The Parkinson's disease related mutant VPS35 (D620N) amplifies the LRRK2 response to endolysosomal stress.

The identification of multiple genes linked to Parkinson's disease (PD) invites the question as to how they may co-operate. We have generated isogenic cell lines that inducibly express either wild-type or a mutant form of the retromer component VPS35 (D620N), which has been linked to PD. This has enabled us to test proposed effects of this mutation in a setting where the relative expression reflects the physiological occurrence. We confirm that this mutation compromises VPS35 association with the WASH complex, but find no defect in WASH recruitment to endosomes, nor in the distribution of lysosomal receptors, cation-independent mannose-6-phosphate receptor and Sortilin. We show VPS35 (D620N) enhances the activity of the Parkinson's associated kinase LRRK2 towards RAB12 under basal conditions. Furthermore, VPS35 (D620N) amplifies the LRRK2 response to endolysosomal stress resulting in enhanced phosphorylation of RABs 10 and 12. By comparing different types of endolysosomal stresses such as the ionophore nigericin and the membranolytic agent l-leucyl-l-leucine methyl ester, we are able to dissociate phospho-RAB accumulation from membrane rupture.

Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.

The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here we show that its lysosomal degradation is dependent on ubiquitylation at Lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4 and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4+ T cells and in cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype, but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive, nor a localization-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4, or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.

Microtubule association of TRIM3 revealed by differential extraction proteomics.

The microtubule network is formed from polymerised tubulin subunits and associating proteins, which govern microtubule dynamics and a diverse array of functions. To identify novel microtubule-binding proteins, we have developed an unbiased biochemical assay, which relies on the selective extraction of cytosolic proteins from U2OS cells, while leaving behind the microtubule network. Candidate proteins are linked to microtubules by their sensitivities to the depolymerising drug nocodazole or the microtubule-stabilising drug taxol, which is quantitated by mass spectrometry. Our approach is benchmarked by co-segregation of tubulin and previously established microtubule-binding proteins. We then identify several novel candidate microtubule-binding proteins, from which we have selected the ubiquitin E3 ligase tripartite motif-containing protein 3 (TRIM3) for further characterisation. We map TRIM3 microtubule binding to its C-terminal NHL-repeat region. We show that TRIM3 is required for the accumulation of acetylated tubulin, following treatment with taxol. Furthermore, loss of TRIM3 partially recapitulates the reduction in nocodazole-resistant microtubules characteristic of α-tubulin acetyltransferase 1 (ATAT1) depletion. These results can be explained by a decrease in ATAT1 following depletion of TRIM3 that is independent of transcription.

ISGylation-independent protection of cell growth by USP18 following interferon stimulation.

Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signalling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation, a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non-catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity.

FBXL4 ubiquitin ligase deficiency promotes mitophagy by elevating NIX levels.

Selective autophagy of mitochondria, mitophagy, is linked to mitochondrial quality control and as such is critical to a healthy organism. We have used a CRISPR/Cas9 approach to screen human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and upon acute mitochondrial depolarization. We identify two cullin-RING ligase substrate receptors, VHL and FBXL4, as the most profound negative regulators of basal mitophagy. We show that these converge, albeit via different mechanisms, on control of the mitophagy adaptors BNIP3 and BNIP3L/NIX. FBXL4 restricts NIX and BNIP3 levels via direct interaction and protein destabilization, while VHL acts through suppression of HIF1α-mediated transcription of BNIP3 and NIX. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study contributes to an understanding of the aetiology of early-onset mitochondrial encephalomyopathy that is supported by analysis of a disease-associated mutation. We further show that the compound MLN4924, which globally interferes with cullin-RING ligase activity, is a strong inducer of mitophagy, thus providing a research tool in this context and a candidate therapeutic agent for conditions linked to mitochondrial dysfunction.

STAM and Hrs interact sequentially with IFN-α Receptor to control spatiotemporal JAK-STAT endosomal activation.

