Bomapin is a redox-sensitive nuclear serpin that affects responsiveness of myeloid progenitor cells to growth environment

<p>Abstract</p> <p>Background</p> <p>Haematopoiesis is a process of formation of mature blood cells from hematopoietic progenitors in bone marrow. Haematopoietic progenitors are stimulated by growth factors and cytokines to proliferate and differentiate, and they die via apoptosis when these factors are depleted. An aberrant response to growth environment may lead to haematological disorders. Bomapin (serpinb10) is a hematopoietic- and myeloid leukaemia-specific protease inhibitor with unknown function.</p> <p>Results</p> <p>We found that the majority of naturally expressed bomapin was located in the nucleus. Both the natural and recombinant bomapin had a disulfide bond which linked the only two bomapin cysteines: one located in the CD-loop and the other near the C-terminus. Computer modelling showed that the cysteines are distant in the reduced bomapin, but can easily be disulfide-linked without distortion of the overall bomapin structure. Low-level ectopic expression of bomapin in bomapin-deficient K562 cells resulted in about 90% increased cell proliferation under normal growth conditions. On the other hand, antisense-downregulation of natural bomapin in U937 cells resulted in a decreased cell proliferation. Bomapin C395S mutant, representing the reduced form of the serpin, had no effect on cell proliferation, suggesting that the disulfide bond-linked conformation of bomapin is biologically important. The bomapin-dependent effect was specific for myeloid cells, since ectopic expression of the serpin in HT1080 cells did not change cell proliferation. In contrast to the survival-promoting activity of bomapin in cells cultured under optimal growth conditions, bomapin enhanced cell apoptosis following growth factor withdrawal.</p> <p>Conclusions</p> <p>We propose that bomapin is a redox-sensitive nuclear serpin that augments proliferation or apoptosis of leukaemia cells, depending on growth factors availability.</p

Studies of protein structure, dynamics and protein-ligand interactions using NMR spectroscopy

In the first part of the thesis, protein-ligand interactions were investigated using the chaperone LcrH, from Yersinia as target protein. The structure of a peptide encompassing the amphipathic domain (residue 278-300) of the protein YopD from Yersinia was determined by NMR in 40% TFE. The structure of YopD278-300 is a well defined α-helix with a β-turn at the C-terminus of the helix capping the structure. This turn is crucial for the structure as peptides lacking the residues involved in the turn are unstructured. NMR relaxation indicates that the peptide is not monomeric. This is supported by intermolecular NOEs found from residue Phe280 to Ile288 and Val292 indicative of a multimeric structure with the helical structures oriented in an antiparallel manner with hydrophobic residues forming the oligomer. The interaction with the chaperone LcrH was confirmed by 1H relaxation experiments and induced chemical shift changes in the peptide Protein-ligand interactions were investigated further in the second paper using a different approach. A wide range of substances were used in screening for affinity against the chaperones PapD and FimC from uropathogenic Escherichia coli using 1H relaxation NMR experiments, surface plasmon resonance and 19F NMR. Fluorine NMR proved to be advantageous as compared to proton NMR as it is straight forward to identify binding ligands due to the well resolved 19F NMR spectra. Several compounds were found to interact with PapD and FimC through induced line-broadening and chemical shift changes for the ligands. Data corroborate well with surface plasmon resonance and proton NMR experiments. However, our results indicate the substances used in this study to have poor specificity for PapD and FimC as the induced chemical shift is minor and hardly no competitive binding is observed. Paper III and IV is an investigation of the structural features of the allergenic 2S albumin Ber e 1 from Brazil nut. Ber e 1 is a 2S albumin previously identified as the major allergen of Brazil nut. Recent studies have demonstrated that endogenous Brazil nut lipids are required for an immune response to occur in vivo. The structure was obtained from 3D heteronuclear NMR experiments followed by simulated annealing using the software ARIA. Interestingly, the common fold of the 2S albumin family, described as a right-handed super helix with the core composed of a helix bundle, is not found in Ber e 1. Instead the C-terminal region is participating in the formation of the core between helix 3, 4 and 5. The dynamic properties of Ber e 1 were investigated using 15N relaxation experiments and data was analyzed using the model-free approach. The analysis showed that a few residues in the loop between helix 2 and 3 experience decreased mobility, compared to the rest of the loop. This is consistent with NOE data as long range NOEs were found from the loop to the core region of the protein. The anchoring of this loop is a unique feature of Ber e 1, as it is not found in any other structures of 2S albumins. Chemical shift mapping of Ber e 1 upon the addition of lipid extract from Brazil nut identified 4 regions in the protein where chemical shift perturbations were detected. Interestingly, all four structural clusters align along a cleft in the structure formed by helix 1-3 on one side and helix 4-5 on the other. This cleft is big enough to encompass a lipid molecule. It is therefore tempting to speculate whether this cleft is the lipid binding epitope in Ber e 1

Solution structure, copper binding and backbone dynamics of recombinant Ber e 1 : the major allergen from brazil nut

