p62 overexpression induces TDP-43 cytoplasmic mislocalisation, aggregation and cleavage and neuronal death.

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.

ALS-associated TBK1 variant p.G175S is defective in phosphorylation of p62 and impacts TBK1-mediated signalling and TDP-43 autophagic degradation.

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.

The 'dark matter' of ubiquitin-mediated processes: opportunities and challenges in the identification of ubiquitin-binding domains.

Ubiquitin modifications of target proteins act to localise, direct and specify a diverse range of cellular processes, many of which are biomedically relevant. To allow this diversity, ubiquitin modifications exhibit remarkable complexity, determined by a combination of polyubiquitin chain length, linkage type, numbers of ubiquitin chains per target, and decoration of ubiquitin with other small modifiers. However, many questions remain about how different ubiquitin signals are specifically recognised and transduced by the decoding ubiquitin-binding domains (UBDs) within ubiquitin-binding proteins. This review briefly outlines our current knowledge surrounding the diversity of UBDs, identifies key challenges in their discovery and considers recent structural studies with implications for the increasing complexity of UBD function and identification. Given the comparatively low numbers of functionally characterised polyubiquitin-selective UBDs relative to the ever-expanding variety of polyubiquitin modifications, it is possible that many UBDs have been overlooked, in part due to limitations of current approaches used to predict their presence within the proteome. Potential experimental approaches for UBD discovery are considered; web-based informatic analyses, Next-Generation Phage Display, deubiquitinase-resistant diubiquitin, proximity-dependent biotinylation and Ubiquitin-Phototrap, including possible advantages and limitations. The concepts discussed here work towards identifying new UBDs which may represent the 'dark matter' of the ubiquitin system.

An FTLD-associated SQSTM1 variant impacts Nrf2 and NF-κB signalling and is associated with reduced phosphorylation of p62.

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/p62 (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.

Second-harmonic generation imaging of collagen in ancient bone.

Second-harmonic generation imaging (SHG) captures triple helical collagen molecules near tissue surfaces. Biomedical research routinely utilizes various imaging software packages to quantify SHG signals for collagen content and distribution estimates in modern tissue samples including bone. For the first time using SHG, samples of modern, medieval, and ice age bones were imaged to test the applicability of SHG to ancient bone from a variety of ages, settings, and taxa. Four independent techniques including Raman spectroscopy, FTIR spectroscopy, radiocarbon dating protocols, and mass spectrometry-based protein sequencing, confirm the presence of protein, consistent with the hypothesis that SHG imaging detects ancient bone collagen. These results suggest that future studies have the potential to use SHG imaging to provide new insights into the composition of ancient bone, to characterize ancient bone disorders, to investigate collagen preservation within and between various taxa, and to monitor collagen decay regimes in different depositional environments.

5-HT(6) receptor agonists and antagonists enhance learning and memory in a conditioned emotion response paradigm by modulation of cholinergic and glutamatergic mechanisms.

BACKGROUND AND PURPOSE: 5-HT(6) receptors are abundant in the hippocampus, nucleus accumbens and striatum, supporting their role in learning and memory. Selective 5-HT(6) receptor antagonists produce pro-cognitive effects in several learning and memory paradigms while 5-HT(6) receptor agonists have been found to enhance and impair memory. EXPERIMENTAL APPROACH: The conditioned emotion response (CER) paradigm was validated in rats. Then we examined the effect of the 5-HT(6) receptor antagonist, EMD 386088 (10 mg·kg(-1) , i.p.), and agonists, E-6801 (2.5 mg·kg(-1) , i.p.) and EMD 386088 (5 mg·kg(-1) , i.p.) on CER-induced behaviour either alone or after induction of memory impairment by the muscarinic receptor antagonist, scopolamine (0.3 mg·kg(-1) , i.p) or the NMDA receptor antagonist, MK-801 (0.1 mg·kg(-1) , i.p). KEY RESULTS: Pairing unavoidable foot shocks with a light and tone cue during CER training induced a robust freezing response, providing a quantitative index of contextual memory when the rat was returned to the shock chamber 24 h later. Pretreatment (-20 min pre-training) with scopolamine or MK-801 reduced contextual freezing 24 h after CER training, showing production of memory impairment. Immediate post-training administration of 5-HT(6) receptor antagonist, SB-270146, and agonists, EMD 386088 and E-6801, had little effect on CER freezing when given alone, but all significantly reversed scopolamine- and MK-801-induced reduction in freezing. CONCLUSION AND IMPLICATIONS Both the 5-HT(6) receptor agonists and antagonist reversed cholinergic- and glutamatergic-induced deficits in associative learning. These findings support the therapeutic potential of 5-HT(6) receptor compounds in the treatment of cognitive dysfunction, such as seen in Alzheimer's disease and schizophrenia.

