Biology subjects

Guenther, F.

Publications and source records attributed to Guenther, F..

6 recordsLinked to original sources

bioRxiv2025

USP30 inhibition improves mitochondrial health through both PINK1-dependent and independent mechanisms

Mitochondrial dysfunction is a key feature of many pathologies, including Parkinsons disease. The selective vulnerability of dopaminergic neurons is thought to be influenced by mitochondrial dysfunction and mutations in the mitophagy regulating proteins PINK1 and Parkin that are known to cause early-onset Parkinsonism in an autosomal recessive manner. Augmentation of mitophagy through inhibition of USP30 may be a viable therapeutic strategy for a number of diseases including Parkinsons. USP30 inhibition has been demonstrated to augment PINK1/PRKN mitophagy but also potentiate basal mitophagy to support the removal of dysfunctional mitochondria. Therefore, long-term de-regulation of mitophagy has been proposed to lead to mitochondrial depletion. We have used an integrated approach across cell lines, primary neurons and iPSC-derived dopaminergic neuronal cultures to assess the short and long-term effects of USP30 inhibition on mitochondrial health and neuronal activity. We investigated the dependence of USP30 inhibition phenotypes on the PINK1/Parkin pathway using genetic ablation and in iPSC-derived neurons from Parkinsons patients with PINK1 or PRKN mutations. Loss of USP30 through CRISPR/Cas9-mediated knockout resulted in increased basal and depolarisation-induced mitophagy in SH-SY5Y cells. Loss of USP30 or pharmacological inhibition altered mitochondrial morphology and led to increases in membrane potential and ATP levels with decreased oxygen consumption, suggesting that USP30 loss results in a more efficient mitochondrial network. These changes in morphology were found to be independent of PINK1 or Parkin. Chronic pharmacological inhibition of USP30 or CRISPRi-mediated knockdown of USP30 did not impact dopaminergic neuronal activity, as assessed by electrophysiological profiling, but did potentiate depolarisation-induced mitophagy in primary and iPSC-derived neuronal cultures. We observed minimal changes in mitophagy levels in iPSC-derived dopaminergic neurons from Parkinsons patients with PINK1 or PRKN mutations that were independent of the ability to produce p65Ub. Importantly, within this experimental paradigm, pharmacological USP30 inhibition increased depolarisation-induced mitophagy in both PINK1 and PRKN patients to the same extent as control neurons. These results support a role for USP30 in modulating the trigger threshold for mitophagy and suggest that USP30 inhibitors may be beneficial in patients with impairments in PINK1/Parkin-mediated mitophagy.

neuroscience↗

bioRxiv2024

Structural Dynamics of the Ubiquitin Specific Protease USP30 in Complex with a Cyanopyrrolidine-Containing Covalent Inhibitor

Inhibition of the mitochondrial deubiquitinating enzyme USP30 is neuroprotective and presents therapeutic opportunities for the treatment of idiopathic Parkinsons Disease and mitophagy-related disorders. We have integrated structural and quantitative proteomics with biochemical assays to decipher the mode of action of covalent USP30 inhibition by a small molecule containing a cyanopyrrolidine reactive group, USP30-I-1. The inhibitor demonstrated high potency and selectivity for endogenous USP30 in neuroblastoma cells. Enzyme kinetics and Hydrogen Deuterium eXchange mass spectrometry (HDX-MS) infers that the inhibitor binds tightly to regions surrounding the USP30 catalytic cysteine and positions itself to form a binding pocket along the thumb and palm domains of the protein, thereby interfering its interaction with ubiquitin substrates. A comparison to a non-covalent USP30 inhibitor containing a benzosulfonamide scaffold revealed a slightly different binding mode closer to the active site Cys77, which may provide the molecular basis for improved selectivity towards USP30 against other members of the DUB enzyme family. Our results highlight advantages in developing covalent inhibitors, such as USP30-I-1, for targeting USP30 as treatment of disorders with impaired mitophagy.

biochemistry↗

bioRxiv2022

Structural Premise of Selective Deubiquitinase USP30 Inhibition by Small-Molecule Benzosulfonamides

