ORCID Profile
0000-0002-2181-9491
Current Organisations
Murdoch University
,
Perron Institute for Neurological and Translational Science
,
University of Western Australia
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Publications
Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70
Publisher: Springer Science and Business Media LLC
Date: 2004
DOI: 10.1379/CSC-26R.1
Abstract: The high-affinity ligand-binding form of unactivated steroid receptors exists as a multicomponent complex that includes heat shock protein (Hsp)90 one of the immunophilins cyclophilin 40 (CyP40), FKBP51, or FKBP52 and an additional p23 protein component. Assembly of this heterocomplex is mediated by Hsp70 in association with accessory chaperones Hsp40, Hip, and Hop. A conserved structural element incorporating a tetratricopeptide repeat (TPR) domain mediates the interaction of the immunophilins with Hsp90 by accommodating the C-terminal EEVD peptide of the chaperone through a network of electrostatic and hydrophobic interactions. TPR cochaperones recognize the EEVD structural motif common to both Hsp90 and Hsp70 through a highly conserved cl domain. In the present study, we investigated in vitro the molecular interactions between CyP40 and FKBP52 and other stress-related components involved in steroid receptor assembly, namely Hsp70 and Hop. Using a binding protein-retention assay with CyP40 fused to glutathione S-transferase immobilized on glutathione-agarose, we have identified the constitutively expressed form of Hsp70, heat shock cognate (Hsc)70, as an additional target for CyP40. Deletion mapping studies showed the binding determinants to be similar to those for CyP40-Hsp90 interaction. Furthermore, a mutational analysis of CyP40 cl domain residues confirmed the importance of this motif in CyP40-Hsc70 interaction. Additional residues thought to mediate binding specificity through hydrophobic interactions were also important for Hsc70 recognition. CyP40 was shown to have a preference for Hsp90 over Hsc70. Surprisingly, FKBP52 was unable to compete with CyP40 for Hsc70 binding, suggesting that FKBP52 discriminates between the TPR cochaperone-binding sites in Hsp90 and Hsp70. Hop, which contains multiple units of the TPR motif, was shown to be a direct competitor with CyP40 for Hsc70 binding. Similar to Hop, CyP40 was shown not to influence the adenosine triphosphatase activity of Hsc70. Our results suggest that CyP40 may have a modulating role in Hsc70 as well as Hsp90 cellular function.
The endoplasmic reticulum‐associated protein, OS‐9, behaves as a lectin in targeting the immature calcium‐sensing receptor
Publisher: Wiley
Date: 05-06-2017
DOI: 10.1002/JCP.25957
Abstract: The mechanisms responsible for the processing and quality control of the calcium‐sensing receptor (CaSR) in the endoplasmic reticulum (ER) are largely unknown. In a yeast two‐hybrid screen of the CaSR C‐terminal tail (residues 865–1078), we identified osteosarcoma‐9 (OS‐9) protein as a binding partner. OS‐9 is an ER‐resident lectin that targets misfolded glycoproteins to the ER‐associated degradation (ERAD) pathway through recognition of specific N ‐glycans by its mannose‐6‐phosphate receptor homology (MRH) domain. We show by confocal microscopy that the CaSR and OS‐9 co‐localize in the ER in COS‐1 cells. In immunoprecipitation studies with co‐expressed OS‐9 and CaSR, OS‐9 specifically bound the immature form of wild‐type CaSR in the ER. OS‐9 also bound the immature forms of a CaSR C‐terminal deletion mutant and a C677A mutant that remains trapped in the ER, although binding to neither mutant was favored over wild‐type receptor. OS‐9 binding to immature CaSR required the MRH domain of OS‐9 indicating that OS‐9 acts as a lectin most likely to target misfolded CaSR to ERAD. Our results also identify two distinct binding interactions between OS‐9 and the CaSR, one involving both C‐terminal domains of the two proteins and the other involving both N‐terminal domains. This suggests the possibility of more than one functional interaction between OS‐9 and the CaSR. When we investigated the functional consequences of altered OS‐9 expression, neither knockdown nor overexpression of OS‐9 was found to have a significant effect on CaSR cell surface expression or CaSR‐mediated ERK1/2 phosphorylation.