BACKGROUND: The 2S albumin Ber e 1 is the major allergen in Brazil nuts. Previous findings indicated that the protein alone does not cause an allergenic response in mice, but the addition of components from a Brazil nut lipid fraction were required. Structural details of Ber e 1 may contribute to the understanding of the allergenic properties of the protein and its potential interaction partners. METHODOLOGY/PRINCIPAL FINDINGS: The solution structure of recombinant Ber e 1 was solved using NMR spectroscopy and measurements of the protein back bone dynamics at a residue-specific level were extracted using (15)N-spin relaxation. A hydrophobic cavity was identified in the structure of Ber e 1. Using the paramagnetic relaxation enhancement property of Cu(2+) in conjunction with NMR, it was shown that Ber e 1 is able to specifically interact with the divalent copper ion and the binding site was modeled into the structure. The IgE binding region as well as the copper binding site show increased dynamics on both fast ps-ns timescale as well as slower µs-ms timescale. CONCLUSIONS/SIGNIFICANCE: The overall fold of Ber e 1 is similar to other 2S albumins, but the hydrophobic cavity resembles that of a homologous non-specific lipid transfer protein. Ber e 1 is the first 2S albumin shown to interact with Cu(2+) ions. This Cu(2+) binding has minimal effect on the electrostatic potential on the surface of the protein, but the charge distribution within the hydrophobic cavity is significantly altered. As the hydrophobic cavity is likely to be involved in a putative lipid interaction the Cu(2+) can in turn affect the interaction that is essential to provoke an allergenic response

ICP analysis of Cu²⁺ content.

<p>ICP analysis of Cu²⁺ content.</p

Electrostatic surface potential of the Ber e 1 in absence and presence of Cu²⁺.

<p>a) Electrostatic surface potential of the Ber e 1 at pH 7 in absence of Cu²⁺. b) Electrostatic surface potential of the Ber e 1 at pH 7 in presence of Cu²⁺. The entry of the hydrophobic cavity is highlighted with a yellow square. The protein shows an overall positive charge on the side of the protein that comprises the hypervariable loop and the entry to the hydrophobic cavity. In contrast, the other side of the protein, in particular helix 1a and most of helix 1b and 2, is negatively charged. The presence of histidines, taken together with the slow dynamics and the overall negative charge between helix 1b and 2, suggests that Cu²⁺ would enter the molecule from the negatively charged side of the protein. However, binding of Cu²⁺ only to a small degree changed the net surface charge on the helix 1b-2 side of the protein. The largest difference in surface potential is observed within the hydrophobic cavity; tuning the surface potential inside the cavity from neutral to more positive charge.</p

Theroretical pepsin cleavage sites and solvent exposed residues.

<p>a) Theoretical pepsin cleavage sites (red) mapped on the tertiary structure of Ber e 1. b) Exposed surface area of the N and H^N atoms in the backbone of Ber e 1. Residues able to undergo pepsin cleavage are highlighted in red. The secondary structure elements, as well as the cysteine linkage are indicated in the top of the figure. Most of the theoretical pepsin cleavage sites are buried within the α-helices. The only surface exposed pepsin cleavage sites are located in the non-native loop and the C-terminal, and cleavage at these positions would not disrupt the integrity of the structure.</p

Structural statistics of the 12 lowest energy structures.

<p>Structural statistics of the 12 lowest energy structures.</p

Ber e 1 copper interaction.

<p>a) Paramagnetic copper relaxation enhancement experiment on Ber e 1. The spectrum shown in black is recorded in the absence of copper, whereas the spectrum shown in red has copper added in a 1∶1 (Cu²⁺:Ber e 1) stoichiometry. N-H groups in the backbone affected by paramagnetic relaxation enhancement by the addition of Cu²⁺ in a 1∶1 ration are HIS 20, CYS 21, ARG 22, TYR 24, GLU 43, HIS 45, SER 47, GLU 48, CYS 49, and GLN 52. In addition N-H groups form sidechains (s.c) of GLN 11, 13, 28 and 83 are also affected by Cu²⁺ at this stoichiometric ratio. b) A model of the copper atom positioning in Ber e 1, based on the nearby residues identified in (a). The N-H backbone groups that are bleached are indicated in the structure as orange rods. c) Due to the slow dynamics around the copper binding site, the copper atom is engulfed into the core of the protein. Interestingly, its position is very close to the bottom of the hydrophobic cavity.</p

Solution structure of Ber e 1.

<p>a) Stereo view of the backbone of the 12 lowest energy structures after energy minimization. b) Cartoon representation of the Ber e 1 structure, with the different structure elements colored as follows: green = helix 1a, cyan = helix 1b, yellow = helix 2, pink = helix 3, blue = helix 4. c) Location of the hydrophobic cavity in Ber e 1. The structure has been rotated 90 degrees with respect to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046435#pone-0046435-g001" target="_blank">figure 1a and b</a>.</p

Ber e 1 backbone dynamics.

<p>a) Residue-specific overall tumbling time (τ_m) values for 77 of 114 residues. The relatively uniform τ_m values indicate isotropic tumbling of Ber e 1. b) Order parameter, S², of the N-H bond vector on a per residue basis. An S² value of 1 equals a completely rigid N-H vector, whereas an S² value of 0 implies complete rotational freedom of the N-H vector. Some flexibility is observed in the hypervariable loop, and the N- and C-terminal, as well as the non-native loop show high flexibility. c) R_ex parameter, showing the residues where µs-ms dynamics could be identified. Slow dynamics is largely located to the interface of helix 1b and 2, as well as at the end of helix 3, leading into the hypervariable loop. The secondary structure elements, as well as the cysteine linkage are indicated in the top of each figure.</p