Limited proteolysis as a tool to probe the tertiary conformation of dysferlin and structural consequences of patient missense variant L344P

Woolger, Natalie; Bournazos, Adam; Sophocleous, Reece A.; Evesson, Frances J.; Lek, Angela; Driemer, Birgit; Sutton, R. Bryan; Cooper, Sandra T.

doi:10.1074/jbc.m117.790014

Cited by 8 publications

(8 citation statements)

References 50 publications

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“…However, the low increase of dysferlin expression suggests that the ubiquitin proteasome system is not the main pathway of dysferlin degradation. These results are in agreement with a previous work by Woolger et al in which they demonstrate that inhibition of autophagy had greater effect than the proteasome inhibitor in increasing levels of dysferlin expression [26]. Mutant-dysferlin is mainly degraded by autophagy while wild type-dysferlin is degraded by UPS [27].…”

Section: Discussionsupporting

confidence: 93%

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Proteasome Inhibitors Reduce Thrombospondin-1 Release in Human Dysferlin-Deficient Myotubes

Fernández‐Simón

Lleixà

Suárez‐Calvet

et al. 2019

Preprint

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Background: Dysferlin is a type-II transmembrane protein and the causative gene of dysferlinopathies, which are characterized by absence or marked reduction in dysferlin protein and muscle weakness. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. The muscle biopsy of patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Release of thrombospondin-1 (TSP-1) by dysferlin deficient muscle has been reported as a possible factor of the inflammation observed in the muscle of both human and mouse models of dysferlinopathy. It has also been reported that treatment with vitamin D3 enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic implication in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patient carrying two missense mutationsMethods: Dysferlin deficient myotubes were treated with EB1089, a vitamin D3 analog, oprozomib and ixazomib to assess proteasome inhibition. Western blot was performed to analyze the effect of the different treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using Enzyme Linked Immunosorbent Assay to analyze the effect of these drugs on its release.A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury. Data were analyzed using a one-way ANOVA test followed by by Tukey post hoc test and analysis of variance. Ap≤0.05 was considered statistically significant. Results : Treatment with proteasome inhibitors and EB1089 resulted in a slight increase of dysferlin expression which was accompanied by a low increase of myogenin expression. Also, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes from a dysferlinopathy patient. However, the increase of dysferlin had no effect on the repair of muscle membrane after injury. Conclusions: Our findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.

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Section: Discussionsupporting

confidence: 93%

“…53% of patients have nonsense mutations, 43% missense mutations and 4% small in-frame insertions or deletions [1]. While nonsense mutations produce a complete absence of the protein, a residual expression of dysferlin protein is detected in primary myotubes from patients carrying missense mutations [8][9].…”

Section: Introductionmentioning

confidence: 99%

Proteasome Inhibitors Reduce Thrombospondin-1 Release in Human Dysferlin-Deficient Myotubes

Fernández‐Simón

Lleixà

Suárez‐Calvet

et al. 2019

Preprint

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“…Figure 3 shows that not only lysines but also arginines are located on both sides of the mTSPO. Both types of residues are selectively digested by trypsin; thus, we took advantage of this to perform a limited proteolysis digestion that has been previously used to probe the conformational changes of proteins [20,21].…”

Section: Resultsmentioning

confidence: 99%

“…Proteoliposomes containing recombinant mTSPO were incubated with various concentrations of [ 3 H]-PK 11195 (SA, 83.5 Ci/mmol, Perkin Elmer SAS, Courtaboeuf, France) in phosphate buffer saline (PBS). The bound ligand was quantified by liquid scintillation spectrometry after filtration on Whatman Filters GF/C (Sigma-Aldrich, Saint Quentin Fallavier, France) according to published protocols [20,25]. The K d and B max values were determined by fitting the saturation curves with the following equation Ligand bound = ( B max × Ligand )/( K d + Ligand ).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

Iatmanen-Harbi

Senicourt

Papadopoulos

et al. 2019

IJMS

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The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

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“…To further probe the conformational changes of SARS-CoV-2 M pro , we performed a limited proteolysis assay by trypsin digestion (28). The cleavage pattern indicated a greater degree of protection of SARS-CoV-2 M pro from trypsin digestion at higher concentrations of TFQ ( Fig.…”

Section: Mechanism Of Action Of Tfq Against Sars-cov-2 M Promentioning

confidence: 99%

Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 main protease by tafenoquinein vitro

Chen

Yang

Huang

et al. 2020

Preprint

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the current pandemic, coronavirus disease 2019 (COVID-19), has taken a huge toll on human lives and the global economy. Therefore, effective treatments against this disease are urgently needed. Here, we established a fluorescence resonance energy transfer (FRET)-based high-throughput screening platform to screen compound libraries to identify drugs targeting the SARS-CoV-2 main protease (Mpro), in particular those which are FDA-approved, to be used immediately to treat patients with COVID-19. Mpro has been shown to be one of the most important drug targets among SARS-related coronaviruses as impairment of Mpro blocks processing of viral polyproteins which halts viral replication in host cells. Our findings indicate that the anti-malarial drug tafenoquine (TFQ) induces significant conformational change in SARS-CoV-2 Mpro and diminishes its protease activity. Specifically, TFQ reduces the alpha-helical content of Mpro, which converts it into an inactive form. Moreover, TFQ greatly inhibits SARS-CoV-2 infection in cell culture system. Hence, the current study provides a mechanistic insight into the mode of action of TFQ against SARS-CoV-2 Mpro. Moreover, the low clinical toxicity of TFQ and its strong antiviral activity against SARS-CoV-2 should warrant further testing in clinical trials.

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Limited proteolysis as a tool to probe the tertiary conformation of dysferlin and structural consequences of patient missense variant L344P

Cited by 8 publications

References 50 publications

Proteasome Inhibitors Reduce Thrombospondin-1 Release in Human Dysferlin-Deficient Myotubes

Proteasome Inhibitors Reduce Thrombospondin-1 Release in Human Dysferlin-Deficient Myotubes

Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 main protease by tafenoquinein vitro

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