2,217,222 research outputs found
Structure-function studies of the Parkinsonism-linked protein DJ-1
No full textDJ-1 is a multifunctional protein linked to familial Parkinson’s disease. DJ-1 has been suggested to exert its cytoprotective function, in part, by acting as a copper carrier that can sequester the reactive metal and/or provide the copper cofactor for the activation of the Cu-Zn superoxide dismutase (SOD1). Using absorption spectroscopy and mass spectrometry, we found that DJ-1 binds one Cu(I) ion per DJ-1 homodimer. The structure of Cu(I)-bound DJ-1 reveals a new biscysteinate metal binding motif formed by juxtaposed Cys-53 at the homodimer interface. We calculated a subfemtomolar dissociation constant (Kd = 6.41 x 10-16 M) for Cu(I) that supports the physiological intracellular retention of the metal. Cu(I)-bound DJ-1 was not capable of interacting and activating SOD1 in vitro. We posit that DJ-1 sequester copper to protect against metal-induced cytotoxicity. Our results illuminate the molecular basis on how disease-linked mutations that impairs homodimerisation could disrupt the metal binding site.
In the second part of this dissertation, we sought to determine the impact of a Parkinsonism-linked A107P mutation on DJ-1 structure and glyoxalase activity. The A107P variant abrogates the ability of DJ-1 to protect against glyoxal-induced cytotoxicity and carboxymethyllysine protein modification. A crystal structure of DJ-1 C106S variant with glycerol and sulphate bound in the active site suggests that Ala-107 is critical for the stabilization of the transition state of the nucleophilic addition step. In our hands, the protein levels of DJ-1 A107P mutant in SH-SH5Y cells were ostensibly similar to the wild-type level but reduced levels were found in HEK 293E and MEF cells. Using CD and NMR spectroscopy, we found that the structural defect caused by the mutation extends beyond the active site. The A107P mutation resulted in a remarkable misfolding of the protein providing a basis for the reduced intracellular protein level and the abrogation of enzymatic activity
DJ-1 in Parkinson's Disease: Clinical Insights and Therapeutic Perspectives.
No full textMutations in the protein DJ-1 cause autosomal recessive forms of Parkinson's disease (PD) and oxidized DJ-1 is found in the brains of idiopathic PD individuals. While several functions have been ascribed to DJ-1 (most notably protection from oxidative stress), its contribution to PD pathogenesis is not yet clear. Here we provide an overview of the clinical research to date on DJ-1 and the current state of knowledge regarding DJ-1 characterization in the human brain. The relevance of DJ-1 as a PD biomarker is also discussed, as are studies exploring DJ-1 as a possible therapeutic target for PD and neurodegeneration
Rethinking live electronic music: a DJ perspective
No full textThe author critiques the conventional understanding of live electronic music through empirical research on his own DJ practice and investigates others working in the field. In reviewing the opinions of theorists and practitioners in both the live electronic music genre and DJ-ing he argues against the body/machine dialectic that has determined much of the thinking in the former. The author forms a notion of the DJ as a real-time composer working beyond traditional binary distinctions who brings the human body and machine into a mutual relationship. Through practice-led research he charts an investigation beginning in physical human gesture and culminating in digital machine repetition. He concludes that mechanical and digital repetition do not obscure human agency in the production of live works and that this concern is imaginary
Exploring mitochondrial contact sites in DJ-1 overexpressing cells
No full textreservedDJ-1 è una piccola proteina di 189 amminoacidi altamente conservata ed espressa in modo ubiquitario. Nell’uomo DJ-1 è codificata dal gene PARK7, il quale era stato correlato nel 2003 a forme autosomiali recessive del morbo di Parkinson familiare. Ancora non è chiaro il contributo che DJ-1 ha nell’insorgenza del Parkinson poiché l’elevato numero di funzioni fisiologiche diverse attribuite a questa proteina rende complicata la comprensione del legame tra le sue mutazioni e la patologia.
DJ-1 funziona da sensore dello stress ossidativo ma può anche partecipare direttamente nella neutralizzazione di ROS come scavenger. Inoltre, la proteina può svolgere funzioni di chaperonina, proteasi, regolatore trascrizionale, proteina legante RNA, regolatore delle funzioni mitocondriali e regolatore autofagico, oltre a poter essere coinvolta nella regolazione dell’omeostasi delle catecolamine.
