The endoplasmic reticulum-associated protein, OS-9, behaves as a lectin in targeting the immature calcium-sensing receptor.

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 Changing Presentation of Paget's Disease of Bone in Australia, A High Prevalence Region.

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.

Bafilomycin A1 Attenuates Osteoclast Acidification and Formation, Accompanied by Increased Levels of SQSTM1/p62 Protein.

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/p62, 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/p62 protein, providing further mechanistic insight into the effect of V-ATPase inhibition in osteoclasts.

The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling.

A yeast two-hybrid screen performed to identify binding partners of the CaR (calcium-sensing receptor) intracellular tail identified the adaptor protein 14-3-3θ as a novel binding partner that bound to the proximal membrane region important for CaR expression and signalling. The 14-3-3θ protein directly interacted with the CaR tail in pull-down studies and FLAG-tagged CaR co-immunoprecipitated with EGFP (enhanced green fluorescent protein)-tagged 14-3-3θ when co-expressed in HEK (human embryonic kidney)-293 or COS-1 cells. The interaction between the CaR and 14-3-3θ did not require a putative binding site in the membrane-proximal region of the CaR tail and was independent of PKC (protein kinase C) phosphorylation. Confocal microscopy demonstrated co-localization of the CaR and EGFP-14-3-3θ in the ER (endoplasmic reticulum) of HEK-293 cells that stably expressed the CaR (HEK-293/CaR cells), but 14-3-3θ overexpression had no effect on membrane expression of the CaR. Overexpression of 14-3-3θ in HEK-293/CaR cells attenuated CaR-mediated Rho signalling, but had no effect on ERK (extracellular-signal-regulated kinase) 1/2 signalling. Another isoform identified from the library, 14-3-3ζ, exhibited similar behaviour to that of 14-3-3θ with respect to CaR tail binding, cellular co-localization and impact on receptor-mediated signalling. However, unlike 14-3-3θ, this isoform, when overexpressed, significantly reduced CaR plasma membrane expression. Results indicate that 14-3-3 proteins mediate CaR-dependent Rho signalling and may modulate the plasma membrane expression of the CaR.

Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling.

The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

The SQSTM1/p62 UBA domain regulates Ajuba localisation, degradation and NF-κB signalling function.

The LIM-domain containing protein Ajuba and the scaffold protein SQSTM1/p62 regulate signalling of NF-κB, a transcription factor involved in osteoclast differentiation and survival. The ubiquitin-associated domain of SQSTM1/p62 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/p62 inhibits this activation in an UBA domain-dependent manner. SQSTM1/p62 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/p62 (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/p62 reduces the amount of Ajuba present in the nucleus. SQSTM1/p62 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/p62 expression, such as osteoclasts in Paget's disease of bone and various cancers, SQSTM1/p62 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/p62 observed in cancer may enhance Ajuba-mediated cancer cell survival.

Proteasome inhibitors impair RANKL-induced NF-kappaB activity in osteoclast-like cells via disruption of p62, TRAF6, CYLD, and IkappaBalpha signaling cascades.

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.

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.

UNLABELLED: Sequestosome 1/p62 (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. INTRODUCTION: Sequestosome 1/p62 (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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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 chaperone function of cyclophilin 40 maps to a cleft between the prolyl isomerase and tetratricopeptide repeat domains.

Cyclophilin 40 (CyP40), an immunophilin cochaperone present in steroid receptor-Hsp90 complexes, contains an N-terminal peptidylprolyl isomerase (PPIase) domain separated from a C-terminal Hsp90-binding tetratricopeptide repeat (TPR) domain by a 30-residue linker. To map CyP40 chaperone function, CyP40 deletion mutants were prepared and analysed for chaperone activity. CyP40 fragments containing the PPIase domain plus linker or the linker region and the adjoining TPR domain retained chaperone activity, whilst individually, the catalytic and TPR domains were devoid of chaperoning ability. CyP40 chaperone function then, is localized within the linker that forms a binding cleft with potential to accommodate non-native substrates.

A novel homozygous deletion in the calcium-sensing receptor ligand-binding domain associated with neonatal severe hyperparathyroidism.

