| 1. |
- Luheshi, Leila M., et al.
(författare)
-
Sequestration of the A beta Peptide Prevents Toxicity and Promotes Degradation In Vivo
- 2010
-
Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 8:3, s. e1000334-
-
Tidskriftsartikel (refereegranskat)abstract
- Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (A beta) peptide by using a small engineered binding protein (Z(A beta 3)) that binds with nanomolar affinity to A beta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(A beta 3) in the brains of Drosophila melanogaster expressing either A beta(42) or the aggressive familial Alzheimer's disease (AD) associated E22G variant of A beta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer A beta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of A beta aggregation and reveal that Z(A beta 3) not only inhibits the initial association of A beta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.
|
|
| 2. |
- Syed, Zulfeqhar Ali, et al.
(författare)
-
A potential role for Drosophila mucins in development and physiology.
- 2008
-
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:8
-
Tidskriftsartikel (refereegranskat)abstract
- Vital vertebrate organs are protected from the external environment by a barrier that to a large extent consists of mucins. These proteins are characterized by poorly conserved repeated sequences that are rich in prolines and potentially glycosylated threonines and serines (PTS). We have now used the characteristics of the PTS repeat domain to identify Drosophila mucins in a simple bioinformatics approach. Searching the predicted protein database for proteins with at least 4 repeats and a high ST content, more than 30 mucin-like proteins were identified, ranging from 300-23000 amino acids in length. We find that Drosophila mucins are present at all stages of the fly life cycle, and that their transcripts localize to selective organs analogous to sites of vertebrate mucin expression. The results could allow for addressing basic questions about human mucin-related diseases in this model system. Additionally, many of the mucins are expressed in selective tissues during embryogenesis, thus revealing new potential functions for mucins as apical matrix components during organ morphogenesis.
|
|
| 3. |
- Benrick, Anna, 1979, et al.
(författare)
-
A non-conservative polymorphism in the IL-6 signal transducer (IL6ST)/gp130 is associated with myocardial infarction in a hypertensive population.
- 2008
-
Ingår i: Regulatory peptides. - : Elsevier BV. - 0167-0115. ; 146:1-3, s. 189-96
-
Tidskriftsartikel (refereegranskat)abstract
- Inflammation is a key component in the development of atherosclerosis, and myocardial infarction (MI); therefore we investigated the association between an interleukin-6 signal transducer (IL6ST)/gp130 polymorphism, gp130 function and risk of MI. Structural modeling suggested that a non-conservative single nucleotide polymorphism in the gp130, Gly148Arg, can change the stability and functional properties of the molecule. In vitro studies were done with BAF/3 cells lacking endogenous gp130. Cells stably transfected with the gp130 148Arg variant proliferated less and showed slightly lower STAT-3 phosphorylation in response to gp130 stimulation as compared to cells transfected with gp130 148Gly. In a prospectively followed hypertensive cohort we identified 167 patients who suffered a MI during the study and compared them to matched controls (mean age 57 years, 73% males, n=482). Carriers of the 148Arg variant (f(Arg)=0.12) of the gp130 receptor had decreased odds ratio for MI in univariate analysis (0.56, 95% CI 0.34-0.91, p=0.02). In conclusion, a genetically determined structural variant of the IL-6 receptor subunit gp130 is, independently of other known risk factors, associated with decreased risk of MI. The variant is also associated with decreased IL-6 responsiveness and could lead to a configuration change in the gp130 receptor.
|
|
| 4. |
|
|
| 5. |
- Boutajangout, Allal, et al.
(författare)
-
Affibody-Mediated Sequestration of Amyloid beta Demonstrates Preventive Efficacy in a Transgenic Alzheimer's Disease Mouse Model
- 2019
-
Ingår i: Frontiers in Aging Neuroscience. - : Frontiers Media S.A.. - 1663-4365 .- 1663-4365. ; 11
-
Tidskriftsartikel (refereegranskat)abstract
- Different strategies for treatment and prevention of Alzheimer's disease (AD) are currently under investigation, including passive immunization with anti-amyloid beta (anti-A beta) monoclonal antibodies (mAbs). Here, we investigate the therapeutic potential of a novel type of A beta-targeting agent based on an affibody molecule with fundamentally different properties to mAbs. We generated a therapeutic candidate, denoted Z(SYM73)-albumin-binding domain (ABD; 16.8 kDa), by genetic linkage of the dimeric Z(SYM73) affibody for sequestering of monomeric A beta-peptides and an ABD for extension of its in vivo half-life. Amyloid precursor protein (APP)/PS1 transgenic AD mice were administered with Z(SYM73)-ABD, followed by behavioral examination and immunohistochemistry. Results demonstrated rescued cognitive functions and significantly lower amyloid burden in the treated animals compared to controls. No toxicological symptoms or immunology-related side-effects were observed. To our knowledge, this is the first reported in vivo investigation of a systemically delivered scaffold protein against monomeric A beta, demonstrating a therapeutic potential for prevention of AD.
|
|
| 6. |
- Bäckström, Stefan, et al.