Activation of the JAK-STAT pathway by type I interferons (IFNs) requires clathrin-dependent endocytosis of the IFN-α and -ß receptor (IFNAR), indicating a role for endosomal sorting in this process. The molecular machinery that brings the selective activation of IFN-α/ß-induced JAK-STAT signalling on endosomes remains unknown. Here we show that the constitutive association of STAM with IFNAR1 and TYK2 kinase at the plasma membrane prevents TYK2 activation by type I IFNs. IFN-α-stimulated IFNAR endocytosis delivers the STAM-IFNAR complex to early endosomes where it interacts with Hrs, thereby relieving TYK2 inhibition by STAM and triggering signalling of IFNAR at the endosome. In contrast, when stimulated by IFN-ß, IFNAR signalling occurs independently of Hrs as IFNAR is sorted to a distinct endosomal subdomain. Our results identify the molecular machinery that controls the spatiotemporal activation of IFNAR by IFN-α and establish the central role of endosomal sorting in the differential regulation of JAK-STAT signalling by IFN-α and IFN-ß.

Segregation of pathways leading to pexophagy.

Peroxisomes are organelles with key roles in metabolism including long-chain fatty acid production. Their metabolic functions overlap and interconnect with those of mitochondria, with which they share an overlapping but distinct proteome. Both organelles are degraded by selective autophagy processes termed pexophagy and mitophagy. Although mitophagy has received intense attention, the pathways linked to pexophagy and associated tools are less well developed. We have identified the neddylation inhibitor MLN4924 as a potent activator of pexophagy and show that this is mediated by the HIF1α-dependent up-regulation of BNIP3L/NIX, a known adaptor for mitophagy. We show that this pathway is distinct from pexophagy induced by the USP30 deubiquitylase inhibitor CMPD-39, for which we identify the adaptor NBR1 as a central player. Our work suggests a level of complexity to the regulation of peroxisome turnover that includes the capacity to coordinate with mitophagy, via NIX, which acts as a rheostat for both processes.

Protein degradation on the global scale.

Protein degradation occurs through proteasomal, endosomal, and lysosomal pathways. Technological advancements have allowed for the determination of protein copy numbers and turnover rates on a global scale, which has provided an overview of trends and rules governing protein degradation. Sharper chemical and gene-editing tools have enabled the specific perturbation of each degradation pathway, whose effects on protein dynamics can now be comprehensively analyzed. We review major studies and innovation in this field and discuss the interdependence between the major pathways of protein degradation.

Benchmarking a highly selective USP30 inhibitor for enhancement of mitophagy and pexophagy.

The deubiquitylase USP30 is an actionable target considered for treatment of conditions associated with defects in the PINK1-PRKN pathway leading to mitophagy. We provide a detailed cell biological characterization of a benzosulphonamide molecule, compound 39, that has previously been reported to inhibit USP30 in an in vitro enzymatic assay. The current compound offers increased selectivity over previously described inhibitors. It enhances mitophagy and generates a signature response for USP30 inhibition after mitochondrial depolarization. This includes enhancement of TOMM20 and SYNJ2BP ubiquitylation and phosphoubiquitin accumulation, alongside increased mitophagy. In dopaminergic neurons, generated from Parkinson disease patients carrying loss of function PRKN mutations, compound 39 could significantly restore mitophagy to a level approaching control values. USP30 is located on both mitochondria and peroxisomes and has also been linked to the PINK1-independent pexophagy pathway. Using a fluorescence reporter of pexophagy expressed in U2OS cells, we observe increased pexophagy upon application of compound 39 that recapitulates the previously described effect for USP30 depletion. This provides the first pharmacological intervention with a synthetic molecule to enhance peroxisome turnover.

Membrane compartmentalisation of the ubiquitin system.

We now have a comprehensive inventory of ubiquitin system components. Understanding of any system also needs an appreciation of how components are organised together. Quantitative proteomics has provided us with a census of their relative populations in several model cell types. Here, by examining large scale unbiased data sets, we seek to identify and map those components, which principally reside on the major organelles of the endomembrane system. We present the consensus distribution of > 50 ubiquitin modifying enzymes, E2s, E3s and DUBs, that possess transmembrane domains. This analysis reveals that the ER and endosomal compartments have a diverse cast of resident E3s, whilst the Golgi and mitochondria operate with a more restricted palette. We describe key functions of ubiquitylation that are specific to each compartment and relate this to their signature complement of ubiquitin modifying components.

The PINK1 repertoire: Not just a one trick pony.