Studi recenti condotti nel nostro laboratorio hanno esaminato l’effetto che DJ-1 può avere sulla modulazione dei contatti tra organelli cellulari e come la sua concentrazione può influire sul numero e la forma di questi siti di contatto, come quelli tra reticolo endoplasmatico e mitocondri o tra lisosomi e mitocondri. Per visualizzare i contatti, il laboratorio che ho frequentato per il mio progetto di tesi ha sviluppato un nuovo strumento ricombinante, le sonde SPLICS, basate sulla tecnologia della complementazione della fluorescenza bimolecolare (BiFC). Il loro funzionamento sfrutta l’abilità di due frammenti di GFP non fluorescenti di ricostituire la GFP completa quando si trovano sufficientemente vicini, ripristinando così il segnale fluorescente. Questa proprietà è stata utilizzata per visualizzare i siti di contatto tra le membrane di due diversi organelli attraverso il targeting selettivo delle due porzioni non fluorescenti con delle sequenze specifiche di localizzazione. Nel mio progetto di tesi viene indagato come l’overespressione di DJ-1 in cellule di mammifero influenza i siti di contatto tra mitocondri e reticolo endoplasmatico o lisosomi.DJ-1 is a small, highly conserved protein of 189 amino acids, which is ubiquitously expressed. In humans, DJ-1 is encoded by the PARK7 gene, which was first linked to early onset, autosomal recessive familial forms of Parkinson’s disease (PD) in 2003. It is still unclear how DJ-1 contributes to PD pathogenesis: the high number of different physiological functions attributed to DJ-1 represent an important drawback in the comprehension of how its mutations cause PD.
DJ-1 can work as an oxidative or stress level sensor, but it can also directly participate in ROS neutralizations as scavenger. Furthermore, the protein has been shown to function as a molecular chaperone, protease, transcriptional regulator, RNA-binding protein, mitochondrial function regulator and autophagy regulator, and it is involved in regulation of catecholamine homeostasis. Recent studies developed in our lab explores DJ-1 modulation of cell organelles contacts, how its concentration affects the
number and the form of organelles tethering, such as that between endoplasmic reticulum (ER) and mitochondria or lysosomes and mitochondria. To visualize organelle contacts sites the laboratory where I spent my thesis project has developed new recombinant tool, the SPLICS probes, based on the SplitGFP and Bimolecular Fluorescence Complementation (BiFC) technology. Their functioning takes advantage from the ability of two non-fluorescent fragments of the GFP to restore the fully fluorescent signal when they are close enough to reconstitute the full GFP by complementation. This property was exploited to visualize contact sites between the membrane of two different organelles upon the selective targeting the two GFP portions to the membrane of the organelles of interest by the addition of specific localization sequences.
In my thesis project it has been specifically examined how DJ-1 overexpression in mammalian cells affects contact sites between mitochondria and ER or lysosomes
The ColorDex DJ system: a new interface for live music mixing
No full textThis paper describes the design and implementation of a new interface prototype for live music mixing. The ColorDex system employs a completely new operational metaphor which allows the mix DJ to prepare up to six tracks at once, and perform mixes between up to three of those at a time. The basic premises of the design are: 1) Build a performance tool that multiplies the possible choices a DJ has in respect in how and when tracks are prepared and mixed; 2) Design the system in such a way that the tool does not overload the performer with unnecessary complexity, and 3) Make use of novel technology to make the performance of live music mixing more engaging for both the performer and the audience. The core components of the system are: A software program to load, visualize and playback digitally encoded tracks; the HDDJ device (built chiefly out of a repurposed hard disk drive), which provides tactile manipulation of the playback speed and position of tracks; and the Cubic Crossfader, a wireless sensor cube that controls of the volume of individual tracks, and allows the DJ to mix these in interesting ways
hpDJ: An automated DJ with floorshow feedback
No full textMany radio stations and nightclubs employ Disk-Jockeys (DJs) to provide a continuous uninterrupted stream or “mix” of dance music, built from a sequence of individual song-tracks. In the last decade, commercial pre-recorded compilation CDs of DJ mixes have become a growth market. DJs exercise skill in deciding an appropriate sequence of tracks and in mixing 'seamlessly' from one track to the next. Online access to large-scale archives of digitized music via automated music information retrieval systems offers users the possibility of discovering many songs they like, but the majority of consumers are unlikely to want to learn the DJ skills of sequencing and mixing. This paper describes hpDJ, an automatic method by which compilations of dance-music can be sequenced and seamlessly mixed by computer, with minimal user involvement. The user may specify a selection of tracks, and may give a qualitative indication of the type of mix required. The resultant mix can be presented as a continuous single digital audio file, whether for burning to CD, or for play-out from a personal playback device such as an iPod, or for play-out to rooms full of dancers in a nightclub. Results from an