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

2021 ◽  
Vol 5 (2) ◽  
pp. e202101287
Author(s):  
Emma V Rusilowicz-Jones ◽  
Francesco G Barone ◽  
Fernanda Martins Lopes ◽  
Elezabeth Stephen ◽  
Heather Mortiboys ◽  
...  
Keyword(s):  
Parkinson Disease ◽  
Dopaminergic Neurons ◽  
Enzymatic Assay ◽  
Pharmacological Intervention ◽  
Loss Of Function ◽  
Biological Characterization ◽  
Mitochondrial Depolarization ◽  
Synthetic Molecule

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.

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

2021 ◽  
Author(s):  
Emma V. Rusilowicz-Jones ◽  
Francesco G. Barone ◽  
Fernanda Martins Lopes ◽  
Elizabeth Stephen ◽  
Heather Mortiboys ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Pulmonary Fibrosis ◽  
Dopaminergic Neurons ◽  
Enzymatic Assay ◽  
Pharmacological Intervention ◽  
Loss Of Function ◽  
Synthetic Molecule

AbstractThe deubiquitylase USP30 is an actionable target considered for treatment of conditions associated with defects in the PINK1/Parkin pathway leading to mitophagy. These include Parkinson’s disease and pulmonary fibrosis. We provide a detailed cell biological characterisation of a benzenesulphonamide 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 following mitochondrial depolarisation. This includes enhancement of TOM20 and SYNJ2BP ubiquitylation and phosphoubiquitin accumulation, alongside increased mitophagy. In dopaminergic neurons, generated from Parkinson’s disease patients carrying loss of function Parkin 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 U20S 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.

scholarly journals Multiple Pathways of LRRK2-G2019S/Rab10 Interaction in Dopaminergic Neurons

2021 ◽  
pp. 1-16
Author(s):  
Alison Fellgett ◽  
C. Adam Middleton ◽  
Jack Munns ◽  
Chris Ugbode ◽  
David Jaciuch ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Dopaminergic Neurons ◽  
Genetic Interaction ◽  
In Vitro Assays ◽  
Rab Proteins ◽  
Circadian Activity ◽  
Loss Of Function ◽  
Lrrk2 G2019s

Background: Inherited mutations in the LRRK2 protein are the common causes of Parkinson’s disease, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10. Objective: To determine if Rab10 is necessary for LRRK2-induced pathophysiological responses in the neurons that control movement, vision, circadian activity, and memory. These four systems were chosen because they are modulated by dopaminergic neurons in both humans and flies. Methods: LRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, retinal neurophysiology, circadian activity pattern (‘sleep’), and courtship memory determined in aged flies. Results: Rab10 loss-of-function rescued LRRK2-G2019S induced bradykinesia and retinal signaling deficits. Rab10 knock-down, however, did not rescue the marked sleep phenotype which results from dopaminergic LRRK2-G2019S. Courtship memory is not affected by LRRK2, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons. Conclusion: We conclude that, in Drosophila dopaminergic neurons, Rab10 is involved in some, but not all, LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinson’s disease.

scholarly journals Physical and Biological Characterization of Ferromagnetic Fiber Networks: Effect of Fibrin Deposition on Short-Term In Vitro Responses of Human Osteoblasts

2015 ◽  
Vol 21 (3-4) ◽  
pp. 463-474 ◽  
Author(s):  
Rose L. Spear ◽  
Brajith Srigengan ◽  
Suresh Neelakantan ◽  
Wolfram Bosbach ◽  
Roger A. Brooks ◽  
...  
Keyword(s):  
Biological Characterization ◽  
Fibrin Deposition ◽  
Short Term ◽  
Human Osteoblasts ◽  
Fiber Networks

scholarly journals In Vitro Biological Characterization of DCUN1D5 in DNA Damage Response

2012 ◽  
Vol 13 (8) ◽  
pp. 4157-4162 ◽  
Author(s):  
Wei Guo ◽  
Guo-Jun Li ◽  
Hong-Bo Xu ◽  
Jie-Shi Xie ◽  
Tai-Ping Shi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Biological Characterization ◽  
Damage Response

scholarly journals Dysbindin deficiency Alters Cardiac BLOC-1 Complex and Myozap Levels in Mice

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2390
Author(s):  
Ankush Borlepawar ◽  
Nesrin Schmiedel ◽  
Matthias Eden ◽  
Lynn Christen ◽  
Alexandra Rosskopf ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Cardiomyocyte Hypertrophy ◽  
Loss Of Function ◽  
Interaction Partners ◽  
Lysosome Related Organelles ◽  
The Stability ◽  
Schizophrenia Susceptibility

Dysbindin, a schizophrenia susceptibility marker and an essential constituent of BLOC-1 (biogenesis of lysosome-related organelles complex-1), has recently been associated with cardiomyocyte hypertrophy through the activation of Myozap-RhoA-mediated SRF signaling. We employed sandy mice (Dtnbp1_KO), which completely lack Dysbindin protein because of a spontaneous deletion of introns 5–7 of the Dtnbp1 gene, for pathophysiological characterization of the heart. Unlike in vitro, the loss-of-function of Dysbindin did not attenuate cardiac hypertrophy, either in response to transverse aortic constriction stress or upon phenylephrine treatment. Interestingly, however, the levels of hypertrophy-inducing interaction partner Myozap as well as the BLOC-1 partners of Dysbindin like Muted and Pallidin were dramatically reduced in Dtnbp1_KO mouse hearts. Taken together, our data suggest that Dysbindin’s role in cardiomyocyte hypertrophy is redundant in vivo, yet essential to maintain the stability of its direct interaction partners like Myozap, Pallidin and Muted.