PTEN-induced kinase 1 (PINK1) is a Parkinson's disease gene that acts as a sensor for mitochondrial damage. Its best understood role involves phosphorylating ubiquitin and the E3 ligase Parkin (PRKN) to trigger a ubiquitylation cascade that results in selective clearance of damaged mitochondria through mitophagy. Here we focus on other physiological roles of PINK1. Some of these also lie upstream of Parkin but others represent autonomous functions, for which alternative substrates have been identified. We argue that PINK1 orchestrates a multi-arm response to mitochondrial damage that impacts on mitochondrial architecture and biogenesis, calcium handling, transcription and translation. We further discuss a role for PINK1 in immune signalling co-ordinated at mitochondria and consider the significance of a freely diffusible cleavage product, that is constitutively generated and degraded under basal conditions.

USP28 deletion and small-molecule inhibition destabilizes c-MYC and elicits regression of squamous cell lung carcinoma.

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient's 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.

The deubiquitylase USP9X controls ribosomal stalling.

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.

USP30 sets a trigger threshold for PINK1-PARKIN amplification of mitochondrial ubiquitylation.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition.

Data mining for traffic information.

Modern cell biology is now rich with data acquired at the whole genome and proteome level. We can add value to this data through integration and application of specialist knowledge. To illustrate, we will focus on the SNARE and RAB proteins; key regulators of intracellular fusion specificity and organelle identity. We examine published mass spectrometry data to gain an estimate of protein copy number and organelle distribution in HeLa cells for each family member. We also survey recent global CRISPR/Cas9 screens for essential genes from these families. We highlight instances of co-essentiality with other genes across a large panel of cell lines that allows for the identification of functionally coherent clusters. Examples of such correlations include RAB10 with the SNARE protein Syntaxin4 (STX4) and RAB7/RAB21 with the WASH and the CCC (COMMD/CCDC22/CCDC93) complexes, both of which are linked to endosomal recycling pathways.

New aspects of USP30 biology in the regulation of pexophagy.

Mitochondria and peroxisomes have a number of features in common: they each play interconnected roles in fatty acid and reactive oxygen species (ROS) metabolism and, once damaged, need to be removed by specialized autophagic mechanisms, termed mitophagy and pexophagy, respectively. Both processes can use ubiquitin as an initiating signal but whereas mitophagy has been extensively studied, pexophagy remains rather poorly understood. Our recent work, along with a new study from Kim and colleagues, has shed light on the molecular mechanism of pexophagy and the importance of reversible ubiquitination in its regulation. Collectively, these studies highlight the physiological role of the deubiquitinase USP30 in suppressing the turnover of peroxisomes. Abbreviations: ROS: reactive oxygen species; DUB: deubiquitinase or deubiquitylase; USP: ubiquitin specific protease; PINK1: PTEN induced kinase 1; CAT: catalase; KO: knock-out; SQSTM1/p62: sequestosome 1; LIR: LC3 interacting region; GFP: green fluorescent protein; RFP: red fluorescent protein; CRISPR: Clustered Regularly Interspaced Short Palendromic Repeat.

Publisher Correction: Breaking the chains: deubiquitylating enzyme specificity begets function.

Figure 2 of the article as originally published contained a graphic editing error, whereby the publisher's redrawn figure wrongly indicated the presence of a Drosophila melanogaster orthologue of ZUP1. This has been corrected in the HTML and PDF versions of the manuscript.

Breaking the chains: deubiquitylating enzyme specificity begets function.

The deubiquitylating enzymes (DUBs, also known as deubiquitylases or deubiquitinases) maintain the dynamic state of the cellular ubiquitome by releasing conjugated ubiquitin from proteins. In light of the many cellular functions of ubiquitin, DUBs occupy key roles in almost all aspects of cell behaviour. Many DUBs show selectivity for particular ubiquitin linkage types or positions within ubiquitin chains. Others show chain-type promiscuity but can select a distinct palette of protein substrates via specific protein-protein interactions established through binding modules outside of the catalytic domain. The ubiquitin chain cleavage mode or chain linkage specificity has been related directly to biological functions. Examples include regulation of protein degradation and ubiquitin recycling by the proteasome, DNA repair pathways and innate immune signalling. DUB cleavage specificity is also being harnessed for analysis of ubiquitin chain architecture that is assembled on specific proteins. The recent development of highly specific DUB inhibitors heralds their emergence as a new class of therapeutic targets for numerous diseases.

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.