early version of this system have been tested on an audience of patrons in a London nightclub, with very favourable results. Subsequent to that experiment, we designed technologies which allow the hpDJ system to monitor the responses of crowds of dancers/listeners, so that hpDJ can dynamically react to those responses from the crowd. The initial intention was that hpDJ would monitor the crowd’s reaction to the song-track currently being played, and use that response to guide its selection of subsequent song-tracks tracks in the mix. In that version, it’s assumed that all the song-tracks existed in some archive or library of pre-recorded files. However, once reliable crowd-monitoring technology is available, it becomes possible to use the crowd-response data to dynamically “remix” existing song-tracks (i.e, alter the track in some way, tailoring it to the response of the crowd) and even to dynamically “compose” new song-tracks suited to that crowd. Thus, the music played by hpDJ to any particular crowd of listeners on any particular night becomes a direct function of that particular crowd’s particular responses on that particular night. On a different night, the same crowd of people might react in a different way, leading hpDJ to create different music. Thus, the music composed and played by hpDJ could be viewed as an “emergent” property of the dynamic interaction between the computer system and the crowd, and the crowd could then be viewed as having collectively collaborated on composing the music that was played on that night. This en masse collective composition raises some interesting legal issues regarding the ownership of the composition (i.e.: who, exactly, is the author of the work?), but revenue-generating businesses can nevertheless plausibly be built from such technologies
DJ-1 in Parkinson’s Disease: Clinical Insights and Therapeutic Perspectives
No full textMutations in the protein DJ-1 cause autosomal recessive forms of Parkinson’s disease (PD) and oxidized DJ-1 is found in the brains of idiopathic PD individuals. While several functions have been ascribed to DJ-1 (most notably protection from oxidative stress), its contribution to PD pathogenesis is not yet clear. Here we provide an overview of the clinical research to date on DJ-1 and the current state of knowledge regarding DJ-1 characterization in the human brain. The relevance of DJ-1 as a PD biomarker is also discussed, as are studies exploring DJ-1 as a possible therapeutic target for PD and neurodegeneration
DJ-1 Molecular Chaperone Activity Depresses Tau Aggregation Propensity through Interaction with Monomers
No full textTau aggregate-bearing lesions are pathological markers and potential mediators of tauopathic neurodegenerative diseases, including Alzheimer’s disease. The molecular chaperone DJ-1 colocalizes with tau pathology in these disorders, but it has been unclear what functional link exists between them. In this study, we examined the consequences of tau/DJ-1 interaction as isolated proteins in vitro. When added to full-length 2N4R tau under aggregation-promoting conditions, DJ-1 inhibited both the rate and extent of filament formation in a concentration-dependent manner. Inhibitory activity was low affinity, did not require ATP, and was not affected by substituting oxidation incompetent missense mutation C106A for wild-type DJ-1. In contrast, missense mutations previously linked to familial Parkinson’s disease and loss of α-synuclein chaperone activity, M26I and E64D, displayed diminished tau chaperone activity relative to wild-type DJ-1. Although DJ-1 directly bound the isolated microtubule-binding repeat region of tau protein, exposure of preformed tau seeds to DJ-1 did not diminish seeding activity in a biosensor cell model. These data reveal DJ-1 to be a holdase chaperone capable of engaging tau as a client in addition to α-synuclein. Our findings support a role for DJ-1 as part of an endogenous defense against the aggregation of these intrinsically disordered proteins
DJ-1 Molecular Chaperone Activity Depresses Tau Aggregation Propensity through Interaction with Monomers
No full textTau aggregate-bearing lesions are pathological markers and potential mediators of tauopathic neurodegenerative diseases, including Alzheimer’s disease. The molecular chaperone DJ-1 colocalizes with tau pathology in these disorders, but it has been unclear what functional link exists between them. In this study, we examined the consequences of tau/DJ-1 interaction as isolated proteins in vitro. When added to full-length 2N4R tau under aggregation-promoting conditions, DJ-1 inhibited both the rate and extent of filament formation in a concentration-dependent manner. Inhibitory activity was low affinity, did not require ATP, and was not affected by substituting oxidation incompetent missense mutation C106A for wild-type DJ-1. In contrast, missense mutations previously linked to familial Parkinson’s disease and loss of α-synuclein chaperone activity, M26I and E64D, displayed diminished tau chaperone activity relative to wild-type DJ-1. Although DJ-1 directly bound the isolated microtubule-binding repeat region of tau protein, exposure of preformed tau seeds to DJ-1 did not diminish seeding activity in a biosensor cell model. These data reveal DJ-1 to be a holdase chaperone capable of engaging tau as a client in addition to α-synuclein. Our findings support a role for DJ-1 as part of an endogenous defense against the aggregation of these intrinsically disordered proteins
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