An ALS-associated variant of the autophagy receptor SQSTM1/p62 reprograms binding selectivity toward the autophagy-related hATG8 proteins.

Recognition of human autophagy-related 8 (hATG8) proteins by autophagy receptors represents a critical step within this cellular quality control system. Autophagy impairment is known to be a pathogenic mechanism in the motor neuron disorder amyotrophic lateral sclerosis (ALS). Overlapping but specific roles of hATG8 proteins belonging to the LC3 and GABARAP subfamilies are incompletely understood, and binding selectivity is typically overlooked. We previously showed that an ALS-associated variant of the SQSTM1/p62 (p62) autophagy receptor bearing an L341V mutation within its ATG8-interacting motif (AIM) impairs recognition of LC3B in vitro, yielding an autophagy-deficient phenotype. Improvements in understanding of hATG8 recognition by AIMs now distinguish LC3-interaction and GABARAP-interaction motifs and predict the effects of L341V substitution may extend beyond loss of function to biasing AIM binding preference. Through biophysical analyses, we confirm impaired binding of the L341V-AIM mutant to LC3A, LC3B, GABARAP, and GABARAPL1. In contrast, p62 AIM interactions with LC3C and GABARAPL2 are unaffected by this mutation. Isothermal titration calorimetry and NMR investigations provided insights into the entropy-driven GABARAPL2/p62 interaction and how the L341V mutation may be tolerated. Competition binding demonstrated reduced association of the L341V-AIM with one hATG8 manifests as a relative increase in association with alternate hATG8s, indicating effective reprogramming of hATG8 selectivity. These data highlight how a single AIM peptide might compete for binding with different hATG8s and suggest that the L341V-AIM mutation may be neomorphic, representative of a disease mechanism that likely extends into other human disorders.

The indirect influence of child play on the association between parent perceptions of the neighborhood environment and sense of community.

The neighborhood is a critical setting that broadly affects health, although specific mechanisms that link relationships between the neighborhood environment and indicators of health are still emerging. Children playing is a simple, but underexplored marker that may explain how the neighborhood built environment is related to a psychological sense of community. A telephone survey was conducted among parents with children in the Greater Phoenix Metropolitan area (n = 251) to gather information on perceptions of the built environment, parent attitudes and physical activity behaviors, children's play, and sense of community in the neighborhood. Results of a structural equation modeling analysis (SEM) indicated that children playing in the neighborhood partially explained the relationship between perceptions of the built environment and sense of community (ß = 0.031, CI = 0.007-0.067). Parent perceptions of the built environment were positively associated with both children playing (ß = 0.229, CI = 0.120-0.341) and sense of community (ß = 0.360, CI = 0.220-0.505), and children playing (ß = 0.135, CI = 0.027-0.243) and parent attitudes (ß = 0.440, CI = 0.319-0.546) were positively related to sense of community. As planners and community advocates consider strategies to build a sense of community within neighborhoods, the importance of favorable environmental conditions that facilitate children's play should be considered.

Factor XII and kininogen asymmetric assembly with gC1qR/C1QBP/P32 is governed by allostery.

The contact system is composed of factor XII (FXII), prekallikrein (PK), and cofactor high-molecular-weight kininogen (HK). The globular C1q receptor (gC1qR) has been shown to interact with FXII and HK. We reveal the FXII fibronectin type II domain (FnII) binds gC1qR in a Zn2+-dependent fashion and determined the complex crystal structure. FXIIFnII binds the gC1qR trimer in an asymmetric fashion, with residues Arg36 and Arg65 forming contacts with 2 distinct negatively charged pockets. gC1qR residues Asp185 and His187 coordinate a Zn2+ adjacent to the FXII-binding site, and a comparison with the ligand-free gC1qR crystal structure reveals the anionic G1-loop becomes ordered upon FXIIFnII binding. Additional conformational changes in the region of the Zn2+-binding site reveal an allosteric basis for Zn2+ modulation of FXII binding. Mutagenesis coupled with surface plasmon resonance demonstrate the gC1qR Zn2+ site contributes to FXII binding, and plasma-based assays reveal gC1qR stimulates coagulation in a FXII-dependent manner. Analysis of the binding of HK domain 5 (HKD5) to gC1qR shows only 1 high-affinity binding site per trimer. Mutagenesis studies identify a critical G3-loop located at the center of the gC1qR trimer, suggesting steric occlusion as the mechanism for HKD5 asymmetric binding. Gel filtration experiments reveal that gC1qR clusters FXII and HK into a higher-order 500-kDa ternary complex. These results support the conclusion that extracellular gC1qR can act as a chaperone to cluster contact factors, which may be a prelude for initiating the cascades that drive bradykinin generation and the intrinsic pathway of coagulation.

