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Find video protocols related to scientific articles indexed in Pubmed.
Chronochemistry in neurodegeneration.
Front Mol Neurosci
PUBLISHED: 01-01-2014
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The problem of distinguishing causes from effects is not a trivial one, as illustrated by the science fiction writer Isaac Asimov in a novel dedicated to an imaginary compound with surprising "chronochemistry" properties. The problem is particularly important when trying to establish the etiology of diseases. Here, we discuss how the problem reflects on our understanding of disease using two specific examples: Alzheimer's disease (AD) and Friedreich's ataxia (FRDA). We show how the fibrillar aggregates observed in AD were first denied any interest, then to assume a central focus, and to finally recess to be considered the dead-end point of the aggregation pathway. This current view is that the soluble aggregates formed along the aggregation pathway rather than the mature amyliod fiber are the causes of disease, Similarly, we illustrate how the identification of causes and and effects have been important in the study of FRDA. This disease has alternatively been considered as the consequence of oxidative stress, iron precipitation or reduction of iron-sulfur cluster protein context. We illustrate how new tools have recently been established which allow us to follow the development of the disease. We hope that this review may inspire similar studies in other scientific disciplines.
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Ferredoxin competes with bacterial frataxin in binding to the desulfurase IscS.
J. Biol. Chem.
PUBLISHED: 07-09-2013
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The bacterial iron-sulfur cluster (isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly.
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Cluster and Fold Stability of E. coli ISC-Type Ferredoxin.
PLoS ONE
PUBLISHED: 01-01-2013
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Iron-sulfur clusters are essential protein prosthetic groups that provide their redox potential to several different metabolic pathways. Formation of iron-sulfur clusters is assisted by a specialised machine that comprises, among other proteins, a ferredoxin. As a first step to elucidate the precise role of this protein in cluster assembly, we have studied the factors governing the stability and the dynamic properties of E. coli ferredoxin using different spectroscopic techniques. The cluster-loaded protein is monomeric and well structured with a flexible C-terminus but is highly oxygen sensitive so that it readily loses the cluster leading to an irreversible unfolding under aerobic conditions. This process is slowed down by reducing conditions and high ionic strengths. NMR relaxation experiments on the cluster-loaded protein also show that, once the cluster is in place, the protein forms a globular and relatively rigid domain. These data indicate that the presence of the iron-sulfur cluster is the switch between a functional and a non-functional state.
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The role of CyaY in iron sulfur cluster assembly on the E. coli IscU scaffold protein.
PLoS ONE
PUBLISHED: 05-04-2011
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Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S](2+) and [4Fe4S](2+) but directly affects enzymatic activity.
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Mycobacterium tuberculosis WhiB1 is an essential DNA-binding protein with a nitric oxide-sensitive iron-sulfur cluster.
Biochem. J.
PUBLISHED: 10-09-2010
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Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. In the present study it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however, in the presence of apo-WhiB1, transcription was severely inhibited, irrespective of the presence or absence of the CRP (cAMP receptor protein) Rv3676, which is known to activate whiB1 expression. Footprinting suggested that autorepression of whiB1 is achieved by apo-WhiB1 binding at a region that overlaps the core promoter elements. A model incorporating regulation of whiB1 expression in response to nitric oxide and cAMP is discussed with implications for sensing two important signals in establishing M. tuberculosis infections.
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Structural bases for the interaction of frataxin with the central components of iron-sulphur cluster assembly.
Nat Commun
PUBLISHED: 06-08-2010
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Reduced levels of frataxin, an essential protein of as yet unknown function, are responsible for causing the neurodegenerative pathology Friedreichs ataxia. Independent reports have linked frataxin to iron-sulphur cluster assembly through interactions with the two central components of this machinery: desulphurase Nfs1/IscS and the scaffold protein Isu/IscU. In this study, we use a combination of biophysical methods to define the structural bases of the interaction of CyaY (the bacterial orthologue of frataxin) with the IscS/IscU complex. We show that CyaY binds IscS as a monomer in a pocket between the active site and the IscS dimer interface. Recognition does not require iron and occurs through electrostatic interactions of complementary charged residues. Mutations at the complex interface affect the rates of enzymatic cluster formation. CyaY binding strengthens the affinity of the IscS/IscU complex. Our data suggest a new paradigm for understanding the role of frataxin as a regulator of IscS functions.
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Of the vulnerability of orphan complex proteins: the case study of the E. coli IscU and IscS proteins.
Protein Expr. Purif.
PUBLISHED: 03-18-2010
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IscS and IscU, the two central protein components of the iron sulfur cluster assembly machinery, form a complex that is still relatively poorly characterized. In an attempt to standardize the purification of these proteins for structural studies we have developed a protocol to produce them individually in high concentration and purity. We show that IscS is a rather robust protein as long as it is produced in a PLP loaded form and that this co-factor is essential for fold stability and enzyme activity. In contrast to previous evidence, we also propose that, in contrast with previous evidence, IscU is a thermodynamically stable protein with a well defined fold but, when produced in isolation, is a complex-orphan protein that is prone to unfolding if not stabilised by a co-factor or a protein partner. Our work will facilitate further structural and functional studies of these proteins and eventually lead to a better understanding of the whole machinery.
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The N-terminus of mature human frataxin is intrinsically unfolded.
FEBS J.
PUBLISHED: 10-16-2009
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Frataxin is a highly conserved nuclear-encoded mitochondrial protein whose deficiency is the primary cause of Friedreichs ataxia, an autosomal recessive neurodegenerative disease. The frataxin structure comprises a well-characterized globular domain that is present in all species and is preceded in eukaryotes by a non-conserved N-terminal tail that contains the mitochondrial import signal. Little is known about the structure and dynamic properties of the N-terminal tail. Here, we show that this region is flexible and intrinsically unfolded in human frataxin. It does not alter the iron-binding or self-aggregation properties of the globular domain. It is therefore very unlikely that this region could be important for the conserved functions of the protein.
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Bacterial frataxin CyaY is the gatekeeper of iron-sulfur cluster formation catalyzed by IscS.
Nat. Struct. Mol. Biol.
PUBLISHED: 02-13-2009
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Frataxin is an essential mitochondrial protein whose reduced expression causes Friedreichs ataxia (FRDA), a lethal neurodegenerative disease. It is believed that frataxin is an iron chaperone that participates in iron metabolism. We have tested this hypothesis using the bacterial frataxin ortholog, CyaY, and different biochemical and biophysical techniques. We observe that CyaY participates in iron-sulfur (Fe-S) cluster assembly as an iron-dependent inhibitor of cluster formation, through binding to the desulfurase IscS. The interaction with IscS involves the iron binding surface of CyaY, which is conserved throughout the frataxin family. We propose that frataxins are iron sensors that act as regulators of Fe-S cluster formation to fine-tune the quantity of Fe-S cluster formed to the concentration of the available acceptors. Our observations provide new perspectives for understanding FRDA and a mechanistic model that rationalizes the available knowledge on frataxin.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.