(författare)
-
Crystallization and preliminary studies of the DNA-binding runt domain of AML1.
- 2001
-
Ingår i: Acta Crystallogr D Biol Crystallogr. - 0907-4449. ; 57:Pt 2, s. 269-71
-
Tidskriftsartikel (refereegranskat)abstract
- The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.
|
|
| 7. |
- Bäckström, Stefan, et al.
(författare)
-
The RUNX1 Runt domain at 1.25A resolution : a structural switch and specifically bound chloride ions modulate DNA binding.
- 2002
-
Ingår i: J Mol Biol. - 0022-2836. ; 322:2, s. 259-72
-
Tidskriftsartikel (refereegranskat)abstract
- The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.
|
|
| 8. |
- Dincbas-Renqvist, Vildan, et al.
(författare)
-
Thermodynamics of folding, stabilization, and binding in an engineered protein--protein complex.
- 2004
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 126:36, s. 11220-30
-
Tidskriftsartikel (refereegranskat)abstract
- We analyzed the thermodynamics of a complex protein-protein binding interaction using the (engineered) Z(SPA)(-)(1) affibody and it's Z domain binding partner as a model. Free Z(SPA)(-)(1) exists in an equilibrium between a molten-globule-like (MG) state and a completely unfolded state, wheras a well-ordered structure is observed in the Z:Z(SPA)(-)(1) complex. The thermodynamics of the MG state unfolding equilibrium can be separated from the thermodynamics of binding and stabilization by combined analysis of isothermal titration calorimetry data and a separate van't Hoff analysis of thermal unfolding. We find that (i) the unfolding equilibrium of free Z(SPA)(-)(1) has only a small influence on effective binding affinity, that (ii) the Z:Z(SPA)(-)(1) interface is inconspicuous and structure-based energetics calculations suggest that it should be capable of supporting strong binding, but that (iii) the conformational stabilization of the MG state to a well-ordered structure in the Z:Z(SPA)(-)(1) complex is associated with a large change in conformational entropy that opposes binding.
|
|
| 9. |
|
|
| 10. |
- Dogan, Jakob, et al.
(författare)
-
Thermodynamics of folding and binding in an affibody:affibody complex.
- 2006
-
Ingår i: Journal of molecular biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 359:5, s. 1305-15
-
Tidskriftsartikel (refereegranskat)abstract
- Affibody binding proteins are selected from phage-displayed libraries of variants of the 58 residue Z domain. Z(Taq) is an affibody originally selected as a binder to Taq DNA polymerase. The anti-Z(Taq) affibody was selected as a binder to Z(Taq) and the Z(Taq):anti-Z(Taq) complex is formed with a dissociation constant K(d)=0.1 microM. We have determined the structure of the Z(Taq):anti-Z(Taq) complex as well as the free state structures of Z(Taq) and anti-Z(Taq) using NMR. Here we complement the structural data with thermodynamic studies of Z(Taq) and anti-Z(Taq) folding and complex formation. Both affibody proteins show cooperative two-state thermal denaturation at melting temperatures T(M) approximately 56 degrees C. Z(Taq):anti-Z(Taq) complex formation at 25 degrees C in 50 mM NaCl and 20 mM phosphate buffer (pH 6.4) is enthalpy driven with DeltaH degrees (bind) = -9.0 (+/-0.1) kcal mol(-1)(.) The heat capacity change DeltaC(P) degrees (,bind)=-0.43 (+/-0.01) kcal mol(-1) K(-1) is in accordance with the predominantly non-polar character of the binding surface, as judged from calculations based on changes in accessible surface areas. A further dissection of the small binding entropy at 25 degrees C (-TDeltaS degrees (bind) = -0.6 (+/-0.1) kcal mol(-1)) suggests that a favourable desolvation of non-polar surface is almost completely balanced by unfavourable conformational entropy changes and loss of rotational and translational entropy. Such effects can therefore be limiting for strong binding also when interacting protein components are stable and homogeneously folded. The combined structure and thermodynamics data suggest that protein properties are not likely to be a serious limitation for the development of engineered binding proteins based on the Z domain.
|
|