G-quadruplex ligands mediate downregulation of DUX4 expression.

Abnormal DUX4 expression in skeletal muscles plays a key role in facioscapulohumeral muscular dystrophy (FSHD) pathogenesis, although the molecular mechanisms regulating DUX4 expression are not fully defined. Using bioinformatic analysis of the genomic DUX4 locus, we have identified a number of putative G-quadruplexes (GQs) forming sequences. Their presence was confirmed in synthetic oligonucleotiode sequences derived from the enhancer, promoter and transcript of DUX4 through circular dichroism and nuclear magnetic resonance analysis. We further examined the binding affinity of a naturally occurring GQ stabilizing compound, berberine, to these non-canonical genetic structures using UV-Vis and fluorescence spectroscopy. Subsequent in vitro study in FSHD patient myoblasts indicated that berberine treatment reduced DUX4 expression and also expression of genes normally switched on by DUX4. Further investigation in a mouse model overexpressing exogenous DUX4 confirmed the therapeutic effects of berberine in downregulating DUX4 protein expression, inhibiting muscle fibrosis, and consequently rescuing muscle function. Our data demonstrate for the first time that GQs are present in the DUX4 locus and that the GQ interactive ligand reduces DUX4 expression suggesting potential role of GQs in FSHD pathogenesis. Our work provides the basis of a novel therapeutic strategy for the treatment of FSHD.

Structural basis of the leukocyte integrin Mac-1 I-domain interactions with the platelet glycoprotein Ib.

Cell-surface receptor interactions between leukocyte integrin macrophage-1 antigen (Mac-1, also known as CR3, αMß2, CD11b/CD18) and platelet glycoprotein Ibα (GPIbα) are critical to vascular inflammation. To define the key residues at the binding interface, we used nuclear magnetic resonance (NMR) to assign the spectra of the mouse Mac-1 I-domain and mapped the residues contacting the mouse GPIbα N-terminal domain (GPIbαN) to the locality of the integrin metal ion-dependant adhesion site (MIDAS) surface. We next determined the crystal structures of the mouse GPIbαN and Mac-1 I-domain to 2 Å and 2.5 Å resolution, respectively. The mouse Mac-1 I-domain crystal structure reveals an active conformation that is stabilized by a crystal contact from the α7-helix with a glutamate side chain completing the octahedral coordination sphere of the MIDAS Mg2+ ion. The amino acid sequence of the α7-helix and disposition of the glutamic acid matches the C-terminal capping region α-helix of GPIbα effectively acting as a ligand mimetic. Using these crystal structures in combination with NMR measurements and docking analysis, we developed a model whereby an acidic residue from the GPIbα leucine-rich repeat (LRR) capping α-helix coordinates directly to the Mac-1 MIDAS Mg2+ ion. The Mac-1:GPIbαN complex involves additional interactions consolidated by an elongated pocket flanking the GPIbαN LRR capping α-helix. The GPIbαN α-helix has an HxxxE motif, which is equivalent by homology to RxxxD from the human GPIbαN. Subsequent mutagenesis of residues at this interface, coupled with surface plasmon resonance studies, confirmed the importance of GPIbαN residues H218, E222, and the Mac-1 MIDAS residue T209 to formation of the complex.

The association between leisure time physical activity and happiness: Testing the indirect role of health perception.

AIMS: Research supports the influence of leisure time physical activity (LTPA) on well-being; however, little is known about the mechanisms that drive the relationship. The aim of this study was to investigate the indirect influence of health perception on the association between neighborhood LTPA and happiness. METHODS: Telephone interviews were conducted using random digit dialing among a sample of adult residents aged 18+ (n = 570) to gather information about neighborhood LTPA behaviors, perceptions of health and happiness, and sociodemographics. RESULTS: Results of a structural equation modeling analysis indicated that health had a small significant indirect influence on the relationship between LTPA and happiness. CONCLUSION: Focusing on perceptions offers a holistic conceptualization of how behavior impacts happiness and may offer important insights into how the experience of engaging in LTPA contributes to the subjective evaluation of personal happiness through perceived health.