Neonatal severe hyperparathyroidism (NSHPT) is a life-threatening disorder usually caused by homozygous mutations occurring in the calcium-sensing receptor (CaR) gene. We examined an infant hospitalised with NSHPT for mutations in the CaR gene using heterozygous sequence analysis and confirmed this result by a restriction enzyme assay. Clinical management of this case, which was beset by other complications, involved control of the hypercalcemia and the effects of hyperparathyroidism by a combination of treatments prior to parathyroidectomy performed at 10 months. Mutational analysis demonstrated a homozygous 5 base-pair deletion in the CaR gene located at the 5' end of exon 4 which would result in a severely truncated, non-functional receptor with only the first 164 amino acids of the CaR followed by 23 amino acids of aberrant sequence. This is the first report of an out-of-frame deletion in the extracellular domain of the CaR associated with clinical disease.

Steroid Receptor-Associated Immunophilins: Candidates for Diverse Drug-Targeting Approaches in Disease.

The steroid receptor-associated TPR cochaperones FKBP51, FKBP52, CyP40 and PP5 have non-redundant roles in steroid receptor function that impact steroid hormone-binding affinity, nucleocyoplasmic shuttling and transcriptional activation of target genes in a tissue-specific manner. Aberrant expression of these TPR immunophilins has the potential to cause steroid-based diseases, including breast and prostate cancer, diabetes and metabolic disorders, male and female infertility and major depressive and neurodegenerative disorders. This review summaries the function of these proteins as cochaperones in steroid receptor-Hsp90 complexes and elaborates on their role in alternative, Hsp90-dependent and -independent signalling pathways not involving steroid receptors. The review also extensively covers current knowledge of the link between the steroid receptor-associated immunophilins and human disease. An improved understanding of their mechanisms of action has revealed opportunities for molecular therapies to enhance or inhibit cellular processes under their control that contribute both to human health and disease.

Steroid Receptor-Associated Immunophilins: A Gateway to Steroid Signalling.

The steroid receptor-associated immunophilins FKBP51, FKBP52, CyP40 and PP5 have specific roles in steroid receptor function that impact steroid hormone-binding affinity, nucleocytoplasmic shuttling and transcriptional activation of target genes in a tissue-specific manner. Aberrant expression of these functionally unique immunophilins has the potential to cause steroid-based diseases, including breast and prostate cancer, diabetes and related metabolic disorders, male and female infertility and major depressive disorders. This review addresses the function of these proteins as co-chaperones in steroid receptor-Hsp90 complexes and extensively covers current knowledge of the link between the steroid receptor-associated immunophilins and human disease. An improved understanding of their mechanisms of action has revealed opportunities for molecular therapies to enhance or inhibit cellular processes under immunophilin control that contribute both to human health and disease.

Functional deletion of the calcium-sensing receptor in a case of neonatal severe hyperparathyroidism.

Heterozygous inactivating mutations of the calcium-sensing receptor (CaR) cause familial hypocalciuric hypercalcemia, whereas homozygous or compound heterozygous inactivating mutations normally cause neonatal severe hyperparathyroidism. In a case of neonatal severe hyperparathyroidism characterized by moderately severe hypercalcemia and very high PTH levels, coupled with evidence of hyperparathyroidism and effects on brain development not previously demonstrated, we detected point mutations on separate alleles of the CaR, resulting in premature stop codon substitutions at G94 and R648. This led to severely truncated receptors and an effective so-called knockout of functional CaR. FLAG-tagged, truncated receptors were expressed in HEK293 cells for functional analysis. Confocal microscopy demonstrated cytoplasmic localization of the G94stop receptor, whereas the R648stop receptor was present both in the cytoplasm and associated with the cell membrane. Only the R648stop receptor could be detected by Western analysis. Functional assays in which R648stop and wild-type receptor were cotransfected into HEK293 cells demonstrated a reduction in wild-type Ca(2+)-responsiveness by the R648stop receptor, even at physiological Ca(2+) levels, thus simulating familial hypocalciuric hypercalcemia in relatives of the infant who were heterozygous for the R648stop mutation. The R648stop receptor alone was nonresponsive to Ca(2+). This case contributes to our understanding of the clinical manifestation of a CaR knockout.

