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

Molecular basis of USP7 inhibition by selective small-molecule inhibitors

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice

Performance evaluation of a novel chemiluminescence assay for detection of anti-GBM antibodies: an international multicenter study

Background. Autoantibodies to the non-collagen region (NC1) of the alpha-3 subunit of collagen IV represent a serological hallmark in the diagnosis of Goodpasture's syndrome (GPS). The objective of our study was to carefully analyze the performance characteristics of a novel anti-glomerular basement membrane (GBM) chemiluminescence immunoassay (CIA). Methods. Sera from patients with GPS (n = 90) were collected from four clinical centers. Samples from different disease groups (n = 397) and healthy individuals (n = 400) were used as controls. All samples were tested for anti-GBM antibodies by a rapid, random access CIA (QUANTA Flash (TM) GBM). Most of the samples were also tested using other methods including different commercial anti-GBM IgG assays and research assays for anti-GBM IgA and IgM. Results. The sensitivity and specificity of the novel CIA was 95.6% [95% confidence interval (CI) 89.0-98.8%] and 99.6% (95% CI 98.9-99.9%), respectively. Receiver operating characteristic analysis showed good discrimination between GPS patients and controls. The area under the curve was 0.98 (CI 0.96-1.0). The three anti-GBM antibody-positive samples from the control group were from two healthy individuals and one human immunodeficiency virus (HIV)-infected patient. All three individuals had low levels of anti-GBM antibodies [20, 24 and 25 chemiluminescent unit (CU), cutoff 20 CU]. When the results of the new CIA were compared to other methods, good agreement was observed: 95.8% (kappa = 0.92) versus EliA (TM) GBM, 97.4% (kappa = 0.95) versus both BINDAZYME (TM) Anti-GBM and QUANTA Lite (R) GBM. Anti-GBM IgA was detectable in low concentrations in patients with GPS and was associated with anti-GBM IgG but was less useful in discriminating GPS patients and controls. No discrimination was found for anti-GBM IgM. Conclusion. The novel QUANTA Flash (TM) GBM CIA demonstrated good sensitivity and specificity and had good agreement with other methods. Our data confirm that similar to 5% of patients with GPS do not have detectable levels of anti-GBM antibodies.TransplantationUrology & NephrologySCI(E)4ARTICLE1243-U2952

Transfer of chirality in radical cyclizations. Cyclization of o- haloacrylanilides to oxindoles with transfer of axial chirality to a newly formed stereocenter [6]

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Molecular basis of USP7 inhibition by selective small-molecule inhibitors.

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice