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1.  Allosteric regulation of deubiquitylase activity through ubiquitination 
Ataxin-3, the protein responsible for spinocerebellar ataxia type-3, is a cysteine protease that specifically cleaves poly-ubiquitin chains and participates in the ubiquitin proteasome pathway. The enzymatic activity resides in the N-terminal Josephin domain. An unusual feature of ataxin-3 is its low enzymatic activity especially for mono-ubiquitinated substrates and short ubiquitin chains. However, specific ubiquitination at lysine 117 in the Josephin domain activates ataxin-3 through an unknown mechanism. Here, we investigate the effects of K117 ubiquitination on the structure and enzymatic activity of the protein. We show that covalently linked ubiquitin rests on the Josephin domain, forming a compact globular moiety and occupying a ubiquitin binding site previously thought to be essential for substrate recognition. In doing so, ubiquitination enhances enzymatic activity by locking the enzyme in an activated state. Our results indicate that ubiquitin functions both as a substrate and as an allosteric regulatory factor. We provide a novel example in which a conformational switch controls the activity of an enzyme that mediates deubiquitination.
PMCID: PMC4428445  PMID: 25988170
ataxin-3; SCA3; polyglutamine disease; deubiquitinating enzyme; cysteine protease; ubiquitin; structure
2.  The Challenge of Producing Ubiquitinated Proteins for Structural Studies 
Cells  2014;3(2):639-656.
Protein 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.
PMCID: PMC4092866  PMID: 24926903
ubiquitin; post-translational modification; mono-ubiquitination; isopeptide bond; native chemical ligation; non-enzymatic ubiquitination; enzymatic ubiquitination; in vitro ubiquitination; X-ray crystallography; nuclear magnetic resonance spectroscopy
3.  Enzymatic production of mono-ubiquitinated proteins for structural studies: The example of the Josephin domain of ataxin-3☆ 
FEBS Open Bio  2013;3:453-458.
Protein ubiquitination occurs through formation of an isopeptide bond between the C-terminal glycine of ubiquitin (Ub) and the ɛ-amino group of a substrate lysine residue. This post-translational modification, which occurs through the attachment of single and/or multiple copies of mono-ubiquitin and poly-ubiquitin chains, is involved in crucial cellular events such as protein degradation, cell-cycle regulation and DNA repair. The abnormal functioning of ubiquitin pathways is also implicated in the pathogenesis of several human diseases ranging from cancer to neurodegeneration. However, despite the undoubted biological importance, understanding the molecular basis of how ubiquitination regulates different pathways has up to now been strongly limited by the difficulty of producing the amounts of highly homogeneous samples that are needed for a structural characterization by X-ray crystallography and/or NMR. Here, we report on the production of milligrams of highly pure Josephin mono-ubiquitinated on lysine 117 through large scale in vitro enzymatic ubiquitination. Josephin is the catalytic domain of ataxin-3, a protein responsible for spinocerebellar ataxia type 3. Ataxin-3 is the first deubiquitinating enzyme (DUB) reported to be activated by mono-ubiquitination. We demonstrate that the samples produced with the described method are correctly folded and suitable for structural studies. The protocol allows facile selective labelling of the components. Our results provide an important proof-of-concept that may pave the way to new approaches to the in vitro study of ubiquitinated proteins.
Graphical abstract
•We set up a protocol for large-scale in vitro enzymatic ubiqitination.•This produced milligrams of highly pure mono-ubiquitinated Josephin domain of ataxin-3.•We applied an alternative labelling scheme for the structural characterization of the sample by NMR.•Ubiquitin covalently linked on lysine 117 directly interacts with Josephin but does not alter the overall fold of the protein.
PMCID: PMC3829987  PMID: 24251111
Ubiquitin; Post-translational modification; Isopeptide bond; Josephin; Spinocerebellar ataxia type 3; Machado–Joseph disease; Deubiquitinating enzyme; ATP, adenosine triphosphate; DTT, dithiothreitol; DUB, deubiquitinating enzyme; GST, glutathione-S-transferase; HSQC, heteronuclear single quantum coherence; IAA, iodoacetamide; JosK117-only, Josephin mutant in which all lysines but K117 are mutated; MS/MS tandem, mass spectrometry; NMR, nuclear magnetic resonance; PDB, Protein Data Bank; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Tris–HCl, 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride
4.  The Role of Interruptions in polyQ in the Pathology of SCA1 
PLoS Genetics  2013;9(7):e1003648.
At least nine dominant neurodegenerative diseases are caused by expansion of CAG repeats in coding regions of specific genes that result in abnormal elongation of polyglutamine (polyQ) tracts in the corresponding gene products. When above a threshold that is specific for each disease the expanded polyQ repeats promote protein aggregation, misfolding and neuronal cell death. The length of the polyQ tract inversely correlates with the age at disease onset. It has been observed that interruption of the CAG tract by silent (CAA) or missense (CAT) mutations may strongly modulate the effect of the expansion and delay the onset age. We have carried out an extensive study in which we have complemented DNA sequence determination with cellular and biophysical models. By sequencing cloned normal and expanded SCA1 alleles taken from our cohort of ataxia patients we have determined sequence variations not detected by allele sizing and observed for the first time that repeat instability can occur even in the presence of CAG interruptions. We show that histidine interrupted pathogenic alleles occur with relatively high frequency (11%) and that the age at onset inversely correlates linearly with the longer uninterrupted CAG stretch. This could be reproduced in a cellular model to support the hypothesis of a linear behaviour of polyQ. We clarified by in vitro studies the mechanism by which polyQ interruption slows down aggregation. Our study contributes to the understanding of the role of polyQ interruption in the SCA1 phenotype with regards to age at disease onset, prognosis and transmission.
Author Summary
Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disorder resulting in loss of coordination and balance. It is caused by an expanded repeated DNA sequence (CAG) in the gene ATXN1. The CAG repeat region is normally interrupted by the DNA sequence CAT. Loss of this interruption is believed to cause instability whereby the CAG repeat expands beyond a key threshold resulting, ultimately, in polyglutamine protein aggregation and cell death. Here we examine how interruptions influence pathology in patients and establish a cellular model to support our findings. We distinguish our patients into two sub-groups based on whether or not their expanded CAG repeat stretches contained an interruption. This is not possible with conventional diagnostic techniques. Differentiating the sub-group with no interruptions led to improved accuracy in predicting their age at onset. The other sub-group, with interruptions, reveals a delay in age at onset that shows greater alignment with the longest stretch of CAG repeats. These findings are significant for genetic counselling and prognosis. Our cellular model and in vitro studies confirmed the relationship between disease severity and uninterrupted repeat length and showed that interruptions do not significantly affect the polyglutamine protein aggregation, but do slow down the aggregation rate.
PMCID: PMC3723530  PMID: 23935513
5.  Haemoglobin-based oxygen carriers: research and reality towards an alternative to blood transfusions 
Blood Transfusion  2010;8(Suppl 3):s59-s68.
PMCID: PMC2897202  PMID: 20606751
haemoglobin; oxygen therapy; transfusion; red blood cells

Results 1-5 (5)