Review: unchained maladie - a reassessment of the role of Ubb(+1) -capped polyubiquitin chains in Alzheimer's disease.

Molecular misreading allows the formation of mutant proteins in the absence of gene mutations. A mechanism has been proposed by which a frameshift mutant of the ubiquitin protein, Ubb(+1) , which accumulates in an age-dependent manner as a result of molecular misreading, contributes to neuropathology in Alzheimer's disease (Lam et al. 2000). Specifically, in the Ubb(+1) -mediated proteasome inhibition hypothesis Ubb(+1) 'caps' unanchored (that is, nonsubstrate linked) polyubiquitin chains, which then act as dominant inhibitors of the 26S proteasome. A review of subsequent literature indicates that this original hypothesis is broadly supported, and offers new insights into the mechanisms accounting for the age-dependent accumulation of Ubb(+1) , and how Ubb(+1) -mediated proteasome inhibition may contribute to Alzheimer's disease. Further, recent studies have highlighted a physiological role for free endogenous unanchored polyubiquitin chains in the direct activation of certain protein kinases. This raises the possibility that Ubb(+1) -capped unanchored polyubiquitin chains could also exert harmful effects through the aberrant activation of tau or other ubiquitin-dependent kinases, neuronal NF-κB activity or NF-κB-mediated neuroinflammatory processes.

Review: the ubiquitin-proteasome system: contributions to cell death or survival in neurodegeneration.

The significance of the accumulation of ubiquitin-positive intraneuronal inclusions in the brains of those affected with different neurodegenerative diseases is currently unclear. While one interpretation is that the disease mechanism(s) involves dysfunction of an ubiquitin-mediated process, such as the ubiquitin-proteasome system, the inclusions are also found in surviving neurones, suggesting a possible neuroprotective role. Here we review recent evidence in support of these seemingly opposing notions gleaned from cell and animal models as well as investigations of patient samples, with particular emphasis on studies relevant to Parkinson's disease.

Recent advances in understanding the molecular basis of Paget disease of bone.

Paget disease of bone (PDB) is a relatively common disorder characterised by increased bone turnover within discrete lesions throughout the skeleton. The condition has a strong genetic component, with mutations affecting the SQSTM1 gene that encodes the p62 protein often found in PDB patients, although environmental factors also play an important role in disease aetiology. The precise disease mechanism(s) in familial forms and sporadic forms of PDB is unclear, although defective RANK-NF-kappaB signalling has been suggested to contribute to the increased activity of pagetic osteoclasts in the former. Here, there is a review of recent advances in the understanding of the molecular basis of PDB with particular emphasis on findings since 2008, and focus on newly defined functions of the p62 protein upon which SQSTM1 mutations may impact in the development of the pagetic phenotype.

Proteomic biomarkers for the diagnosis and risk stratification of polycystic ovary syndrome: a systematic review.

