Dr Rosie Staniforth
Tel: 0114 222 2761
Until very recently, our understanding of the causes behind neurodegenerative diseases has been very limited. The outbreak of BSE (commonly referred to as mad cow disease) and the rise of Alzheimer’s has meant that there is increasing pressure to obtain a cure. These two diseases are characterised by the accumulation of fibrous material called "amyloid" in the brain. This substance has been shown to consist of naturally occurring proteins assembled into this unnatural mass. Whether or not this protein-based material is the direct cause for deterioration of brain tissue in patients suffering from these diseases is a matter of some controversy. However, it is clear to all that these proteinaceous fibres are closely associated with the pathology: mutations in genes coding for these proteins cause the early onset of the disease and premature death. Another cause for hope is that this pattern of events may be common to almost all neurodegenerative diseases, including Parkinson’s and Huntington’s, and a large number of other diseases including certain forms of cancer, diabetes and stroke. It is exciting to think that understanding the mechanism of amyloid formation may be a step towards a universal cure since, so far, therapies have only been targeted at alleviating symptoms but cannot prevent or halt the onset of these devastating diseases.
As a biochemist, my interest is in studying the assembly of "normal" proteins into amyloid fibres at a molecular level (fig.1). A first consideration when tackling such a problem is that, unlike other polymers, protein molecules exist as unique three-dimensional structures in vivo (referred to as their folded state). In amyloidogenesis, proteins with totally different folds form fibres with very similar properties suggesting there may be common, structurally analogous intermediates on the assembly route. This also means that considerable conformational changes (unfolding) of the protein molecules are likely to occur either before or after association.
A well-characterised protein with a number of advantageous properties is cystatin C, a protease inhibitor which is associated with a disease causing recurrent stroke (Human Cystatin C Amyloid Angiopathy). I have identified a number of different conformers of this protein which could all be candidate precursors for the assembly process. These include partially folded states of the monomeric protein and a "domain-swapped" dimer. The latter species (fig. 2) is the result of two molecules of cystatin coming together and exchanging part of their chains by unfolding then refolding into a conformation made up of two cystatin-folds, where each protein chain contributes half its constituent amino acids to one fold and half to the other. This is the first time this kind of intertwining of protein chains is observed for an amyloidotic protein. Such a process of assembly results in the formation of extremely stable oligomers and has often been speculated as a convenient mechanism for amyloid fibre formation. Structural characterisation of the different forms of cystatin from monomer to dimer to fibre stretches the boundaries of current technology. We are using a combination of techniques including dynamic NMR (nuclear magnetic resonance) spectroscopy and high-resolution electron microscopy.
Identifying different states of the protein is only the first step in characterising the mechanism of amyloid formation: each species must be put in context within a sequence of molecular events, i.e. a pathway needs to be defined. To do this, we have set up a screen to identify optimum conditions for observing fibre formation in vitro. Time courses will be recorded when the reaction is initiated from a number of different states of the protein including the dimeric form. This will not only establish a mechanism for the first time but it will also provide an experimental system which can be used to screen for potential therapeutic drugs.
Structural biology, mechanism of amyloid formation, cystatins, Alzheimer's disease
Level 3 Modules
MBB310 Assembly of Supramolecular Structures (Module Coordinator)
Level 2 Modules
MBB261 Biochemistry 2
Level 1 Modules
MBB165 Practical Molecular Bioscience 1
Honours and Distinctions
- Dennison AJC, Jones RAL, Staniforth RA & Parnell AJ (2016) Interaction of partially denatured insulin with a DSPC floating lipid bilayer. Soft Matter, 12(3), 824-829. View this article in WRRO
- Davis PJ, Holmes D, Waltho JP & Staniforth RA (2015) Limited Proteolysis Reveals That Amyloids from the 3D Domain-Swapping Cystatin B Have a Non-Native β-Sheet Topology. Journal of Molecular Biology, 427(15), 2418-2434.
- Taler-Verčič A, Kirsipuu T, Friedemann M, Noormägi A, Polajnar M, Smirnova J, Znidarič MT, Zganec M, Skarabot M, Vilfan A, Staniforth RA, Palumaa P & Zerovnik E (2013) The role of initial oligomers in amyloid fibril formation by human stefin B.. Int J Mol Sci, 14(9), 18362-18384.
- Paramore R, Morgan GJ, Davis PJ, Sharma CA, Hounslow A, Taler-Verčič A, Žerovnik E, Waltho JP, Cliff MJ & Staniforth RA (2012) Mapping local structural perturbations in the native state of stefin B (cystatin B) under amyloid forming conditions. Frontiers in Molecular Neuroscience(AUG 2012), 1-30. View this article in WRRO
- Jelinska C, Davis PJ, Kenig M, Zerovnik E, Kokalj SJ, Gunčar G, Turk D, Turk V, Clarke DT, Waltho JP & Staniforth RA (2011) Modulation of contact order effects in the two-state folding of stefins A and B.. Biophys J, 100(9), 2268-2274.
- Žerovnik E, Stoka V, Mirtič A, Gunčar G, Grdadolnik J, Staniforth RA, Turk D & Turk V (2011) Mechanisms of amyloid fibril formation--focus on domain-swapping.. FEBS J, 278(13), 2263-2282.
- Zerovnik E, Staniforth RA & Turk D (2010) Amyloid fibril formation by human stefins: Structure, mechanism & putative functions.. Biochimie, 92(11), 1597-1607.