The role of sequestosome 1/p62 protein in amyotrophic lateral sclerosis and frontotemporal dementia pathogenesis
Publisher: Medknow
Date: 2020
p62 overexpression induces TDP-43 cytoplasmic mislocalisation, aggregation and cleavage and neuronal death
Publisher: Springer Science and Business Media LLC
Date: 06-2021
DOI: 10.1038/S41598-021-90822-2
Abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) that exist on a spectrum of neurodegenerative disease. A hallmark of pathology is cytoplasmic TDP-43 aggregates within neurons, observed in 97% of ALS cases and ~ 50% of FTLD cases. This mislocalisation from the nucleus into the cytoplasm and TDP-43 cleavage are associated with pathology, however, the drivers of these changes are unknown. p62 is invariably also present within these aggregates. We show that p62 overexpression causes TDP-43 mislocalisation into cytoplasmic aggregates, and aberrant TDP-43 cleavage that was dependent on both the PB1 and ubiquitin-associated (UBA) domains of p62. We further show that p62 overexpression induces neuron death. We found that stressors (proteasome inhibition and arsenic) increased p62 expression and that this shifted the nuclear:cytoplasmic TDP-43 ratio. Overall, our study suggests that environmental factors that increase p62 may thereby contribute to TDP-43 pathology in ALS and FTLD.
Molecular Mechanisms Underlying TDP-43 Pathology in Cellular and Animal Models of ALS and FTLD
Publisher: MDPI AG
Date: 29-04-2021
DOI: 10.3390/IJMS22094705
Abstract: Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are neurodegenerative disorders that exist on a disease spectrum due to pathological, clinical and genetic overlap. In up to 97% of ALS cases and ~50% of FTLD cases, the primary pathological protein observed in affected tissues is TDP-43, which is hyperphosphorylated, ubiquitinated and cleaved. The TDP-43 is observed in aggregates that are abnormally located in the cytoplasm. The pathogenicity of TDP-43 cytoplasmic aggregates may be linked with both a loss of nuclear function and a gain of toxic functions. The cellular processes involved in ALS and FTLD disease pathogenesis include changes to RNA splicing, abnormal stress granules, mitochondrial dysfunction, impairments to axonal transport and autophagy, abnormal neuromuscular junctions, endoplasmic reticulum stress and the subsequent induction of the unfolded protein response. Here, we review and discuss the evidence for alterations to these processes that have been reported in cellular and animal models of TDP-43 proteinopathy.
A novel mutation (K378X) in the sequestosome 1 gene associated with increased NF-kappaB signaling and Paget's disease of bone with a severe phenotype.
Publisher: Wiley
Date: 07-2006
DOI: 10.1359/JBMR.060405
Abstract: Sequestosome 1 62 (p62) mutations are associated with PDB however, there are limited data regarding functional consequences. We report a novel mutation in exon 7 (K378X) in a patient with polyostotic Paget's disease of bone. p62 mutants increased NF-kappaB activation and significantly potentiated osteoclast formation and bone resorption in human primary cell cultures. Sequestosome 1 62 (p62) mutations are associated with Paget's disease of bone (PDB) however, there are limited data regarding functional consequences. One report has linked the common P392L mutation in the p62 ubiquitin binding associated (UBA) domain with increases in NF-kappaB activity, a transcription factor essential for osteoclastogenesis. To further clarify the functional impact of p62 mutations associated with PDB, we assessed the effect of p62 mutation (a novel mutation: K378X, and previously reported mutations: P392L and E396X) on RANK-induced NF-kappaB activation and compared this with the effect of wildtype p62. In addition, we studied the effect of p62 mutation on osteoclast formation and bone resorption. We performed co-transfection experiments with expression plasmids for p62 (wildtype or mutated) and RANK and an NF-kappaB luciferase reporter gene. Luciferase activities were recorded after addition of luciferin to cellular lysates. RAW(264.7) cells stably expressing enhanced green fluorescent protein (EGFP)-tagged p62 (wildtype, K378X, or P392L) or EGFP alone were assessed for changes in cell proliferation. Additionally, these cells were stimulated with RANKL to produce osteoclast-like cells (OLCs). Primary human monocytes collected from the K378X-affected patient and a control subject were stimulated to form OLCs and bone resorption data were obtained. The novel mutation introduces a premature stop codon in place of Lys-378 and thereby eliminates the entire p62 UBA domain this and two additional natural mutations (P392L, E396X) increased NF-kappaB activation compared with wildtype p62. Wildtype p62 consistently inhibited NF-kappaB activation compared with empty vector. UBA mutations (K378X and P392L) significantly increased the number of OLCs formed in response to RANKL and also the number of nuclei of the OLCs. K378X-affected human monocytes formed more OLCs with more nuclei and increased bone resorption compared with control monocytes. Our data show that mutation of the p62 UBA domain results in increased activation of NF-kappaB and osteoclast formation and function compared with wildtype p62. These results may partially explain the mechanism by which p62 mutation contributes to the pathogenesis of PDB.