DNA replication initiation in Bacillus subtilis: structural and functional characterization of the essential DnaA-DnaD interaction.

The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA-DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.

Active Transportation: The Role of Parent Attitude, The Physical Environment, and Social Capital.

BACKGROUND: Despite recommendation and confirmed physical activity benefits, participation in active transportation to school (ATS) has continued to decline. This study's purpose was to create and test a model of ATS that is directly explained by the constructs of parent attitude, the physical environment, and social capital controlling for age and gender. METHODS: Participants were parents (N = 248) of children at 6 elementary and 2 middle schools in 1 district in the Southwestern United States. The survey included previously validated behavior, environmental, attitude, and social items (eg, Safe Routes to School Parent Survey/U.S. General Social Survey). Structural equation modeling was used to test the fit of the model and whether parent attitude, the physical environment, and social capital were associated with active transportation. RESULTS: An adjusted measurement model was a good fit for the data. The physical environment (ß = 0.391; P < .01) as well as parent attitude (ß = 0.535; P < .001) were positively associated with ATS. CONCLUSION: This study supports a model of ATS, affirming that parent attitude, the physical environment, and social capital are effective constructs from which to conceptualize associations with walking and biking to school.

Method for the Purification of Endogenous Unanchored Polyubiquitin Chains.

Unanchored polyubiquitin chains are endogenous non-substrate linked ubiquitin polymers which have emerging roles in the control of cellular physiology. We describe an affinity purification method based on an isolated ubiquitin-binding domain, the ZnF_UBP domain of the deubiquitinating enzyme USP5, which permits the selective purification of mixtures of endogenous unanchored polyubiquitin chains that are amenable to downstream molecular analyses. Further, we present methods for detection of unanchored polyubiquitin chains in purified fractions.

ALS-FTLD associated mutations of SQSTM1 impact on Keap1-Nrf2 signalling.

The transcription factor Nrf2 and its repressor protein Keap1 play key roles in the regulation of antioxidant stress responses and both Keap1-Nrf2 signalling and oxidative stress have been implicated in the pathogenesis of the ALS-FTLD spectrum of neurodegenerative disorders. The Keap1-binding partner and autophagy receptor SQSTM1/p62 has also recently been linked genetically to ALS-FTLD, with some missense mutations identified in patients mapping within or close to its Keap1-interacting region (KIR, residues 347-352). Here we report the effects on protein function of four different disease associated mutations of SQSTM1/p62 which affect the KIR region. Only mutations mapping precisely to the KIR (P348L and G351A) were associated with a loss of Keap1 binding in co-immunoprecipitations comparable to wild-type SQSTM1/p62. These selective effects on Keap1 recognition were entirely rational based on protein structural models. Consistent with impaired Keap1 binding, the P348L and G351A KIR mutants showed reduced ability to activate Nrf2 signalling compared to wild-type SQSTM1/p62 in antioxidant response element (ARE)-luciferase reporter assays. The results suggest that SQSTM1 mutations within the KIR of SQSTM1/p62 contribute to aetiology of some cases of ALS-FTLD through a mechanism involving aberrant expression or regulation of oxidative response genes.

Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD.

Growing evidence implicates impairment of autophagy as a candidate pathogenic mechanism in the spectrum of neurodegenerative disorders which includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). SQSTM1, which encodes the autophagy receptor SQSTM1/p62, is genetically associated with ALS-FTLD, although to date autophagy-relevant functional defects in disease-associated variants have not been described. A key protein-protein interaction in autophagy is the recognition of a lipid-anchored form of LC3 (LC3-II) within the phagophore membrane by SQSTM1, mediated through its LC3-interacting region (LIR), and notably some ALS-FTLD mutations map to this region. Here we show that although representing a conservative substitution and predicted to be benign, the ALS-associated L341V mutation of SQSTM1 is defective in recognition of LC3B. We place our observations on a firm quantitative footing by showing the L341V-mutant LIR is associated with a ∼3-fold reduction in LC3B binding affinity and using protein NMR we rationalize the structural basis for the effect. This functional deficit is realized in motor neuron-like cells, with the L341V mutant EGFP-mCherry-SQSTM1 less readily incorporated into acidic autophagic vesicles than the wild type. Our data supports a model in which the L341V mutation limits the critical step of SQSTM1 recruitment to the phagophore. The oligomeric nature of SQSTM1, which presents multiple LIRs to template growth of the phagophore, potentially gives rise to avidity effects which amplify the relatively modest impact of any single mutation on LC3B binding. Over the lifetime of a neuron, impaired autophagy could expose a vulnerability, which ultimately tips the balance from cell survival toward cell death.