BACKGROUND: The exact causes of polycystic ovary syndrome (PCOS) are uncertain, and treatment could be improved. Discovery-based approaches like 'proteomics' may result in faster insights into the causes of PCOS and improved treatment. OBJECTIVES: To identify the number and nature of proteomic biomarkers found in PCOS so far and to identify their diagnostic and therapeutic potential. SEARCH STRATEGY: All published studies on proteomic biomarkers in women with PCOS identified through the MEDLINE (1966-2008), EMBASE (1980-2008) and the ISI web of knowledge (v4.2) databases. SELECTION CRITERIA: The terms 'polycystic ovary syndrome' and 'proteomic', 'proteomics', 'proteomic biomarker' or 'proteomics biomarker' without any limits/restrictions were used. DATA COLLECTION AND ANALYSIS: Original data were abstracted where available and summarised on a separate Microsoft Excel (2007) database for analysis. MAIN RESULTS: Seventeen articles were identified, of which 6 original papers and 1 review article contained original data. Tissues investigated included serum, omental biopsies, ovarian biopsies, follicular fluid and T lymphocytes. Sample sizes ranged from 3 to 30 women. One hundred and forty-eight biomarkers were identified. The biomarkers were involved in many pathways, for example the regulation of fibrinolysis and thrombosis, insulin resistance, immunity/inflammation and the antioxidant pathway. Eleven groups of biomarkers appeared to be independently validated. The individual sensitivities for the diagnosis of PCOS were reported for 11 named biomarkers and ranged from 57 to 100%. AUTHOR'S CONCLUSIONS: Proteomic biomarker discovery in PCOS offers great potential. Current challenges include reproducibility and data analysis. The establishment of a PCOS-specific biomarker data bank and international consensus on the framework of systematic reviews in this field are required.

Disassociation between the effects of amino acids and insulin on signaling, ubiquitin ligases, and protein turnover in human muscle.

We determined the effects of intravenous infusion of amino acids (AA) at serum insulin of 5, 30, 72, and 167 mU/l on anabolic signaling, expression of ubiquitin-proteasome components, and protein turnover in muscles of healthy young men. Tripling AA availability at 5 mU/l insulin doubled incorporation of [1-(13)C]leucine [i.e., muscle protein synthesis (MPS), P < 0.01] without affecting the rate of leg protein breakdown (LPB; appearance of d(5)-phenylalanine). While keeping AA availability constant, increasing insulin to 30 mU/l halved LPB (P < 0.05) without further inhibition at higher doses, whereas rates of MPS were identical to that at 5 mU/l insulin. The phosphorylation of PKB Ser(473) and p70(S6k) Thr(389) increased concomitantly with insulin, but whereas raising insulin to 30 mU/l increased the phosphorylation of mTOR Ser(2448), 4E-BP1 Thr(37/46), or GSK3beta Ser(9) and decreased that of eEF2 Thr(56), higher insulin doses to 72 and 167 mU/l did not augment these latter responses. MAFbx and proteasome C2 subunit proteins declined as insulin increased, with MuRF-1 expression largely unchanged. Thus increasing AA and insulin availability causes changes in anabolic signaling and amounts of enzymes of the ubiquitin-proteasome pathway, which cannot be easily reconciled with observed effects on MPS or LPB.

Temporal changes in the involvement of pyruvate dehydrogenase complex in muscle lactate accumulation during lipopolysaccharide infusion in rats.

A characteristic manifestation of sepsis is muscle lactate accumulation. This study examined any putative (causative) association between pyruvate dehydrogenase complex (PDC) inhibition and lactate accumulation in the extensor digitorum longus (EDL) muscle of rats infused with lipopolysaccharide (LPS), and explored the involvement of increased transcription of muscle-specific pyruvate dehydrogenase kinase (PDK) isoenzymes. Conscious, male Sprague-Dawley rats were infused i.v. with saline (0.4 ml h(-1), control) or LPS (150 mug kg(-1) h(-1)) for 2 h, 6 h or 24 h (n = 6-8). Muscle lactate concentration was elevated after 2, 6 and 24 h LPS infusion. Muscle PDC activity was the same at 2 h and 6 h, but was 65% lower after 24 h of LPS infusion (P < 0.01), when there was a 47% decrease in acetylcarnitine concentration (P < 0.05), and a 24-fold increase in PDK4 mRNA expression (P < 0.001). These changes were preceded by marked increases in tumour necrosis factor-alpha and interleukin-6 mRNA expression at 2 h. The findings indicate that the early (2 and 6 h) elevation in muscle lactate concentration during LPS infusion was not attributable to limited muscle oxygen availability or ATP production (evidenced by unchanged ATP and phosphocreatine (PCr) concentrations) or to PDC inhibition, whereas after 24 h, muscle lactate accumulation appears to have resulted from PDC activation status limiting pyruvate flux, most probably due to cytokine-mediated up-regulation of PDK4 transcription.