The helix 1-3 loop in the glucocorticoid receptor LBD is a regulatory element for FKBP cochaperones
Publisher: The Endocrine Society
Date: 07-2013
DOI: 10.1210/ME.2012-1023
Abstract: The heat-shock protein 90 (Hsp90) cochaperone FK506-binding protein 52 (FKBP52) upregulates, whereas FKBP51 inhibits, hormone binding and nuclear targeting of the glucocorticoid receptor (GR). Decreased cortisol sensitivity in the guinea pig is attributed to changes within the helix 1 to helix 3 (H1-H3) loop of the guinea pig GR (gpGR) ligand-binding domain. It has been proposed that this loop serves as a contact point for FKBP52 and/or FKBP51 with receptor. We examined the role of the H1-H3 loop in GR activation by FKBP52 using a Saccharomyces cerevisiae model. The activity of rat GR (rGR) containing the gpGR H1-H3 loop substitutions was still potentiated by FKBP52, confirming the loop is not involved in primary FKBP52 interactions. Additional assays also excluded a role for other intervening loops between ligand-binding domain helices in direct interactions with FKBP52 associated with enhanced receptor activity. Complementary studies in FKBP51-deficient mouse embryo fibroblasts and HEK293 cells demonstrated that substitution of the gpGR H1-H3 loop residues into rGR dramatically increased receptor repression by FKBP51 without enhancing receptor-FKBP51 interaction and did not alter recruitment of endogenous Hsp90 and the p23 cochaperone to receptor complexes. FKBP51 suppression of the mutated rGR did not require FKBP51 peptidylprolyl cis-trans isomerase activity and was not disrupted by mutation of the FK1 proline-rich loop thought to mediate reciprocal FKBP influences on receptor activity. We conclude that the gpGR-specific mutations within the H1-H3 loop confer global changes within the GR-Hsp90 complex that favor FKBP51 repression over FKBP52 potentiation, thus identifying the loop as an important target for GR regulation by the FKBP cochaperones.
Sequestosome 1 Mutations in Paget's Disease of Bone in Australia: Prevalence, Genotype/Phenotype Correlation, and a Novel Non‐UBA Domain Mutation (P364S) Associated With Increased NF‐κB Signaling With
Publisher: Wiley
Date: 07-2009
DOI: 10.1359/JBMR.090214
Abstract: Previously reported Sequestosome 1(SQSTM1) 62 gene mutations associated with Paget's disease of bone (PDB) cluster in, or cause deletion of, the ubiquitin-associated (UBA) domain. The aims of this study were to examine the prevalence of SQSTM1 mutations in Australian patients, genotype henotype correlations and the functional consequences of a novel point mutation (P364S) located upstream of the UBA. Mutation screening of the SQSTM1 gene was conducted on 49 kindreds with PDB. In addition, 194 subjects with apparently sporadic PDB were screened for the common P392L mutation by restriction enzyme digestion. HEK293 cells stably expressing RANK were co-transfected with expression plasmids for SQSTM1 (wildtype or mutant) or empty vector and a NF-kappaB luciferase reporter gene. GST-SQSTM1 (wildtype and mutant) proteins were used in pull-down assays to compare monoubiquitin-binding ability. We identified SQSTM1 mutations in 12 of 49 families screened (24.5%), comprising 9 families with the P392L mutation and 1 family each with the following mutations: K378X, 390X, and a novel P364S mutation in exon 7, upstream of the UBA. The P392L mutation was found in 9 of 194 (4.6%) patients with sporadic disease. Subjects with SQSTM1 mutations had more extensive disease, but not earlier onset, compared with subjects without mutations. In functional studies, the P364S mutation increased NF-kappaB activation compared with wildtype SQSTM1 but did not reduce ubiquitin binding. This suggests that increased NF-kappaB signaling, but not the impairment of ubiquitin binding, may be essential in the pathogenesis of PDB associated with SQSTM1 mutations.