- Žerovnik E, Staniforth RA & Turk D (2010) Amyloid fibril formation by human stefins: Structure, mechanism & putative functions. Biochimie, 92(11), 1597-1607.
- Milanesi L, Jelinska C, Hunter CA, Hounslow AM, Staniforth RA & Waltho JP (2008) A method for the reversible trapping of proteins in non-native conformations.. Biochemistry, 47(51), 13620-13634.
- Morgan GJ, Giannini S, Hounslow AM, Craven CJ, Zerovnik E, Turk V, Waltho JP & Staniforth RA (2008) Exclusion of the native alpha-helix from the amyloid fibrils of a mixed alpha/beta protein.. J Mol Biol, 375(2), 487-498.
- Zerovnik E, Skarabot M, Skerget K, Giannini S, Stoka V, Jenko-Kokalj S & Staniforth RA (2007) Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology.. Amyloid, 14(3), 237-247.
- Jenko Kokalj S, Guncar G, Stern I, Morgan G, Rabzelj S, Kenig M, Staniforth RA, Waltho JP, Zerovnik E & Turk D (2007) Essential role of proline isomerization in stefin B tetramer formation.. J Mol Biol, 366(5), 1569-1579.
- Cliff MJ, Alizadeh T, Jelinska C, Craven CJ, Staniforth RA & Waltho JP (2006) A thiol labelling competition experiment as a probe for sidechain packing in the kinetic folding intermediate of N-PGK.. J Mol Biol, 364(4), 810-823.
- Reed MAC, Jelinska C, Syson K, Cliff MJ, Splevins A, Alizadeh T, Hounslow AM, Staniforth RA, Clarke AR, Craven CJ & Waltho JP (2006) The denatured state under native conditions: a non-native-like collapsed state of N-PGK.. J Mol Biol, 357(2), 365-372.
- Kenig M, Jenko-Kokalj S, Tusek-Znidaric M, Pompe-Novak M, Guncar G, Turk D, Waltho JP, Staniforth RA, Avbelj F & Zerovnik E (2006) Folding and amyloid-fibril formation for a series of human stefins' chimeras: any correlation?. Proteins, 62(4), 918-927.
- Sanders A, Jeremy Craven C, Higgins LD, Giannini S, Conroy MJ, Hounslow AM, Waltho JP & Staniforth RA (2004) Cystatin forms a tetramer through structural rearrangement of domain-swapped dimers prior to amyloidogenesis.. J Mol Biol, 336(1), 165-178.
- Staniforth RA, Giannini S, Higgins LD, Conroy MJ, Hounslow AM, Jerala R, Craven CJ & Waltho JP (2001) Three-dimensional domain swapping in the folded and molten-globule states of cystatins, an amyloid-forming structural superfamily.. EMBO J, 20(17), 4774-4781.
- Staniforth RA, Dean JL, Zhong Q, Zerovnik E, Clarke AR & Waltho JP (2000) The major transition state in folding need not involve the immobilization of side chains.. Proc Natl Acad Sci U S A, 97(11), 5790-5795.
- Staniforth RA, Giannini S, Bigotti MG, Cutruzzolà F, Travaglini-Allocatelli C & Brunori M (2000) A new folding intermediate of apomyoglobin from Aplysia limacina: stepwise formation of a molten globule.. J Mol Biol, 297(5), 1231-1244.
- Travaglini-Allocatelli C, Cutruzzolà F, Bigotti MG, Staniforth RA & Brunori M (1999) Folding mechanism of Pseudomonas aeruginosa cytochrome c551: role of electrostatic interactions on the hydrophobic collapse and transition state properties.. J Mol Biol, 289(5), 1459-1467.
- Travaglini-Allocatelli C, Cutruzzola F, Bigotti MG, Staniforth RA & Brunori M (1999) Folding mechanism of Pseudomonas aeruginosa cytochrome c(551): Role of electrostatic interactions on the hydrophobic collapse and transition state properties.. FASEB J, 13(7), A1387-A1387.
- Bigotti MG, Allocatelli CT, Staniforth RA, Arese M, Cutruzzolà F & Brunori M (1998) Equilibrium unfolding of a small bacterial cytochrome, cytochrome c551 from Pseudomonas aeruginosa.. FEBS Lett, 425(3), 385-390.
- Staniforth RA, Bigotti MG, Cutruzzolà F, Allocatelli CT & Brunori M (1998) Unfolding of apomyoglobin from Aplysia limacina: the effect of salt and pH on the cooperativity of folding.. J Mol Biol, 275(1), 133-148.
- Staniforth RA, Burston SG, Atkinson T & Clarke AR (1994) Affinity of chaperonin-60 for a protein substrate and its modulation by nucleotides and chaperonin-10.. Biochem J, 300 ( Pt 3), 651-658.
- Staniforth RA, Cortés A, Burston SG, Atkinson T, Holbrook JJ & Clarke AR (1994) The stability and hydrophobicity of cytosolic and mitochondrial malate dehydrogenases and their relation to chaperonin-assisted folding.. FEBS Lett, 344(2-3), 129-135.
- Staniforth RA, Burston SG, Smith CJ, Jackson GS, Badcoe IG, Atkinson T, Holbrook JJ & Clarke AR (1993) The energetics and cooperativity of protein folding: a simple experimental analysis based upon the solvation of internal residues.. Biochemistry, 32(15), 3842-3851.
- Jackson GS, Staniforth RA, Halsall DJ, Atkinson T, Holbrook JJ, Clarke AR & Burston SG (1993) Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle: implications for the mechanism of assisted protein folding.. Biochemistry, 32(10), 2554-2563.