Characterization of biomarkers in polycystic ovary syndrome (PCOS) using multiple distinct proteomic platforms.

A variety of prefractionation methods (including a novel reversed-phase solid-phase-extraction (RP-SPE) combined with SDS-PAGE and proteomic based approaches (e.g., 2-dimensional gel electrophoresis (2DE) and MALDI-TOF mass spectrometry combined with Artificial Neural Network (ANN) bioinformatic tools) were used to investigate the protein/peptide signatures in patients with Polycystic Ovary Syndrome (PCOS). Four potential PCOS biomarkers were identified (complement C4alpha3c and C4gamma and haptoglobin alpha and beta chains).

Principles of proteomics and its applications in cancer.

During the past decade, genomic analyses have been introduced into cancer studies with variable success. It has become recognised, however, that genomic techniques in isolation are insufficient to study the complex pathways of carcinogenesis; this has led to the application of proteomic techniques, which allow for the reliable analysis of complex mixtures of proteins. This article reviews the basic principles of proteomics, methods currently used in proteomics including two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), and the application of proteomics in cancer research. Currently, proteomic technology has been used in two main areas of cancer research: early diagnosis and treatment (included prediction of response to treatment and targeting novel cancer agents). The initial results from both in vitro and in vivo studies are impressive. These technologies, particularly when combined with genomic analyses, will provide valuable insights into the molecular basis of carcinogenesis and the development of more effective anti-cancer therapies.

p62 mutations, ubiquitin recognition and Paget's disease of bone.

Functional analyses of PDB (Paget's disease of bone)-associated mutants of the p62 [also known as SQSTM1 (sequestosome 1)] signalling adaptor protein represent an interesting paradigm for understanding not only the disease mechanism in this skeletal disorder, but also the critical determinants of ubiquitin recognition by an ubiquitin-binding protein. The 11 separate PDB mutations identified to date all affect the C-terminal region of p62 containing the UBA domain (ubiquitin-associated domain), a ubiquitin-binding element. All of these mutations have deleterious effects on ubiquitin binding by p62 in vitro, and there is evidence of an inverse relationship between ubiquitin-binding function and disease severity. The effects on ubiquitin-binding function of most of the mutations can be attributed to either reduced UBA domain stability, and/or the mutations affecting the presumed ubiquitin-binding interface of the UBA domain. However, a subset of the mutations are more difficult to rationalize; several of these affect sequences of p62 outside of the minimal ubiquitin-binding region, providing insights into non-UBA domain sequences within the host protein which mediate ubiquitin-binding affinity. The p62 mutations are presumed to result in activation of (osteoclast) NF-kappaB (nuclear factor kappaB) signalling. Understanding how loss of ubiquitin-binding function of p62 impacts on signal transduction events in osteoclasts will undoubtedly further our understanding of the disease mechanism in PDB at the molecular level.

Loss of ubiquitin binding is a unifying mechanism by which mutations of SQSTM1 cause Paget's disease of bone.

Ubiquitin-associated (UBA) domain mutations of SQSTM1 are an important cause of Paget's disease of bone (PDB), which is a human skeletal disorder characterized by abnormal bone turnover. We previously showed that, when introduced into the full-length SQSTM1 protein, the disease-causing P392L, M404V, G411S, and G425R missense mutations and the E396X truncating mutation (representative of all of the SQSTM1 truncating mutations) cause a generalized loss of monoubiquitin binding and impaired K48-linked polyubiquitin binding at physiological temperature. Here, we show that the remaining three known PDB missense mutations, P387L, S399P, and M404T, have similar deleterious effects on monoubiquitin binding and K48-linked polyubiquitin binding by SQSTM1. The P387L mutation affects an apparently unstructured region at the N terminus of the UBA domain, some five residues from the start of the first helix, which is dispensable for polyubiquitin binding by the isolated UBA domain. Our findings support the proposal that the disease mechanism in PDB with SQSTM1 mutations involves a common loss of ubiquitin binding function of SQSTM1 and implicate a sequence extrinsic to the compact globular region of the UBA domain as a critical determinant of ubiquitin recognition by the full-length SQSTM1 protein.