Mutant SQSTM1/p62 Signaling in Paget's Disease of Bone
Publisher: Elsevier
Date: 2016
Disulfiram Attenuates Osteoclast Differentiation In Vitro: A Potential Antiresorptive Agent
Publisher: Public Library of Science (PLoS)
Date: 30-04-2015
The Changing Presentation of Paget’s Disease of Bone in Australia, A High Prevalence Region
Publisher: Springer Science and Business Media LLC
Date: 07-09-2017
DOI: 10.1007/S00223-017-0312-1
Abstract: Studies from several countries suggest that the incidence of Paget's disease of bone (PDB) and the severity of newly diagnosed cases are declining. The aim of this study was to examine secular changes in clinical presentation of PDB in Australia, which historically had the highest prevalence outside the United Kingdom. The participants were 293 patients (61% male) diagnosed between 1956 and 2013 with details recorded in the database of the Paget's Disease Research Group of Western Australia. The mean age at diagnosis was 62 years (range 28-90) 26% of participants had a family history of PDB and 11% had Sequestosome 1 (SQSTM1) mutations. After adjustment for covariates (SQSTM1 mutation status, family history, country of birth, smoking and dog exposure), there was a significant positive relationship between year of diagnosis and age at diagnosis (P < 0.001) and significant negative relationships between year of diagnosis and both pre-treatment total plasma alkaline phosphatase activity (ALP) and number of involved bones (P < 0.001 for each). Patients with SQSTM1 mutations had more extensive disease (P < 0.001) and higher pre-treatment ALP (P = 0.013). In subgroup analyses, relationships between year of diagnosis and each of age at diagnosis, number of involved bones and ALP were similar in patients with sporadic or familial disease, and in patients with and without SQSTM1 mutations. We conclude that the severity of PDB in Western Australia has declined over recent decades. This is likely to reflect altered exposure to one or more environmental agents involved in pathogenesis.
ALS-FTLD associated mutations of SQSTM1 impact on Keap1-Nrf2 signalling
Publisher: Elsevier BV
Date: 10-2016
The S349T mutation of SQSTM1 links Keap1/Nrf2 signalling to Paget's disease of bone
Publisher: Elsevier BV
Date: 02-2013
DOI: 10.1016/J.BONE.2012.10.023
Abstract: Mutations affecting the Sequestosome 1 (SQSTM1) gene commonly occur in patients with the skeletal disorder Paget's disease of bone (PDB), a condition characterised by defective osteoclast differentiation and function. Whilst most mutations cluster within the ubiquitin-associated (UBA) domain of the SQSTM1 protein, and are associated with dysregulated NFκB signalling, several non-UBA domain mutations have also been identified. Keap1 is a SQSTM1-interacting protein that regulates the levels and activity of the Nrf2 transcription factor. This in turn controls the expression of numerous cytoprotective genes that contribute to the cell's capacity to defend itself against chemical and oxidative stress, through binding to the antioxidant response element (ARE). The PDB-associated S349T mutation maps to the Keap1-interacting region (KIR) of SQSTM1, however the effects of PDB mutant SQSTM1 on Keap1 function have not been investigated. Here we show that unlike other SQSTM1 mutations, the S349T mutation results in neither impaired ubiquitin-binding function in pull-down assays, nor dysregulated NFκB signalling in luciferase reporter assays. Keap1 is expressed in differentiating osteoclast-like cells and the S349T mutation selectively impairs the SQSTM1-Keap1 interaction in co-immunoprecipitations, which molecular modelling indicates results from effects on critical hydrogen bonds required to stabilise the KIR-Keap1 complex. Further, S349T mutant SQSTM1, but not other PDB-associated mutants, showed reduced ability to activate Nrf2 signalling as assessed by ARE-luciferase reporter assays. Thus, SQSTM1-mediated dysregulation of the Keap1-Nrf2 axis, which could potentially lead to aberrant production of oxidative response genes, may contribute to disease aetiology in a subset of PDB patients.
The SQSTM1/p62 UBA domain regulates Ajuba localisation, degradation and NF-κB signalling function
Publisher: Public Library of Science (PLoS)
Date: 04-11-2021
DOI: 10.1371/JOURNAL.PONE.0259556
Abstract: The LIM-domain containing protein Ajuba and the scaffold protein SQSTM1 62 regulate signalling of NF-κB, a transcription factor involved in osteoclast differentiation and survival. The ubiquitin-associated domain of SQSTM1 62 is frequently mutated in patients with Paget’s disease of bone. Here, we report that Ajuba activates NF-κB activity in HEK293 cells, and that co-expression with SQSTM1 62 inhibits this activation in an UBA domain-dependent manner. SQSTM1 62 regulates proteins by targeting them to the ubiquitin-proteasome system or the autophagy-lysosome pathway. We show that Ajuba is degraded by autophagy, however co-expression with SQSTM1 62 (wild type or UBA-deficient) protects Ajuba levels both in cells undergoing autophagy and those exposed to proteasomal stress. Additionally, in unstressed cells co-expression of SQSTM1 62 reduces the amount of Ajuba present in the nucleus. SQSTM1 62 with an intact ubiquitin-associated domain forms holding complexes with Ajuba that are not destined for degradation yet inhibit signalling. Thus, in situations with altered levels and localization of SQSTM1 62 expression, such as osteoclasts in Paget’s disease of bone and various cancers, SQSTM1 62 may compartmentalize Ajuba and thereby impact its cellular functions and disease pathogenesis. In Paget’s, ubiquitin-associated domain mutations may lead to increased or prolonged Ajuba-induced NF-κB signalling leading to increased osteoclastogenesis. In cancer, Ajuba expression promotes cell survival. The increased levels of SQSTM1 62 observed in cancer may enhance Ajuba-mediated cancer cell survival.
An FTLD-associated SQSTM1 variant impacts Nrf2 and NF-κB signalling and is associated with reduced phosphorylation of p62
Publisher: Elsevier BV
Date: 07-2019
DOI: 10.1016/J.MCN.2019.04.001
Abstract: Elevated oxidative stress has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). In response to oxidative stress, the Nrf2 transcription factor activates protective antioxidant genes. A critical regulator of Nrf2 is the inhibitory protein Keap1, which mediates Nrf2 degradation. In response to cellular stress an interaction between Keap1 and SQSTM1 62 (p62), a signalling adaptor protein, allows for increased Nrf2 signalling as it escapes degradation. Mutations in SQSTM1 (encoding p62) are linked with ALS-FTLD. Previously, two ALS-FTLD-associated p62 mutant proteins within the Keap1 interacting region (KIR) of p62 were found to be associated with decreased Keap1-p62 binding and Nrf2 activation. Here we report that a non-KIR domain FTLD-associated variant of p62 (p.R110C), affecting a residue close to the N-terminal PB1 oligomerisation domain, also reduces Keap1-p62 binding in cellulo and thereby reduces Nrf2 activity in reporter assays. Further, we observed that expression of p.R110C increased NF-κB activation compared with wild type p62. Altered signalling appeared to be linked with reduced phosphorylation of p62 at Serine residues -349 and -403. Our results confirm that ALS-FTLD mutations affecting multiple domains of p62 result in a reduced stress response, suggesting that altered stress signalling may directly contribute to the pathology of some ALS-FTLD cases.
Paget disease of bone-associated UBA domain mutations of SQSTM1 exert distinct effects on protein structure and function
Publisher: Elsevier BV
Date: 07-2014
SQSTM1 mutations – Bridging Paget disease of bone and ALS/FTLD
Publisher: Elsevier BV
Date: 07-2014
DOI: 10.1016/J.YEXCR.2014.01.020
Abstract: Paget disease of bone (PDB) is a skeletal disorder common in Western Europe but extremely rare in the Indian subcontinent and Far East. The condition has a strong genetic element with mutations affecting the SQSTM1 gene, encoding the p62 protein, frequently identified. Recently SQSTM1 mutations have also been reported in a small number of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), neurodegenerative disorders in which significant coexistence with PDB has not been previously recognized. Although several SQSTM1 mutations are common to both ALS/FTLD and PDB, many are ALS/FTLD-specific. The p62 protein regulates various cellular processes including NF-κB signaling and autophagy pathways. Here we consider how knowledge of the impact of PDB-associated SQSTM1 mutations (several of which are now known to be relevant for ALS/FTLD) on these pathways, as well as the locations of the mutations within the p62 primary sequence, may provide new insights into ALS/FTLD disease mechanisms.