Immunophilin chaperones in steroid receptor signalling.

The immunophilin cochaperones, cyclophilin 40 (CyP40), FKBP51 and FKBP52 and PP5, a serine/threonine protein phosphatase, have been implicated as modulators of steroid receptor function through their association with Hsp90, a molecular chaperone with a key role in steroid hormone signalling. Although progress towards a satisfying definition for the role of these components in steroid receptor complexes has been slow, recent developments arising from novel approaches in both yeast and mammalian systems, together with available crystal structures for Hsp90 and some of these cochaperones, are beginning to provide important clues about their function. Hsp90, recently identified as a member of the GHKL superfamily of ATPases, is the central player in receptor assembly, an energy-driven process that allows receptor and the immunophilins to be proximally located, or to interact directly, on a Hsp90 scaffold. Immunophilin structure, relative abundance, their binding affinity for Hsp90 and their ability to interact with specific receptors may all contribute to a selective preference of the immunophilins for individual receptors. Association of receptors with different immunophilins leads to differential functional consequences for receptor activity. Observations of glucocorticoid resistance in New World primates, attributed to FKBP51 overexpression and incorporation into glucocorticoid receptor complexes, have provided the first evidence that these cochaperones can control hormone-binding affinity. Application of a yeast model to FKBP52 function in the glucocorticoid receptor system has now provided crucial evidence that this immunophilin enhances receptor transcriptional activity by increasing receptor avidity for hormone through PPIase-mediated conformational changes in the ligand-binding domain. A recent novel finding suggests that hormone binding may induce a functional exchange of immunophilins in receptor complexes and that the modified complex directs receptor to the nucleus.

Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70.

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 clamp 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 clamp 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.

New insights into the role of sequestosome 1/p62 mutant proteins in the pathogenesis of Paget's disease of bone.

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/p62 (SQSTM1/p62) 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/p62 protein are associated with increases in nuclear factor κB activation, osteoclast differentiation, and bone resorption. Although the precise mechanisms by which SQSTM1/p62 mutations contribute to disease pathogenesis and progression are not well defined, it is apparent that as well as affecting nuclear factor κB signaling, SQSTM1/p62 is a master regulator of ubiquitinated protein turnover via autophagy and the ubiquitin-proteasome system. Additional roles for SQSTM1/p62 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/p62 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/p62 protein, with particular emphasis on their relationship to PDB, including challenges associated with translating SQSTM1/p62 research into clinical diagnosis and treatment.

The helix 1-3 loop in the glucocorticoid receptor LBD is a regulatory element for FKBP cochaperones.

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.

The S349T mutation of SQSTM1 links Keap1/Nrf2 signalling to Paget's disease of bone.

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.

Common susceptibility alleles and SQSTM1 mutations predict disease extent and severity in a multinational study of patients with Paget's disease.

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 individuals 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 individuals 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 individuals at high risk of severe disease who might benefit from enhanced surveillance and early intervention.

The role of the calcium-sensing receptor in human disease.

Following the discovery of the calcium-sensing receptor (CaSR) in 1993, its pivotal role in disorders of calcium homeostasis such as Familial Hypocalciuric Hypercalcemia (FHH) was quickly demonstrated. Since then, it has become clear that the CaSR has immense functional versatility largely through its ability to activate many different signaling pathways in a ligand- and tissue-specific manner. This allows the receptor to play diverse and crucial roles in human physiology and pathophysiology, both in calcium homeostasis and in tissues and biological processes unrelated to calcium balance. This review covers current knowledge of the role of the CaSR in disorders of calcium homeostasis (FHH, neonatal severe hyperparathyroidism, autosomal dominant hypocalcemia, primary and secondary hyperparathyroidism, hypercalcemia of malignancy) as well as unrelated diseases such as breast and colorectal cancer (where the receptor appears to play a tumor suppressor role), Alzheimer's disease, pancreatitis, diabetes mellitus, hypertension and bone and gastrointestinal disorders. In addition, it examines the use or potential use of CaSR agonists or antagonists (calcimimetics and calcilytics) and other drugs mediated through the CaSR, in the management of disorders as diverse as hyperparathyroidism, osteoporosis and gastrointestinal disease.