Structural and functional studies of mutations affecting the UBA domain of SQSTM1 (p62) which cause Paget's disease of bone.

Mutations affecting the UBA (ubiquitin-associated) domain of SQSTM1 (Sequestosome 1) (p62) are a common cause of Paget's disease of bone. The missense mutations resolve into those which retain [P392L (Pro(392)-->Leu), G411S] or abolish (M404V, G425R) the ability of the isolated UBA domain to bind Lys-48-linked polyubiquitin. These effects can be rationalized with reference to the solution structure of the UBA domain, which we have determined by NMR spectroscopy. The UBA domain forms a characteristic compact three-helix bundle, with a hydrophobic patch equivalent to that previously implicated in ubiquitin binding by other UBA domains. None of the mutations affect overall folding of the UBA domain, but both M404V and G425R involve residues in the hydrophobic patch, whereas Pro-392 and Gly-411 are more remote. A simple model assuming the isolated UBA domain is functioning as a compact monomer can explain the effects of the mutations on polyubiquitin binding. The P392L and G411S mutations do however have subtle local effects on secondary structure, which may become more relevant in full-length SQSTM1. Identification of the in vivo ubiquitylated substrates of SQSTM1 will be most informative in determining the functional significance of the SQSTM1-ubiquitin interaction, and consequences of the disease-associated mutations.

SQSTM1 and Paget's disease of bone.

Mutations in the Sequestosome 1 gene ( SQSTM1; also known as p62) have recently been identified as the cause of 5q35-linked Paget's disease of bone (PDB). All of the mutations identified to date affect the ubiquitin-associated (UBA) domain of SQSTM1, a region of the protein that binds noncovalently to ubiquitin. In this review we consider the possible functional significance of the SQSTM1-ubiquitin interaction, and consequences of the SQSTM1 UBA domain mutations. Clarification of the in vivo roles of SQSTM1 in bone-cell function will be central to improving our understanding of the molecular pathogenesis of PDB and related conditions.

alpha-synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome.

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease based on mutations in familial cases of the disease and its presence in Lewy bodies. Here we show that over-expression of wild-type human alpha-synuclein is sufficient to induce inclusion formation in SH-SY5Y cells. In this cellular model, proteasome inhibition leads to an increase of alpha-synuclein accumulation in vivo without ubiquitylation. In accordance, we find that in vitro, unmodified alpha-synuclein can be directly degraded by the 20S proteasome. These findings suggest an ubiquitin-independent mechanism of proteasomal degradation for alpha-synuclein and other natively unfolded proteins.

Purification of poly-ubiquitinated proteins by S5a-affinity chromatography.

Poly-ubiquitination, the post-translational covalent conjugation of isopeptide-linked chains of ubiquitin to other target proteins, is the central signal for proteolytic degradation by the 26S proteasome complex. The S5a subunit of the 26S proteasome binds poly-ubiquitin chains containing four or more ubiquitins. We have used an immobilised glutathione-S-transferase (GST)-S5a fusion protein to purify poly-ubiquitinated proteins from mammalian tissues, with the intention of expanding the repertoire of known substrates of the ubiquitin pathway. A complex mixture of poly-ubiquitinated proteins was successfully purified from normal pig brain extract following induction of in vitro ubiquitination. Western blots of two-dimensional gels of this mixture showed at least two diagonal series of ubiquitin-positive spots. Individual spots in each series were separated by approximately 9 kDa suggesting that they represent poly-ubiquitinated proteins with increasing numbers of ubiquitins in the chains. S5a-binding proteins purified from ubiquitination-induced human placental extracts, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualised by Coomassie staining, contained a single major species with an apparent denatured molecular mass of approximately 60 kDa. Edman degradation identified this protein as hHR23B, a human homologue of the Saccharomyces cerevisiae DNA repair protein Rad23p. In this case hHR23B is not ubiquitinated but instead contains an intrinsic ubiquitin-like domain at its N-terminus, through which it interacts with S5a (Hiyama, H., et al., J Biol. Chem. 1999, 274, 28,019-28,025).