Common susceptibility alleles and SQSTM1 mutations predict disease extent and severity in a multinational study of patients with Paget's disease
Publisher: Wiley
Date: 18-10-2013
DOI: 10.1002/JBMR.1975
Abstract: Paget's disease of bone (PDB) has a strong genetic component. Here, we investigated possible associations between genetic variants that predispose to PDB and disease severity. Allelic variants identified as predictors of PDB from genome-wide association studies were analyzed in 1940 PDB patients from the United Kingdom, Italy, Western Australia, and Spain. A cumulative risk allele score was constructed by adding the variants together and relating this to markers of disease severity, alone and in combination with SQSTM1 mutations. In SQSTM1-negative patients, risk allele scores in the highest tertile were associated with a 27% increase in disease extent compared with the lowest tertile (p < 0.00001) with intermediate values in the middle tertile (20% increase p = 0.0007). The effects were similar for disease severity score, which was 15% (p = 0.01) and 25% (p < 0.00001) higher in the middle and upper tertiles, respectively. Risk allele score remained a significant predictor of extent and severity when SQSTM-positive in iduals were included, with an effect size approximately one-third of that observed with SQSTM1 mutations. A genetic risk score was developed by combining information from both markers, which identified subgroups of in iduals with low, medium, and high levels of severity with a specificity of 70% and sensitivity of 55%. Risk allele scores and SQSTM1 mutations both predict extent and severity of PDB. It is possible that with further refinement, genetic profiling may be of clinical value in identifying in iduals at high risk of severe disease who might benefit from enhanced surveillance and early intervention.
Bafilomycin A1 Attenuates Osteoclast Acidification and Formation, Accompanied by Increased Levels of SQSTM1/p62 Protein
Publisher: Wiley
Date: 26-11-2015
DOI: 10.1002/JCB.25442
Abstract: Vacuolar proton pump H + ‐adenosine triphosphatases (V‐ATPases) play an important role in osteoclast function. Further understanding of the cellular and molecular mechanisms of V‐ATPase inhibition is vital for the development of anti‐resorptive drugs specifically targeting osteoclast V‐ATPases. In this study, we observed that bafilomycin A1, a naturally‐occurring inhibitor of V‐ATPases, increased the protein level of SQSTM1 62, a known negative regulator of osteoclast formation. Consistently, we found that bafilomycin A1 diminishes the intracellular accumulation of the acidotropic probe lysotracker in osteoclast‐like cells indicative of reduced acidification. Further, bafilomycin A1 inhibits osteoclast formation with attenuation of cell fusion and multi‐nucleation of osteoclast‐like cells during osteoclast differentiation. Taken together, these data indicate that bafilomycin A1 attenuates osteoclast differentiation in part via increased levels of SQSTM1 62 protein, providing further mechanistic insight into the effect of V‐ATPase inhibition in osteoclasts. J. Cell. Biochem. 117: 1464–1470, 2016. © 2015 Wiley Periodicals, Inc.
Targeted sequencing of DCSTAMP in familial Paget's disease of bone
Publisher: Elsevier BV
Date: 06-2019
Proteasome inhibitors impair RANKL‐induced NF‐κB activity in osteoclast‐like cells via disruption of p62, TRAF6, CYLD, and IκBα signaling cascades
Publisher: Wiley
Date: 13-04-2009
DOI: 10.1002/JCP.21787
Abstract: Proteasome inhibitors represent a promising therapy for the treatment of relapsed and/or refractory multiple myeloma, a disease that is concomitant with osteolysis and enhanced osteoclast formation. While blockade of the proteosome pathway has been recently shown to influence osteoclast formation and function, the precise molecular cascade underlying these effects is presently unclear. Here, we provide evidence that proteasome inhibitors directly impair osteoclast formation and function via the disruption of key RANK-mediated signaling cascades. Disruption of the proteosome pathway using selective inhibitors (MG-132, MG-115, and epoxomicin) resulted in the accumulation of p62 and CYLD, and altered the subcellular targeting and distribution of p62 and TRAF6 in osteoclast-like cells. Proteosome inhibition also blocked RANKL-induced NF-kappaB activation, IkappaBalpha degradation and nuclear translocation of p65. The disruption in RANK-signaling correlated dose-dependently with an impairment in osteoclastogenesis, with relative potency epoxomicin > MG-132 > MG-115 based on equimolar concentrations. In addition, these inhibitors were found to impact osteoclastic microtubule organization and attenuate bone resorption. Based on these data we propose that deregulation of key RANK-mediated signaling cascades (p62, TRAF6, CYLD, and IkappaBalpha) underscores proteasome-mediated inhibition of osteolytic bone conditions.
