbB2, EGFR down-regulation is efficient and insensitive to reduction of ErbB2 levels. Deubiquitinating enzymes (DUBs) may extend protein half-lives by rescuing ubiquitinated substrates from proteasomal degradation or from ubiquitin-dependent lysosomal sorting. Using a siRNA library directed at the full complement of human DUBs, we identified POH1 (also known as Rpn11 or PSMD14), a component of the proteasome lid, as a critical DUB controlling the apparent ErbB2 levels. Moreover, the effects on ErbB2 levels can be reproduced by administration of proteasomal inhibitors such as epoxomicin used at maximally tolerated doses. However, the extent of this apparent loss and specificity for ErbB2 versus EGFR could not be accounted for by changes in transcription or degradation rate. Further investigation revealed that cell surface ErbB2 levels are only mildly affected by POH1 knock-down and that the apparent loss can at least partially be explained by the accumulation of higher molecular weight ubiquitinated forms of ErbB2 that are detectable with an extracellular but not intracellular domain directed antibody. We propose that POH1 may deubiquitinate ErbB2 and that this activity is not necessarily coupled to proteasomal degradation

The Epstein-Barr Virus G-Protein-Coupled Receptor Contributes to Immune Evasion by Targeting MHC Class I Molecules for Degradation

Epstein-Barr virus (EBV) is a human herpesvirus that persists as a largely subclinical infection in the vast majority of adults worldwide. Recent evidence indicates that an important component of the persistence strategy involves active interference with the MHC class I antigen processing pathway during the lytic replication cycle. We have now identified a novel role for the lytic cycle gene, BILF1, which encodes a glycoprotein with the properties of a constitutive signaling G-protein-coupled receptor (GPCR). BILF1 reduced the levels of MHC class I at the cell surface and inhibited CD8+ T cell recognition of endogenous target antigens. The underlying mechanism involves physical association of BILF1 with MHC class I molecules, an increased turnover from the cell surface, and enhanced degradation via lysosomal proteases. The BILF1 protein of the closely related CeHV15 c1-herpesvirus of the Rhesus Old World primate (80% amino acid sequence identity) downregulated surface MHC class I similarly to EBV BILF1. Amongst the human herpesviruses, the GPCR encoded by the ORF74 of the KSHV c2-herpesvirus is most closely related to EBV BILF1 (15% amino acid sequence identity) but did not affect levels of surface MHC class I. An engineered mutant of BILF1 that was unable to activate G protein signaling pathways retained the ability to downregulate MHC class I, indicating that the immune-modulating and GPCR-signaling properties are two distinct functions of BILF1. These findings extend our understanding of the normal biology of an important human pathogen. The discovery of a third EBV lytic cycle gene that cooperates to interfere with MHC class I antigen processing underscores the importance of the need for EBV to be able to evade CD8+ T cell responses during the lytic replication cycle, at a time when such a large number of potential viral targets are expressed

Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral—but not the apical—plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion

In-Cell Biochemistry Using NMR Spectroscopy

Biochemistry and structural biology are undergoing a dramatic revolution. Until now, mostly in vitro techniques have been used to study subtle and complex biological processes under conditions usually remote from those existing in the cell. We developed a novel in-cell methodology to post-translationally modify interactor proteins and identify the amino acids that comprise the interaction surface of a target protein when bound to the post-translationally modified interactors. Modifying the interactor proteins causes structural changes that manifest themselves on the interacting surface of the target protein and these changes are monitored using in-cell NMR. We show how Ubiquitin interacts with phosphorylated and non-phosphorylated components of the receptor tyrosine kinase (RTK) endocytic sorting machinery: STAM2 (Signal-transducing adaptor molecule), Hrs (Hepatocyte growth factor regulated substrate) and the STAM2-Hrs heterodimer. Ubiquitin binding mediates the processivity of a large network of interactions required for proper functioning of the RTK sorting machinery. The results are consistent with a weakening of the network of interactions when the interactor proteins are phosphorylated. The methodology can be applied to any stable target molecule and may be extended to include other post-translational modifications such as ubiquitination or sumoylation, thus providing a long-awaited leap to high resolution in cell biochemistry