Dampening functional levels of the mitochondrial deubiquitylating enzyme USP30 has been suggested as an effective therapeutic strategy against neurodegenerative disorders such as Parkinsons Disease. USP30 inhibition may counteract the deleterious effects of impaired turnover of damaged mitochondria which is inherent to both familial and sporadic forms of the disease. Small-molecule inhibitors targeting USP30 are currently in development, but little is known about their precise nature of binding to the protein. We have integrated biochemical and structural approaches to gain novel mechanistic insights into USP30 inhibition by a small-molecule benzosulfonamide containing compound, 39. Activity-based protein profiling (ABPP) mass spectrometry confirmed target engagement, the high selectivity, and potency of 39 for USP30 against 49 other deubiquitylating enzymes in a neuroblastoma cell line. In vitro characterization of 39 enzyme kinetics infers slow and tight binding behavior, which is comparable with features of covalent modification of USP30. Finally, we blended hydrogen-deuterium exchange mass spectrometry and computational docking to elucidate the molecular architecture and geometry of USP30 complex formation with 39, identifying structural rearrangements at the cleft of the USP30 thumb and palm subdomains. These studies suggest that 39 binds to the thumb-palm cleft that guides the ubiquitin C-terminus into the active site, thereby preventing ubiquitin binding and isopeptide bond cleavage, and confirming its importance in the inhibitory process. Our data will pave the way for the design and development of next-generation inhibitors targeting USP30 and associated deubiquitinylases.

biochemistry↗

bioRxiv2020

A novel USP30 inhibitor recapitulates genetic loss of USP30 and sets the trigger for PINK1-PARKIN amplification of mitochondrial ubiquitylation.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinsons Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. 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 most 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. In this model, 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 and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.

cell biology↗

bioRxiv2019

Genome-wide association meta-analysis for early age-related macular degeneration highlights novel loci and insights for advanced disease

BackgroundAdvanced age-related macular degeneration (AMD) is a leading cause of blindness. While around half of the genetic contribution to advanced AMD has been uncovered, little is known about the genetic architecture of the preceding early stages of the diseases. MethodsTo identify genetic factors for early AMD, we conducted a genome-wide association meta-analysis with 14,034 early AMD cases and 91,214 controls from 11 sources of data including data from the International AMD Genomics Consortium (IAMDGC) and the UK Biobank (UKBB). We ascertained early AMD via color fundus photographs by manual grading for 10 sources and by using an automated machine learning approach for >170,000 images from UKBB. We searched for significant genetic loci in a genome-wide association screen (P<5x10-8) based on the meta-analysis of the 11 sources and via a candidate approach based on 13 suggestive early AMD variants from Holliday et al 2013 (P<0.05/13, additional 3,432 early AMD cases and 11,235 controls). For the novel AMD regions, we conducted in-silico follow-up analysis to prioritize causal genes and pathway analyses. ResultsWe identified 11 loci for early AMD, 9 novel and 2 known for early AMD. Most of these 11 loci overlapped with known advanced AMD loci (near ARMS2/HTRA1, CFH, APOE, C2, C3, CETP, PVRL2, TNFRSF10A, VEGFA), except two that were completely novel to any AMD. Among the 17 genes within the two novel loci, in-silico functional annotation suggested CD46 and TYR as the most likely responsible genes. We found the presence or absence of an early AMD effect to distinguish known pathways of advanced AMD genetics (complement/lipid pathways or extracellular matrix metabolism, respectively). ConclusionsOur data on early AMD genetics provides a resource comparable to the existing data on advanced AMD genetics, which enables a joint view. Our large GWAS on early AMD identified novel loci, highlighted shared and distinct genetics between early and advanced AMD and provides insights into AMD etiology. The ability of early AMD effects to differentiate the major pathways for advanced AMD underscores the biological relevance of a joint view on early and advanced AMD genetics.

genetics↗

bioRxiv2019

Chances and challenges of machine learning based disease classification in genetic association studies illustrated on age-related macular degeneration

Imaging technology and machine learning algorithms for disease classification set the stage for high-throughput phenotyping and promising new avenues for genome-wide association studies (GWAS). Despite emerging algorithms, there has been no successful application in GWAS so far. We established machine learning based disease classification in genetic association analysis as a misclassification problem. To evaluate chances and challenges, we performed a GWAS based on automated classification of age-related macular degeneration (AMD) in UK Biobank (images from 135,500 eyes; 68,400 persons). We quantified misclassification of automatically derived AMD in internal validation data (images from 4,001 eyes; 2,013 persons) and developed a maximum likelihood approach (MLA) to account for it when estimating genetic association. We demonstrate that our MLA guards against bias and artefacts in simulation studies. By combining a GWAS on automatically derived AMD classification and our MLA in UK Biobank data, we were able to dissect true association (ARMS2/HTRA1, CFH) from artefacts (near HERC2) and to identify eye color as relevant source of misclassification. On this example of AMD, we are able to provide a proof-of-concept that a GWAS using machine learning derived disease classification yields relevant results and that misclassification needs to be considered in the analysis. These findings generalize to other phenotypes and also emphasize the utility of genetic data for understanding misclassification structure of machine learning algorithms.

genetics↗