Preparation and Biological Evaluation of Si-Substituted HA Ceramics with Controlled Composition

2007 ◽  
Vol 361-363 ◽  
pp. 1059-1062
Author(s):  
Mickael Palard ◽  
J. Combes ◽  
Eric Champion ◽  
Didier Bernache-Assollant
Keyword(s):  
Thermal Treatment ◽  
Biological Evaluation ◽  
Precipitation Process ◽  
Biological Characterization ◽  
Hydroxyapatite Ceramics

This work aimed at preparing dense and monophasic silicated hydroxyapatite ceramics over the range 0 ≤ x ≤ 1.0 mol of silicon. The synthesis of the powder via an aqueous precipitation process followed by an adapted thermal treatment showed that it was possible to obtain dense single-phased apatite ceramics containing up to 0.6 mol of silicon. The in vitro biological characterization of these materials was performed.

scholarly journals Characterization of a Spontaneous 9.5-Kilobase-Deletion Mutant of Murine Gammaherpesvirus 68 Reveals Tissue-Specific Genetic Requirements for Latency

2002 ◽  
Vol 76 (13) ◽  
pp. 6532-6544 ◽  
Author(s):  
Eric T. Clambey ◽  
Herbert W. Virgin ◽  
Samuel H. Speck
Keyword(s):  
Deletion Mutant ◽  
Latent Infection ◽  
Loss Of Function ◽  
Murine Gammaherpesvirus ◽  
Peritoneal Cells ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (γHV68 [also known as MHV-68]) establishes a latent infection in mice, providing a small-animal model with which to identify host and viral factors that regulate gammaherpesvirus latency. While γHV68 establishes a latent infection in multiple tissues, including splenocytes and peritoneal cells, the requirements for latent infection within these tissues are poorly defined. Here we report the characterization of a spontaneous 9.5-kb-deletion mutant of γHV68 that lacks the M1, M2, M3, and M4 genes and eight viral tRNA-like genes. Previously, this locus has been shown to contain the latency-associated M2, M3, and viral tRNA-like genes. Through characterization of this mutant, we found that the M1, M2, M3, M4 genes and the viral tRNA-like genes are dispensable for (i) in vitro replication and (ii) the establishment and maintenance of latency in vivo and reactivation from latency following intraperitoneal infection. In contrast, following intranasal infection with this mutant, there was a defect in splenic latency at both early and late times, a phenotype not observed in peritoneal cells. These results indicate (i) that there are different genetic requirements for the establishment of latency in different latent reservoirs and (ii) that the genetic requirements for latency depend on the route of infection. While some of these phenotypes have been observed with specific mutations in the M1 and M2 genes, other phenotypes have never been observed with the available γHV68 mutants. These studies highlight the importance of loss-of-function mutations in defining the genetic requirements for the establishment and maintenance of herpesvirus latency.

Biological characterization of charge isomers of human growth hormone

1988 ◽  
Vol 118 (1) ◽  
pp. 14-21 ◽  
Author(s):  
A. Skottner ◽  
A. Forsman ◽  
B. Skoog ◽  
J. L. Kostyo ◽  
C. M. Cameron ◽  
...  
Keyword(s):  
Biological Activity ◽  
Biological Activities ◽  
Human Growth ◽  
Proteolytic Processing ◽  
Biological Characterization ◽  
Growth Promoting ◽  
Rat Adipose Tissue ◽  
Insulin Like Activity

Abstract. Since deamidation of the human GH molecule may alter the manner and extent to which the hormone is cleaved by proteases, and since it has been repeatedly suggested that proteolytic processing is required for the expression of certain of the activities of GH, the present study was conducted to determine whether the biological activity profiles of more acidic forms of human GH are altered. Three charge isomers, GH-b, GH-c and GH-d, representing primarily deamidated forms, were isolated from a native human GH preparation (Crescormon®) in amounts adequate for characterization of their biological activities. All three were essentially equipotent in a radioimmunoassay for human GH. When assessed for growth-promoting activity in the hypophysectomized rat, the isomers were again equipotent with each other and with the GH preparation from which they were derived. The charge isomers also had significant in vitro insulin-like activity on isolated rat adipose tissue and diabetogenic activity in the ob/ob mouse. Thus, the biological activity profiles of these charge isomers of human GH do not differ greatly from one another.

In vitro biological characterization of macroporous 3D Bonelike® structures prepared through a 3D machining technique

2009 ◽  
Vol 29 (3) ◽  
pp. 930-935 ◽  
Author(s):  
M.S. Laranjeira ◽  
A.G. Dias ◽  
J.D. Santos ◽  
M.H. Fernandes
Keyword(s):  
Biological Characterization
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