SilE is an intrinsically disordered periplasmic "molecular sponge" involved in bacterial silver resistance.

Ag(+) resistance was initially found on the Salmonella enetrica serovar Typhimurium multi-resistance plasmid pMG101 from burns patients in 1975. The putative model of Ag(+) resistance, encoded by the sil operon from pMG101, involves export of Ag(+) via an ATPase (SilP), an effluxer complex (SilCFBA) and a periplasmic chaperon of Ag(+) (SilE). SilE is predicted to be intrinsically disordered. We tested this hypothesis using structural and biophysical studies and show that SilE is an intrinsically disordered protein in its free apo-form but folds to a compact structure upon optimal binding to six Ag(+) ions in its holo-form. Sequence analyses and site-directed mutagenesis established the importance of histidine and methionine containing motifs for Ag(+) -binding, and identified a nucleation core that initiates Ag(+) -mediated folding of SilE. We conclude that SilE is a molecular sponge for absorbing metal ions.

Mass spectrometry insights into a tandem ubiquitin-binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin.

Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate-conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker-optimized ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnF-UBP domain in tandem with a linkage-selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48-linked polyubiquitin chains. Utilizing native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48-linked ubiquitin dimers. Furthermore, MS/MS analyses indicate that the t-UBD, when applied as an affinity enrichment reagent, can be used to favor the purification of endogenous unanchored Lys48-linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain-selective binding in nonbiological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the 'ubiquitome'. All MS data have been deposited in the ProteomeXchange with identifier PXD004059 (http://proteomecentral.proteomexchange.org/dataset/PXD004059).

Correction: Enantiopure titanocene complexes--direct evidence for paraptosis in cancer cells.

Correction for 'Enantiopure titanocene complexes--direct evidence for paraptosis in cancer cells' by Melchior Cini et al., Metallomics, 2016, DOI: 10.1039/c5mt00297d.

Enantiopure titanocene complexes--direct evidence for paraptosis in cancer cells.

Tolerated by normal tissues, anti-cancer therapies based on titanium compounds are limited by low efficacy/selectivity and lack of understanding of their mode(s) of action. In vitro antitumour activity and mode of cell death incurred by enantiopure TiCl2{η-C5H4CHEt(2-MeOPh)}2 (abbreviated Cp(R)2TiCl2) has been investigated. The in vitro anti-tumour activity of Cp(R)2TiCl2 is selective for cancer cells; in clonogenic assays, (S,S)-Cp(R)2TiCl2 was twice as effective at inhibiting colony formation than other stereoisomers after 24 h exposure. HPLC, MS and NMR techniques determined hydrolysis of Cp(R)2TiCl2; data strongly correlate with soluble [Cp(R)2Ti(OH)(OH2)](+) being the biological trigger. Treatment of cells with Cp(R)2TiCl2 provoked extensive cytoplasmic vacuolization, endoplasmic reticulum (ER) swelling and activation of MAPKinase signal transduction, consistent with ligand-induced paraptosis, type III cell death, which is morphologically distinct from, and independent of apoptosis. Indeed, distinct from cisplatin, Cp(R)2TiCl2 failed to perturb cell cycle dynamics, induce γH2AX foci or evoke apoptosis in MDA-MB-468 and HCT-116 cells.

A Fluorescence Resonance Energy Transfer Assay For Monitoring α- Synclein Aggregation in a Caenorhabditis Elegans Model For Parkinson's Disease.