Prognosis for splicing factor PRPF8 retinitis pigmentosa, novel mutations and correlation between human and yeast phenotypes
Publisher: Hindawi Limited
Date: 15-03-2010
DOI: 10.1002/HUMU.21236
Abstract: PRPF8-retinitis pigmentosa is said to be severe but there has been no overview of phenotype across different mutations. We screened RP patients for PRPF8 mutations and identified three new missense mutations, including the first documented mutation outside exon 42 and the first de novo mutation. This brings the known RP-causing mutations in PRPF8 to nineteen. We then collated clinical data from new and published cases to determine an accurate prognosis for PRPF8-RP. Clinical data for 75 PRPF8-RP patients were compared, revealing that while the effect on peripheral retinal function is severe, patients generally retain good visual acuity in at least one eye until the fifth or sixth decade. We also noted that prognosis for PRPF8-RP differs with different mutations, with p.H2309P or p.H2309R having a worse prognosis than p.R2310K. This correlates with the observed difference in growth defect severity in yeast lines carrying the equivalent mutations, though such correlation remains tentative given the limited number of mutations for which information is available. The yeast phenotype is caused by lack of mature spliceosomes in the nucleus, leading to reduced RNA splicing function. Correlation between yeast and human phenotypes suggests that splicing factor RP may also result from an underlying splicing deficit.
New insights into the role of sequestosome 1/p62 mutant proteins in the pathogenesis of Paget's disease of bone.
Publisher: The Endocrine Society
Date: 08-2013
DOI: 10.1210/ER.2012-1034
Abstract: Paget's disease of bone (PDB) is characterized by focal areas of aberrant and excessive bone turnover, specifically increased bone resorption and disorganized bone formation. Germline mutations in the sequestosome 1 62 (SQSTM1 62) gene are common in PDB patients, with most mutations affecting the ubiquitin-associated domain of the protein. In vitro, osteoclast precursor cells expressing PDB-mutant SQSTM1 62 protein are associated with increases in nuclear factor κB activation, osteoclast differentiation, and bone resorption. Although the precise mechanisms by which SQSTM1 62 mutations contribute to disease pathogenesis and progression are not well defined, it is apparent that as well as affecting nuclear factor κB signaling, SQSTM1 62 is a master regulator of ubiquitinated protein turnover via autophagy and the ubiquitin-proteasome system. Additional roles for SQSTM1 62 in the oxidative stress-induced Keap1/Nrf2 pathway and in caspase-mediated apoptosis that were recently reported are potentially relevant to the pathogenesis of PDB. Thus, SQSTM1 62 may serve as a molecular link or switch between autophagy, apoptosis, and cell survival signaling. The purpose of this review is to outline recent advances in understanding of the multiple pathophysiological roles of SQSTM1 62 protein, with particular emphasis on their relationship to PDB, including challenges associated with translating SQSTM1 62 research into clinical diagnosis and treatment.
