1,721,353 research outputs found
Chemical shift assignment of the alternative scaffold protein IscA
Full text linkThe IscA protein (11.5 kDa) is an essential component of the iron sulphur cluster biogenesis machine. In bacteria, the machine components are clustered in operons, amongst which the most important is the isc operon. Bacterial IscA has direct homologues also in eukaryotes. Like the protein IscU, IscA is thought to assist cluster formation as an alternative scaffold protein which receives the cluster before transferring it further to the final acceptors. Several crystal structures have been published. They all report an IscA dimeric form, although the packing of the protomers in the dimers differs amongst structures. No solution studies have currently been reported. Here we report the 1H, 13C and 15N backbone and side-chain chemical shift assignments of the cluster-free E. coli IscA as a starting point for further studies of the structure and functions of this still poorly characterized protein. We show that IscA exists in solution as an equilibrium between different species. Spectrum assignment was thus challenging given the heterogeneous nature of the sample but doable through judicious choice of selective labelling and concentration dependent studies
How an Enzyme Can Be a Non-Enzyme
No full textAbstractThe crystal structure of the endonuclease, ribonuclease III, in complex with a double-stranded RNA fragment reveals the mechanism that allows for switching between two functional forms of the enzyme
Glutamine repeats: Structural hypotheses and neurodegeneration
No full textA growing number of neurodegenerative diseases are caused by expansion of CAG trinucleotide repeats coding for polyglutamine. The presence of intranuclear inclusions in the affected neuronal cells has suggested a mechanism for pathogenesis based on protein misfolding and aggregation. Detailed understanding of these phenomena is therefore crucial in order to rationalize different phases of the diseases. In the past decade, a few studies have focused on the structural properties of polyglutamine and on the molecular bases of the aggregation process. Most of these studies have been performed on polyglutamine peptides and protein models. Only one report is currently available on the characterization of a full-length polyglutamine protein. The structural hypotheses resulting from these studies are reviewed here
Kaleidoscopic protein–protein interactions in the life and death of ataxin-1: new strategies against protein aggregation
No full textUnderstanding how proteins protect themselves from aberrant aggregation is of primary interest for understanding basic biology, protein biochemistry, and human disease. We discuss the paradigmatic example of ataxin-1 (Atx1), the protein responsible for neurodegenerative spinocerebellar ataxia type 1 (SCA1). This disease is part of the increasing family of pathologies caused by protein aggregation and misfolding. We discuss the importance of protein–protein interactions not only in the nonpathological function of Atx1 but also in protecting the protein from aggregation and misfolding. The lessons learned from Atx1 may lead to a more general understanding of the cell's protective strategies against aggregation. The obtained knowledge may suggest a new perspective for designing specific therapeutic strategies for the cure of misfolding diseases
The challenge of producing ubiquitinated proteins for structural studies
No full textProtein ubiquitination is an important post-translational modification involved in several essential signalling pathways. It has different effects on the target protein substrate, i.e., it can trigger the degradation of the protein in the proteasome, change the interactions of the modified protein with its partners, or affect its localization and activity. In order to understand the molecular mechanisms underlying the consequences of protein ubiquitination, scientists have to face the challenging task of producing ubiquitinated proteins for structural characterization with X-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy. These techniques require milligrams of homogeneous samples of high purity. The strategies proposed so far for the production of ubiquitinated proteins can be divided into two groups, i.e., chemical (or non-enzymatic) and enzymatic methodologies. In this review, we summarize the still very sparse examples available in the literature that describe successful production of ubiquitinated proteins amenable for biochemical and structural studies, and discuss advantages and disadvantages of the techniques proposed. We also give a perspective of the direction in which the field might evolve
The missing links to link ubiquitin: Methods for the enzymatic production of polyubiquitin chains
Full text linkAbstractAttachment of ubiquitin (Ub) as monoUb and polyUb chains of different lengths and linkages to proteins plays a dominant role in very different regulatory mechanisms. Therefore, the study of polyUb chains has assumed a central interest in biochemistry and structural biology. An essential step necessary to allow in vitro biochemical and structural studies of polyUbs is the production of their chains in high quantities and purity. This is not always an easy task and can be achieved both enzymatically and chemically. Previous reviews have covered chemical cross-linking exhaustively. In this review, we concentrate on the different approaches developed so far for the enzymatic production of different Ub chains. These strategies permit a certain flexibility in the production of chains with various linkages and lengths. We critically describe the available methods and comment on advantages and limitations. It is clear that the field is mature to study most of the possible links, but some more work needs to be done to complete the picture and to exploit the current methodologies for understanding in full the Ub code
Chronochemistry in neurodegeneration
Full text linkThe problem of distinguishing causes from effects is not a trivial one, as illustrated by the science fiction writer Isaac Asimov in novels dedicated to an imaginary compound with surprising ‘chronochemistry’ properties. The problem is particularly important when trying to establish the aetiology of diseases. Here, we discuss how the problem reflects on our understanding of disease using two specific examples: Alzheimer’s disease and Friedreich’s ataxia. We show how the fibrillar aggregates observed in Alzheimer’s disease 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 fibre are the causes of disease, Similarly, we illustrate how the identification of causes and effects have been important in the study of Friedreich’s ataxia. 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 been recently developed which allow us to follow the development of the disease. We hope that this review may inspire similar studies in other scientific disciplines
The scaffold protein IscU retains a structured conformation in the Fe-S cluster assembly complex
No full textIscU and IscS are two essential proteins in the machine responsible for the biogenesis of iron-sulfur clusters, prosthetic groups that are involved in several essential functions. The scaffold protein IscU is the temporary acceptor of the cluster that results when the protein forms a 110 kDa complex with the desulfurase IscS. In the absence of zinc, which stabilises the folded state, IscU is present in solution in equilibrium between a structured and an unstructured form. It has been suggested that IscS preferentially binds unstructured IscU, although crystal structures indicate otherwise. To learn more about the IscS-IscU complex, we have used advanced solution NMR techniques to observe directly the state of fold of IscS-bound IscU. We present unambiguous evidence that IscU is folded in the complex and that the unstructured form does not bind to IscS. Our data correlate with several observations and explain an IscU-related pathology.</p
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