The aggregation of α-synuclein (Syn or S) to form insoluble fibrils is important in the pathogenesis of Parkinson's disease, but key risk factors remain ill-defined. We have developed Fluorescence Resonance Energy Transfer (FRET)-based assays for α-synuclein aggregation, using Green Fluorescent Protein variants Cerulean (C) or Venus (V), fused to each other (CV, VC) or to human synuclein (SC, SV etc). Bacterially expressed proteins were purified to homogeneity, and C-terminal fusions SC and SV largely retained their ability to aggregate in vitro. FRET signals from mixtures of SC and SV were used to monitor aggregation. These fusion genes were linked to the C. elegans unc-54 myosin promoter to generate integrated transgenic strains. Increased FRET signals, indicative of S aggregation, were observed following treatment of unc-54::SC + unc-54::SV double transgenic worms with low concentrations of mercury or chlorpyrifos, or with RNAi against hsp-70 and hip-1. Opposite changes in Yellow Fluorescent Protein (YFP) fluorescence in an unc-54::SV strain (NL5901) are likely to reflect FRET from Yellow Fluorescent Protein to aggregates of Syn fusion protein. This could provide the basis for a high throughput screening assay, which could be used for studying the effects of toxic chemicals and environmental pollutants on the aggregation of proteins such as Syn in vivo.

The role of short RNA loops in recognition of a single-hairpin exon derived from a mammalian-wide interspersed repeat.

Splice-site selection is controlled by secondary structure through sequestration or approximation of splicing signals in primary transcripts but the exact role of even the simplest and most prevalent structural motifs in exon recognition remains poorly understood. Here we took advantage of a single-hairpin exon that was activated in a mammalian-wide interspersed repeat (MIR) by a mutation stabilizing a terminal triloop, with splice sites positioned close to each other in a lower stem of the hairpin. We first show that the MIR exon inclusion in mRNA correlated inversely with hairpin stabilities. Employing a systematic manipulation of unpaired regions without altering splice-site configuration, we demonstrate a high correlation between exon inclusion of terminal tri- and tetraloop mutants and matching tri-/tetramers in splicing silencers/enhancers. Loop-specific exon inclusion levels and enhancer/silencer associations were preserved across primate cell lines, in 4 hybrid transcripts and also in the context of a distinct stem, but only if its loop-closing base pairs were shared with the MIR hairpin. Unlike terminal loops, splicing activities of internal loop mutants were predicted by their intramolecular Watson-Crick interactions with the antiparallel strand of the MIR hairpin rather than by frequencies of corresponding trinucleotides in splicing silencers/enhancers. We also show that splicing outcome of oligonucleotides targeting the MIR exon depend on the identity of the triloop adjacent to their antisense target. Finally, we identify proteins regulating MIR exon recognition and reveal a distinct requirement of adjacent exons for C-terminal extensions of Tra2α and Tra2ß RNA recognition motifs.

Ubiquitin-binding domains: mechanisms of ubiquitin recognition and use as tools to investigate ubiquitin-modified proteomes.

Ubiquitin-binding domains (UBDs) are modular units found within ubiquitin-binding proteins that mediate the non-covalent recognition of (poly)ubiquitin modifications. A variety of mechanisms are employed in vivo to achieve polyubiquitin linkage and chain length selectivity by UBDs, the structural basis of which have in some instances been determined. Here, we review current knowledge related to ubiquitin recognition mechanisms at the molecular level and explore how such information has been exploited in the design and application of UBDs in isolation or artificially arranged in tandem as tools to investigate ubiquitin-modified proteomes. Specifically, we focus on the use of UBDs to directly purify or detect (poly)ubiquitin-modified proteins and more broadly for the targeted manipulation of ubiquitin-mediated processes, highlighting insights into ubiquitin signalling that have been provided.

Ligand selectivity in stabilising tandem parallel folded G-quadruplex motifs in human telomeric DNA sequences.

Biophysical studies of ligand interactions with three human telomeric repeat sequences (d(AGGG(TTAGGG)n, n = 3, 7 and 11)) show that an oxazole-based 'click' ligand, which induces parallel folded quadruplexes, preferentially stabilises longer telomeric repeats providing evidence for selectivity in binding at the interface between tandem quadruplex motifs.

A targeted oligonucleotide enhancer of SMN2 exon 7 splicing forms competing quadruplex and protein complexes in functional conditions.

The use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES) anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE) sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5' end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs.