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Stephen P Bottomley

Publications and source records attributed to Stephen P Bottomley.

At least 19 recordsLinked to original sources
PubMed2006

Serpin acceleration of amyloid fibril formation: a role for accessory proteins.

Protein aggregation underlies an increasing number of human diseases. Recent experiments have shown that the aggregation reaction is exquisitely specific involving particular interactions between non-native proteins. However, aggregation of certain proteins, for example beta-amyloid, in vivo leads to the recruitment of other proteins into the aggregate. Antichymotrypsin, a non-fibril forming protein, is always observed to be associated with beta-amyloid plaques in Alzheimer's sufferers. The role of antichymotrypsin is controversial with studies showing it can either accelerate or inhibit the aggregation reaction. To investigate the role of antichymotrypsin in fibrillogenesis we have studied its interaction with apolipoprotein C-II, a well characterized model system for the study of fibrillogenesis. Our data demonstrate that sub-stoichiometric amounts of antichymotrypsin and its alternate structural forms can dramatically accelerate the aggregation of apolipoprotein C-II, whereas the presence of alpha(1)-antitrypsin, a structural homologue of antichymotrypsin, cannot. Sedimentation velocity experiments show more apolipoprotein C-II fibrils were formed in the presence of antichymotrypsin. Using pull-down assays and immuno-gold labeling we demonstrate an interaction between antichymotrypsin and apolipoprotein C-II fibrils that specifically occurs during fibrillogenesis. Taken together these data demonstrate an interaction between antichymotrypsin and apolipoprotein C-II that accelerates fibrillogenesis and indicates a specific role for accessory proteins in protein aggregation.

Amyloid↗
PubMed2006

Molecular gymnastics: serpin structure, folding and misfolding.

The native state of serpins represents a long-lived intermediate or metastable structure on the serpin folding pathway. Upon interaction with a protease, the serpin trap is sprung and the molecule continues to fold into a more stable conformation. However, thermodynamic stability can also be achieved through alternative, unproductive folding pathways that result in the formation of inactive conformations. Our increasing understanding of the mechanism of protease inhibition and the dynamics of native serpin structures has begun to reveal how evolution has harnessed the actual process of protein folding (rather than the final folded outcome) to elegantly achieve function. The cost of using metastability for function, however, is an increased propensity for misfolding.

Animals↗
PubMed2006

X-ray crystal structure of MENT: evidence for functional loop-sheet polymers in chromatin condensation.

Most serpins are associated with protease inhibition, and their ability to form loop-sheet polymers is linked to conformational disease and the human serpinopathies. Here we describe the structural and functional dissection of how a unique serpin, the non-histone architectural protein, MENT (Myeloid and Erythroid Nuclear Termination stage-specific protein), participates in DNA and chromatin condensation. Our data suggest that MENT contains at least two distinct DNA-binding sites, consistent with its simultaneous binding to the two closely juxtaposed linker DNA segments on a nucleosome. Remarkably, our studies suggest that the reactive centre loop, a region of the MENT molecule essential for chromatin bridging in vivo and in vitro, is able to mediate formation of a loop-sheet oligomer. These data provide mechanistic insight into chromatin compaction by a non-histone architectural protein and suggest how the structural plasticity of serpins has adapted to mediate physiological, rather than pathogenic, loop-sheet linkages.

Animals↗
PubMed2006

Tracking the unfolding pathway of a multirepeat protein via tryptophan scanning: evidence of localized instability in the mitochondrial import receptor Tom70.

The tetratricopeptide repeat (TPR) is a degenerate 34-amino acid repeating motif that forms a repeating helix-turn-helix structure and is a well characterized mediator of protein-protein interactions. Recently, a biophysical investigation on one naturally occurring TPR protein, Tom70, found that the mitochondrial receptor displayed an unusual three-state unfolding pathway, distinct from the two-state model usually displayed by TPR proteins. To investigate this unusual behavior, we undertook a tryptophan-scanning analysis of Tom70, where both native and engineered tryptophan residues are used as fluorescent reporters to monitor the range of local and global unfolding events that comprise the unfolding pathway of Tom70. Specifically, seven Tom70 variants were constructed, each with a single tryptophan residue in each of the seven TPR repeats of Tom70. By combining equilibrium and kinetic fluorescent unfolding assays, with circular dichroism experiments, our study reveals that the unusual folding pathway of Tom70 is a consequence of the unfolding of two separate, autonomous TPR arrays, with the less stable region appearing to account for the low structural stability of Tom70.

Amino Acid Motifs↗
PubMed2006

The two-stage pathway of ataxin-3 fibrillogenesis involves a polyglutamine-independent step.

The aggregation of ataxin-3 is associated with spinocerebellar ataxia type 3, which is characterized by the formation of intraneuronal aggregates. However, the mechanism of aggregation is currently not well understood. Ataxin-3 consists of a folded Josephin domain followed by two ubiquitin-interacting motifs and a C-terminal polyglutamine tract, which in the non-pathological form is less than 45 residues in length. We demonstrate that ataxin-3 with 64 glutamines (at(Q64)) undergoes a two-stage aggregation. The first stage involves formation of SDS-soluble aggregates, and the second stage results in formation of SDS-insoluble aggregates via the poly(Q) region. Both these first and second stage aggregates display typical amyloid-like characteristics. Under the same conditions at(Q15) and at(QHQ) undergo a single step aggregation event resulting in SDS-soluble aggregates, which does not involve the polyglutamine tract. These aggregates do not convert to the SDS-insoluble form. These observations demonstrate that ataxin-3 has an inherent capacity to aggregate through its non-polyglutamine domains. However, the presence of a pathological length polyglutamine tract introduces an additional step resulting in formation of a highly stable amyloid-like aggregate.

Ataxin-3↗
PubMed2006

A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production.

BACKGROUND: In the past few years, both automated and manual high-throughput protein expression and purification has become an accessible means to rapidly screen and produce soluble proteins for structural and functional studies. However, many of the commercial vectors encoding different solubility tags require different cloning and purification steps for each vector, considerably slowing down expression screening. We have developed a set of E. coli expression vectors with different solubility tags that allow for parallel cloning from a single PCR product and can be purified using the same protocol. RESULTS: The set of E. coli expression vectors, encode for either a hexa-histidine tag or the three most commonly used solubility tags (GST, MBP, NusA) and all with an N-terminal hexa-histidine sequence. The result is two-fold: the His-tag facilitates purification by immobilised metal affinity chromatography, whilst the fusion domains act primarily as solubility aids during expression, in addition to providing an optional purification step. We have also incorporated a TEV recognition sequence following the solubility tag domain, which allows for highly specific cleavage (using TEV protease) of the fusion protein to yield native protein. These vectors are also designed for ligation-independent cloning and they possess a high-level expressing T7 promoter, which is suitable for auto-induction. To validate our vector system, we have cloned four different genes and also one gene into all four vectors and used small-scale expression and purification techniques. We demonstrate that the vectors are capable of high levels of expression and that efficient screening of new proteins can be readily achieved at the laboratory level. CONCLUSION: The result is a set of four rationally designed vectors, which can be used for streamlined cloning, expression and purification of target proteins in the laboratory and have the potential for being adaptable to a high-throughput screening.

Base Sequence↗
PubMed2006

The REFOLD database: a tool for the optimization of protein expression and refolding.

A large proportion of proteins expressed in Escherichia coli form inclusion bodies and thus require renaturation to attain a functional conformation for analysis. In this process, identifying and optimizing the refolding conditions and methodology is often rate limiting. In order to address this problem, we have developed REFOLD, a web-accessible relational database containing the published methods employed in the refolding of recombinant proteins. Currently, REFOLD contains >300 entries, which are heavily annotated such that the database can be searched via multiple parameters. We anticipate that REFOLD will continue to grow and eventually become a powerful tool for the optimization of protein renaturation. REFOLD is freely available at http://refold.med.monash.edu.au.

Databases, Protein↗
PubMed2006

Purification of polyglutamine proteins.

The misfolding and formation of fibrillar-like aggregates by polyglutamine proteins is believed to be a key factor in the development of the neurodegenerative polyglutamine diseases; however, relatively little is known about structural and conformational aspects of polyglutamine-induced misfolding and aggregation. This is largely attributable to the fact that polyglutamine proteins have proved difficult to purify in quantities suitable for biochemical and biophysical analyses, thus limiting the extent to which the proteins can be conformationally characterized. Recent advances, however, have seen the development of a number of protocols enabling the expression and purification of these proteins in more significant quantities. In this report, we describe a purification protocol for ataxin-3, which, in its polyglutamine-expanded form, causes Machado-Joseph disease. Purification of different length ataxin-3 variants, including one of pathological length, is facilitated by an N-terminal hexa-histidine tag, which enables binding to a nickel-chelated agarose resin. A key issue that arose during purification was the undesirable proteolysis of ataxin-3 by a trace contaminant protease. We solved this problem by the addition of a benzamidine-binding step during purification, which greatly reduced the level of proteases present. We found that the inclusion of this step had a significant positive impact on the quality of the purified protein product. We also inactivated trace amounts of proteases during experiments by the addition of specific protease inhibitors. Finally, we also describe initial structural and functional analyses that confirm the integrity of the purified protein.

Ataxin-3↗
PubMed2005

The murine orthologue of human antichymotrypsin: a structural paradigm for clade A3 serpins.

Antichymotrypsin (SERPINA3) is a widely expressed member of the serpin superfamily, required for the regulation of leukocyte proteases released during an inflammatory response and with a permissive role in the development of amyloid encephalopathy. Despite its biological significance, there is at present no available structure of this serpin in its native, inhibitory state. We present here the first fully refined structure of a murine antichymotrypsin orthologue to 2.1 A, which we propose as a template for other antichymotrypsin-like serpins. A most unexpected feature of the structure of murine serpina3n is that it reveals the reactive center loop (RCL) to be partially inserted into the A beta-sheet, a structural motif associated with ligand-dependent activation in other serpins. The RCL is, in addition, stabilized by salt bridges, and its plane is oriented at 90 degrees to the RCL of antitrypsin. A biochemical and biophysical analysis of this serpin demonstrates that it is a fast and efficient inhibitor of human leukocyte elastase (ka: 4 +/- 0.9 x 10(6) m(-1) s(-)1) and cathepsin G (ka: 7.9 +/- 0.9 x 10(5) m(-1) s(-)1) giving a spectrum of activity intermediate between that of human antichymotrypsin and human antitrypsin. An evolutionary analysis reveals that residues subject to positive selection and that have contributed to the diversity of sequences in this sub-branch (A3) of the serpin superfamily are essentially restricted to the P4-P6' region of the RCL, the distal hinge, and the loop between strands 4B and 5B.

Amino Acid Sequence↗
PubMed2005

REFOLD: an analytical database of protein refolding methods.

The expression and harvesting of proteins from insoluble inclusion bodies by solubilization and refolding is a technique commonly used in the production of recombinant proteins. To bring clarity to the large and widespread quantity of published protein refolding data, we have recently established the REFOLD database (http://refold.med.monash.edu.au), which is a freely available, open repository for protocols describing the refolding and purification of recombinant proteins. Refolding methods are currently published in many different formats and resources--REFOLD provides a standardized system for the structured reporting and presentation of these data. Furthermore, data in REFOLD are readily accessible using a simple search function, and the database also enables analyses which identify and highlight particular trends between suitable refolding and purification conditions and specific protein properties. This information may in turn serve to facilitate the rational design and development of new refolding protocols for novel proteins. There are approximately 200 proteins currently listed in REFOLD, and it is anticipated that with the continued contribution of data by researchers this number will grow significantly, thus strengthening the emerging trends and patterns and making this database a valuable tool for the scientific community.

Databases, Protein↗
PubMed2005

Dopamine promotes alpha-synuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway.

Dopamine (DA) and alpha-synuclein (alpha-SN) are two key molecules associated with Parkinson's disease (PD). We have identified a novel action of DA in the initial phase of alpha-SN aggregation and demonstrate that DA induces alpha-SN to form soluble, SDS-resistant oligomers. The DA:alpha-SN oligomeric species are not amyloidogenic as they do not react with thioflavin T and lack the typical amyloid fibril structures as visualized with electron microscopy. Circular dichroism studies indicate that in the presence of lipid membranes DA interacts with alpha-SN, causing an alteration to the structure of the protein. Furthermore, DA inhibited the formation of iron-induced alpha-SN amyloidogenic aggregates, suggesting that DA acts as a dominant modulator of alpha-SN aggregation. These observations support the paradigm emerging for other neurodegenerative diseases that the toxic species is represented by a soluble oligomer and not the insoluble fibril.

Amyloid↗
PubMed2005

Antagonism of antiviral and allogeneic activity of a human public CTL clonotype by a single altered peptide ligand: implications for allograft rejection.

Alloreactive T lymphocytes are central mediators of graft-versus-host disease and allograft rejection. A public CTL clonotype with specificity for the alloantigens HLA-B*4402 and B*4405 is often expanded to large numbers in healthy HLA-B*0801(+) individuals, driven by cross-reactive stimulation with the common, persistent herpesvirus EBV. Since such alloreactive memory CTL expansions have the potential to influence transplantation outcome, altered peptide ligands (APLs) of the target HLA-B*0801-binding EBV peptide, FLRGRAYGL, were screened as specific antagonists for this immunodominant clonotype. One APL, FLRGRFYGL, exerted powerful antagonism of a prototypic T cell clone expressing this immunodominant TCR when costimulated with target cells presenting HLA-B*0801(FLRGRAYGL). Significantly, this APL also reduced the lysis of allogeneic target cells expressing HLA-B*4402 by up to 99%. The affinities of the agonist and antagonist complexes for the public TCR, measured using solution and solid-phase assays, were 8 and 138 muM, respectively. Surprisingly, the half-life of the agonist and antagonist complexes was similar, yet the association rate for the antagonist complex was significantly slower. These observations were further supported by structural studies that suggested a large conformational hurdle was required to ligate the immunodominant TCR to the HLA-B*0801 antagonist complex. By defining an antagonist APL against an immunodominant alloreactive TCR, these findings raise the prospect of exploiting such peptides to inhibit clinical alloreactivity, particularly against clonal T cell expansions that react with alloantigens.

Antigens, Viral↗
PubMed2005

The high resolution crystal structure of the human tumor suppressor maspin reveals a novel conformational switch in the G-helix.

Maspin is a serpin that acts as a tumor suppressor in a range of human cancers, including tumors of the breast and lung. Maspin is crucial for development, because homozygous loss of the gene is lethal; however, the precise physiological role of the molecule is unclear. To gain insight into the function of human maspin, we have determined its crystal structure in two similar, but non-isomorphous crystal forms, to 2.1- and 2.8-A resolution, respectively. The structure reveals that maspin adopts the native serpin fold in which the reactive center loop is expelled fully from the A beta-sheet, makes minimal contacts with the core of the molecule, and exhibits a high degree of flexibility. A buried salt bridge unique to maspin orthologues causes an unusual bulge in the region around the D and E alpha-helices, an area of the molecule demonstrated in other serpins to be important for cofactor recognition. Strikingly, the structural data reveal that maspin is able to undergo conformational change in and around the G alpha-helix, switching between an open and a closed form. This change dictates the electrostatic character of a putative cofactor binding surface and highlights this region as a likely determinant of maspin function. The high resolution crystal structure of maspin provides a detailed molecular framework to elucidate the mechanism of function of this important tumor suppressor.

Animals↗
PubMed2005

PFD: a database for the investigation of protein folding kinetics and stability.

We have developed a new database that collects all protein folding data into a single, easily accessible public resource. The Protein Folding Database (PFD) contains annotated structural, methodological, kinetic and thermodynamic data for more than 50 proteins, from 39 families. A user-friendly web interface has been developed that allows powerful searching, browsing and information retrieval, whilst providing links to other protein databases. The database structure allows visualization of folding data in a useful and novel way, with a long-term aim of facilitating data mining and bioinformatics approaches. PFD can be accessed freely at http://pfd.med.monash.edu.au.

Databases, Protein↗
PubMed2005

A protein family under 'stress' - serpin stability, folding and misfolding.

The native fold of inhibitory serpins (serpin proteinase inhibitors) is metastable and therefore does not represent the most stable conformation that the primary sequence encodes for. The most stable form is adopted when the reactive centre loop (RCL) inserts, as the fourth strand, into the A b -sheet. Currently a serpin can adopt at least four more stable conformations, termed the cleaved, delta, latent and polymeric states. The accessibility of these alternative low energy folds renders the serpin molecule susceptible to mutations that can result in dysfunction and pathology. Here, we discuss the means by which the serpin can attain and preserve this metastable conformation. We also consider the triggers for misfolding to these more stable states and the mechanisms by which it occurs.

Animals↗
PubMed2005

Functional insights from the distribution and role of homopeptide repeat-containing proteins.

Expansion of "low complex" repeats of amino acids such as glutamine (Poly-Q) is associated with protein misfolding and the development of degenerative diseases such as Huntington's disease. The mechanism by which such regions promote misfolding remains controversial, the function of many repeat-containing proteins (RCPs) remains obscure, and the role (if any) of repeat regions remains to be determined. Here, a Web-accessible database of RCPs is presented. The distribution and evolution of RCPs that contain homopeptide repeats tracts are considered, and the existence of functional patterns investigated. Generally, it is found that while polyamino acid repeats are extremely rare in prokaryotes, several eukaryote putative homologs of prokaryote RCP-involved in important housekeeping processes-retain the repetitive region, suggesting an ancient origin for certain repeats. Within eukarya, the most common uninterrupted amino acid repeats are glutamine, asparagines, and alanine. Interestingly, while poly-Q repeats are found in vertebrates and nonvertebrates, poly-N repeats are only common in more primitive nonvertebrate organisms, such as insects and nematodes. We have assigned function to eukaryote RCPs using Online Mendelian Inheritance in Man (OMIM), the Human Reference Protein Database (HRPD), FlyBase, and Wormpep. Prokaryote RCPs were annotated using BLASTp searches and Gene Ontology. These data reveal that the majority of RCPs are involved in processes that require the assembly of large, multiprotein complexes, such as transcription and signaling.

Amino Acid Sequence↗
PubMed2005

Serpins 2005 - fun between the beta-sheets. Meeting report based upon presentations made at the 4th International Symposium on Serpin Structure, Function and Biology (Cairns, Australia).

Serpins are the largest family of protease inhibitors and are fundamental for the control of proteolysis in multicellular eukaryotes. Most eukaryote serpins inhibit serine or cysteine proteases, however, noninhibitory members have been identified that perform diverse functions in processes such as hormone delivery and tumour metastasis. More recently inhibitory serpins have been identified in prokaryotes and unicellular eukaryotes, nevertheless, the precise molecular targets of these molecules remains to be identified. The serpin mechanism of protease inhibition is unusual and involves a major conformational rearrangement of the molecule concomitant with a distortion of the target protease. As a result of this requirement, serpins are susceptible to mutations that result in polymerization and conformational diseases such as the human serpinopathies. This review reports on recent major discoveries in the serpin field, based upon presentations made at the 4th International Symposium on Serpin Structure, Function and Biology (Cairns, Australia).

Animals↗