Activate or inhibit? Implications of autophagy modulation as a therapeutic strategy for alzheimer’s disease
Publisher: MDPI AG
Date: 14-09-2020
DOI: 10.3390/IJMS21186739
Abstract: Neurodegenerative diseases result in a range of conditions depending on the type of proteinopathy, genes affected or the location of the degeneration in the brain. Proteinopathies such as senile plaques and neurofibrillary tangles in the brain are prominent features of Alzheimer’s disease (AD). Autophagy is a highly regulated mechanism of eliminating dysfunctional organelles and proteins, and plays an important role in removing these pathogenic intracellular protein aggregates, not only in AD, but also in other neurodegenerative diseases. Activating autophagy is gaining interest as a potential therapeutic strategy for chronic diseases featuring protein aggregation and misfolding, including AD. Although autophagy activation is a promising intervention, over-activation of autophagy in neurodegenerative diseases that display impaired lysosomal clearance may accelerate pathology, suggesting that the success of any autophagy-based intervention is dependent on lysosomal clearance being functional. Additionally, the effects of autophagy activation may vary significantly depending on the physiological state of the cell, especially during proteotoxic stress and ageing. Growing evidence seems to favour a strategy of enhancing the efficacy of autophagy by preventing or reversing the impairments of the specific processes that are disrupted. Therefore, it is essential to understand the underlying causes of the autophagy defect in different neurodegenerative diseases to explore possible therapeutic approaches. This review will focus on the role of autophagy during stress and ageing, consequences that are linked to its activation and caveats in modulating this pathway as a treatment.
Functional and structural consequences of TBK1 missense variants in frontotemporal lobar degeneration and amyotrophic lateral sclerosis
Publisher: Elsevier BV
Date: 11-2022
DOI: 10.1016/J.NBD.2022.105859
Abstract: Mutations in the Tank-binding kinase 1 (TBK1) gene were identified in 2015 in in iduals with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). They account for ∼3-4% of cases. To date, over 100 distinct mutations, including missense, nonsense, deletion, insertion, duplication, and splice-site mutations have been reported. While nonsense mutations are predicted to cause disease via haploinsufficiency, the mechanisms underlying disease pathogenesis with missense mutations is not fully elucidated. TBK1 is a kinase involved in neuroinflammation, which is commonly observed in these diseases. TBK1 also phosphorylates key autophagy mediators, thereby regulating proteostasis, a pathway that is dysregulated in ALS-FTLD. Recently, several groups have characterised various missense mutations with respect to their effects on the phosphorylation of known TBK1 substrates, TBK1 homodimerization, interaction with optineurin, and the regulation of autophagy and neuroinflammatory pathways. Further, the effects of either global or conditional heterozygous knock-out of Tbk1, or the heterozygous or homozygous knock-in of ALS-FTLD associated mutations, alone or when crossed with the SOD1 G93A classical ALS mouse model or a TDP-43 mouse model, have been reported. In this review we summarise the known functional effects of TBK1 missense mutations. We also present novel modelling data that predicts the structural effects of missense mutations and discuss how they correlate with the known functional effects of these mutations.
ALS-associated TBK1 variant p.G175S is defective in phosphorylation of p62 and impacts TBK1-mediated signalling and TDP-43 autophagic degradation
Publisher: Elsevier BV
Date: 10-2020
DOI: 10.1016/J.MCN.2020.103539
Abstract: Mutations affecting SQSTM1 coding for p62 and TANK-Binding Kinase 1 (TBK1) have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TBK1 is a serine-threonine kinase that regulates p62's activity as an autophagy receptor via phosphorylation and also has roles in neuroinflammatory signalling pathways. The mechanisms underlying ALS and FTLD pathogenesis as a result of TBK1 mutations are incompletely understood, however, loss of TBK1 function can lead to dysregulated autophagy and mitophagy. Here, we report that an ALS-associated TBK1 variant affecting the kinase domain, p.G175S, is defective in phosphorylation of p62 at Ser-403, a modification critical for regulating its ubiquitin-binding function, as well as downstream phosphorylation at Ser-349. Consistent with these findings, expression of p.G175S TBK1 was associated with decreased induction of autophagy compared to wild type and reduced degradation of the ALS-linked protein TDP-43. Expression of wild type TBK1 increased NF-κB signalling ~300 fold in comparison to empty vector cells, whereas p.G175S TBK1 was unable to promote NF-κB signalling above levels observed in empty vector transfected cells. We also noted a hitherto unknown role for TBK1 as a suppressor of oxidative stress (Nrf2) signalling and show that p.G175S TBK1 expressing cells lose this inhibitory function. Our data suggest that TBK1 ALS mutations may broadly impair p62-mediated cell signalling, which ultimately may reduce neuronal survival, in addition TDP-43 was not efficiently degraded, together these effects may contribute to TBK1 mutation associated ALS and FTLD pathogenesis.
Related Organisations
Perron Institute For Neurological And Translational Science
Location: Australia
Related Funding Activities
No related grants have been discovered for Sarah Lyn Rea.